Pharmaceutical composition for preventing or treating nervous system tumors and application thereof

Abstract

The invention discloses a pharmaceutical composition for preventing or treating nervous system tumors and application of the pharmaceutical composition. The pharmaceutical composition comprises adult stem cells and isoquinoline alkaloids in a pharmaceutically effective amount. According to the present invention, the nervous system tumor treatment effect of the pharmaceutical composition containing the adult stem cells and the isoquinoline alkaloid is proved for the first time, the adult stem cells enhance the anti-tumor effect of the isoquinoline alkaloid, and the isoquinoline alkaloid reduces the risk of the adult stem cells in promoting the invasion / metastasis of the nervous system tumor.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a pharmaceutical composition for preventing or treating nervous system tumors and applications thereof. Background Art

[0002] Nervous system tumors include primary intracranial tumors originating from parenchymal cells, primary intracranial tumors originating from non-brain parenchymal cells, and metastatic tumors. Central nervous system tumors are a common type of nervous system tumors. Among them, tumors originating from brain glial cells (including astrocytes, oligodendrocytes, and ependymal cells) are called gliomas. According to the malignancy of gliomas, they are further divided into grades I to VI: grades I and II are collectively referred to as low-grade gliomas, and grades III to IV are collectively referred to as high-grade gliomas (malignant gliomas). Grade IV glioma is also called glioblastoma (glioblastoma multiforme), which is the most common (accounting for about 50% of all gliomas) and most malignant tumor in the brain, with astrocytes as the main component.

[0003] Malignant glioblastoma (GBM) is the most common and most aggressive primary malignant brain tumor in humans. Due to its high invasiveness, clinical treatment is difficult and has a high mortality rate. The current clinical treatment method for GBM is maximal surgical resection, followed by radiotherapy and chemotherapy. GBM is classified as a grade 4 central nervous system (CNS) tumor by the World Health Organization (WHO). The median survival of patients is only about 14 months, and the prognosis is usually poor. Specifically, surgery has the defects of being highly damaging to tissues or organs, prone to recurrence, and difficult to completely remove the lesions. Chemotherapy drugs, on the other hand, have the disadvantages of being highly damaging and prone to drug resistance.

[0004] Chemotherapy drugs such as 5-fluorouracil, temozolomide, and paclitaxel have certain efficacy in the treatment of glioblastoma, but they also have disadvantages such as side effects, limited efficacy, and individual differences. Among them, most anticancer drugs are difficult to enter the brain due to the low permeability of the blood-brain barrier (BBB), and are prone to drug resistance. After entering the brain, the targeting is poor and there are many adverse reactions. In clinical practice, the drug dose needs to be increased to obtain an effective therapeutic concentration in the brain.

[0005] Studies in recent years have shown that MSCs have certain clinical application prospects in the treatment of tumor diseases, immune regulation and tissue regeneration. Taking advantage of the multi-differentiation characteristics, current research mainly uses MSCs as seed cells for tissue repair and applies them to cell regeneration and tissue repair engineering. At the same time, MSCs are easy to extract in vitro, have low immunogenicity, repair specific tissues in vivo, and show strong immunoregulatory effects. Among them, MSCs can regulate a variety of immune functions including T and B lymphocytes, natural killer cells and antigen presenting cells, but currently, the general application is gene-edited adult stem cells.

[0006] Moreover, current studies have shown that MSCs have tumor tropism, can be "attracted" by tumor cells to the tumor lesion site, and can significantly inhibit the occurrence of tumors. With the discovery of its chemotactic effect on tumor cells and its low immunogenicity, MSCs are expected to become an ideal carrier for anti-tumor drugs, thereby achieving targeted treatment of tumor cells. However, different studies have different conclusions on the role of MSCs in the occurrence and development of tumor cells. Some researchers believe that MSCs have a promoting effect on the occurrence, development and deterioration of tumors at specific stages. Therefore, there has been controversy over the dual biological effects of MSCs in promoting or inhibiting tumors.

[0007] Defects and tumorigenic effects of MSCs in the treatment of GBM: MSCs are currently considered to be an important endogenous component in the formation of the tumor microenvironment, but their relationship with the occurrence and development of GBM in the brain and the formation of the tumor microenvironment is still unclear. Studies have shown that tumor cells interact with MSCs in the tumor microenvironment, reprogramming MSCs with anti-tumor effects, showing a tumor-promoting phenotype, thereby causing tumor growth. On the contrary, GBM cells also differentiate into chondrocytes and adipocytes under the influence of MSCs, and in rare cases differentiate into osteocytes, affecting the stability of the brain environment and the health of the body of GBM patients. At the same time, after co-culture of MSCs and GBM, the invasiveness of tumor cells in vitro is increased. Studies have shown that GBM and MSCs can form functional syncytia and enhance each other's migration ability. The interaction between MSCs and GBM can also occur through microvesicle exchange, which ultimately causes MSCs to acquire tumor-like characteristics, including enhanced migration and invasion capabilities. These characteristics are consistent with the epithelial-mesenchymal transition (EMT)-like phenotype, and its development is associated with increased tumor invasiveness. In addition, MSCs have multidirectional differentiation potential and may transform into tumor cells under certain conditions, thereby increasing the risk of cancer in patients. The malignant transformation of MSCs can be regulated not only by the interaction between GBM and MSCs, but also by cytokines secreted by other cells in the tumor microenvironment.

[0008] Current status of research on the treatment of central nervous system tumors (such as GBM) with isoquinoline alkaloids: Recent studies have shown that tetrandirne (Tet), a member of the isoquinoline alkaloids, effectively increases blood-brain barrier (BBB) ​​transport in vitro by inhibiting overexpressed P-glycoprotein on the blood-brain barrier (BBB). The latest study found that Tet can significantly inhibit the invasion of GBM cells in vitro. However, most of the studies found so far are in vitro experiments related to tetrandirne, and no in vivo experiments related to tetrandirne have been found, especially no in vivo experiments combining tetrandirne with adult stem cells have been found.

[0009] In summary, malignant proliferation and invasion are the characteristics of central nervous system tumor (especially GBM) cells. Although some studies have shown that adult stem cells (such as MSCs) have an inhibitory effect on the proliferation of central nervous system tumors (especially GBM), they may promote the migration and invasion of tumor cells. However, there are currently few studies on the combined use of adult stem cells with other drugs or treatment methods. Therefore, the purpose of the present invention is to explore the effect of the combined use of isoquinoline alkaloids and adult stem cells on the proliferation and invasion of central nervous system tumor cells, aiming to provide more strategies for the treatment of central nervous system tumors, and to provide ideas for combined medication for subsequent immunotherapy. Summary of the invention

[0010] Based on the defects of the prior art, the technical solution provided by the present invention is as follows:

[0011] In a first aspect, the present invention provides a pharmaceutical composition for preventing or treating nervous system tumors, comprising a pharmaceutically effective amount of adult stem cells and isoquinoline alkaloids.

[0012] Preferably, the adult stem cells are adult stem cells of human or animal origin.

[0013] More preferably, the adult stem cells include at least one of mesenchymal stem cells, neural stem cells, hematopoietic stem cells, and vascular endothelial stem cells originating from human or animal tissues.

[0014] Preferably, the isoquinoline alkaloids include bisbenzylisoquinoline and / or benzylisoquinoline.

[0015] More preferably, the isoquinoline alkaloid is at least one of tertrine, nortertrine, thalidomide, thalidomide, chelidonine, berberine, lycorine, vincristine, liesinine, neferine and sinomenine.

[0016] Preferably, the nervous system tumor comprises a central nervous system tumor.

[0017] More preferably, the central nervous system tumor comprises a glioma.

[0018] Further preferably, the glioma comprises glioblastoma.

[0019] Preferably, the pharmaceutical composition is administered via at least one of the nasocerebral administration route, the ocular administration route, the oral administration route, and the injection administration route.

[0020] In a second aspect, the present invention provides a pharmaceutical composition for preventing or treating a nervous system tumor for use in preparing a drug for preventing or treating a nervous system tumor. Preferably, the nervous system tumor includes a central nervous system tumor. More preferably, the central nervous system tumor includes a glioma. Further preferably, the glioma includes a glioblastoma.

[0021] The beneficial effects of the present invention include at least:

[0022] (1) The present invention demonstrates for the first time the efficacy of a pharmaceutical composition comprising adult stem cells and isoquinoline alkaloids (especially Tet) in treating nervous system tumors (especially GBM), wherein not only do adult stem cells enhance the anti-tumor effect of isoquinoline alkaloids, but isoquinoline alkaloids also reduce the risk of adult stem cells promoting the invasion / metastasis of nervous system tumors (especially GBM).

[0023] In particular, the present invention reveals the interaction between homologous mesenchymal stem cells (MSCs) and glioblastoma (GBM), deeply studies the effects of MSCs on the affinity, proliferation, migration and invasion of GBM cells, and reveals that MSCs can both protect and repair the nervous system cells around tumor cells in the treatment of GBM.

[0024] (2) The present invention is the first to implement the administration of adult stem cells and isoquinoline alkaloids (especially Tet) via the nose-to-brain route, allowing the drug to directly bypass the blood-brain barrier and enter the brain, significantly improving the drug concentration in the brain and the therapeutic effect.

[0025] (3) The present invention establishes in vivo and in vitro experimental models that are closer to actual clinical situations. In particular, the present invention uses mice with normal immune function and homologous GBM cell lines to establish in vivo and in vitro experimental models that are closer to actual clinical situations, which can better demonstrate the therapeutic effect of the pharmaceutical composition of the present invention.

[0026] Other features and advantages of the present invention will be described in detail in the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The main purpose of the embodiment of the present invention is shown.

[0028] Figure 2 The overall design framework of the embodiment of the present invention is shown.

[0029] Figure 3 The Transwell experiment showed that MSCs had affinity for GBM.

[0030] Figure 4 Shows the transfection efficiency of MSCs.

[0031] Figure 5 Shows fluorescence tracking of mouse MSCs cells in vitro.

[0032] Figure 6 Shows fluorescence tracking of mouse-derived MSCs cells in vivo.

[0033] Figure 7 A line graph showing the effects of different treatment conditions on the proliferation rate of GBM cells.

[0034] Figure 8 Shown are the effects of the control group or MSCs-conditioned medium group on GBM cell migration.

[0035] Fig. 9 The mRNA expression levels related to EMT invasion are shown.

[0036] Fig.10 Show the expression levels of proteins related to EMT invasion.

[0037] Fig.11 Shows the effects of different Tet drug concentrations and treatment times on GBM cell viability.

[0038] Fig.12 A graph showing the mRNA expression level of vimentin.

[0039] Fig.13 The mRNA expression levels of β-catenin are shown.

[0040] Fig.14 A graph showing the mRNA expression level of N-cadherin.

[0041] Fig.15 The mRNA expression levels of E-cadherin are shown.

[0042] Fig.16 The expression levels of EMT and invasion-related proteins in the control group, 20 μmol / L Tet group, 20 μmol / L Te+MSCs group, and MSCs group are shown.

[0043] Fig.17The results showed that in the established subcutaneous tumor-bearing mouse model, the GBM tumor weights in the 20 mg / Kg Tet, 20 mg / Kg Tet+MSCs and MSCs groups were significantly decreased, but the effect of the Tet and MSCs combination group was significantly better than that of the other groups.

[0044] Fig.18 Figure showing immunohistochemistry of subcutaneous tumor tissue.

[0045] Fig.19 Images showing H&E staining of intracranial tumors.

[0046] Fig. 20 The mRNA expression levels of vimentin are shown.

[0047] Fig.21 The mRNA expression levels of β-catenin are shown.

[0048] Fig. 22 The mRNA expression levels of N-cadherin are shown.

[0049] Fig.23 The mRNA expression levels of E-cadherin are shown.

[0050] Fig.24 The histone expression levels of the control group, 20 mg / Kg Tet, 20 mg / Kg Tet+MSCs group and MSCs are shown.

[0051] Fig.25 Shows the immunohistochemistry and quantification of intracranial GBM transplanted tumors. DETAILED DESCRIPTION

[0052] In order to make the purpose, technical scheme and beneficial effects of the present invention clearer, the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments. Examples of the embodiments are shown in the accompanying drawings. It should be understood that the specific embodiments described in the following embodiments of the present invention are only exemplary descriptions of the specific embodiments of the present invention, and are intended to be used to explain the present invention, but do not constitute a limitation of the present invention.

[0053] In the present invention, the terms "comprise", "include", etc. may be open, semi-closed or closed.

[0054] Unless defined otherwise, all technical and scientific terms used in the present invention have the same meaning as commonly understood by one of ordinary skill in the art.

[0055] The endpoints and any values ​​of the ranges disclosed in the present invention are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values ​​(such as ± 5%). For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be regarded as specifically disclosed in this article. In the description of the present invention, unless otherwise specified, the meaning of "multiple / multiple" and similar words is two / kinds or more than two / kinds.

[0056] In a first aspect, the present invention provides a pharmaceutical composition for preventing or treating nervous system tumors, comprising a pharmaceutically effective amount of adult stem cells and isoquinoline alkaloids.

[0057] In the present invention, adult stem cells such as mesenchymal stem cells (MSCs) are a kind of multipotent stem cells derived from the mesoderm, which have self-renewal and multidirectional differentiation capabilities. MSCs can not only be directly isolated from the bone marrow, but also be derived from non-hematopoietic multipotent stem cells obtained from tissues such as the umbilical cord, fat, placenta and muscle, which can differentiate into various cells such as adipocytes, osteoblasts and chondrocytes in vivo and in vitro. Adult stem cells have a tendency to the damaged area, and adult stem cells can protect and repair the nervous system cells around tumor cells. Therefore, the tendency of adult stem cells to tumor tissue can improve the targeting of treatment, and adult stem cells (especially through the nose and brain administration route) have application value in the treatment of nervous system tumors.

[0058] In the present invention, MSCs that meet the definition of the International Society for Cellular Therapy (ISCT) are selected, and these cells have self-renewal and multidirectional differentiation potential. In some preferred embodiments, the patient's own MSCs (or homologous adult stem cells) are used to reduce immune rejection. In other embodiments, if autologous cells are not available or insufficient in number, allogeneic MSCs (or heterologous adult stem cells) can be used.

[0059] In the present invention, MSCs are cultured and expanded in a sterile, strictly controlled environment, and specific culture medium and growth factors are used to promote the proliferation of MSCs and maintain their undifferentiated state.

[0060] In some preferred embodiments, the adult stem cells are adult stem cells of human or animal origin.

[0061] In some more preferred embodiments, the adult stem cells include at least one of mesenchymal stem cells, neural stem cells, hematopoietic stem cells, and vascular endothelial stem cells originating from human or animal tissues.

[0062] In some preferred embodiments, the human or animal tissue includes at least one of umbilical cord, umbilical cord blood, bone marrow, fat, muscle, nerve, skin, amnion, and placenta.

[0063] In the present invention, "isoquinoline alkaloids" and "isoquinoline alkaloids" are used interchangeably.

[0064] In the present invention, isoquinoline alkaloids, such as tetrandirne (Tet) in the dibenzylisoquinoline alkaloids, are the main active ingredients of the traditional Chinese medicine tetrandrone, are naturally fat-soluble, and are also called tetrandrin. Tet is a calcium channel blocker with good safety and no obvious toxic and side effects on the body. At the same time, Tet has pharmacological effects such as antipyretic, angiogenesis inhibition, anti-inflammatory and antibacterial, inhibits the proliferation of tumor cells and induces apoptosis, reverses the multidrug resistance of various tumors, reduces the toxic and side effects of chemotherapy drugs, enhances the sensitivity of radiotherapy and chemotherapy to tumor cells, and inhibits angiogenesis and metastasis.

[0065] In the present invention, "isoquinoline alkaloids" are mainly used to kill tumor cells. In some preferred embodiments, the isoquinoline alkaloids include bisbenzylisoquinoline and / or benzylisoquinoline.

[0066] In some more preferred embodiments, the isoquinoline alkaloid is at least one of tertrine, nortertrine, thalidomide, thalidomide, chelidonine, berberine, lycorine, vincristine, liesinine, neferine and sinomenine.

[0067] In some preferred embodiments, the nervous system tumor comprises a central nervous system tumor.

[0068] In some more preferred embodiments, the central nervous system tumor comprises a glioma.

[0069] In some further preferred embodiments, the glioma comprises glioblastoma.

[0070] In some most preferred embodiments, the pharmaceutical composition is a pharmaceutical composition comprising tetrandrine and homologous adult stem cells for in situ treatment of glioblastoma.

[0071] In some preferred embodiments, the pharmaceutical composition is administered via at least one of a nose-brain administration route, an eye administration route, an oral administration route, and an injection administration route.

[0072] In some more preferred embodiments, the injection administration route is direct intracerebral injection under image guidance.

[0073] In some more preferred embodiments, the pharmaceutical composition is administered via a nose-brain administration route.

[0074] In a second aspect, the present invention provides the use of a pharmaceutical composition for preventing or treating a nervous system tumor in the preparation of a medicament for preventing or treating a nervous system tumor. In some preferred embodiments, the nervous system tumor comprises a central nervous system tumor. In some more preferred embodiments, the central nervous system tumor comprises a glioma. In some further preferred embodiments, the glioma comprises a glioblastoma.

[0075] The pharmaceutical composition of the present invention is used to prevent and / or treat nervous system tumors (such as glioblastoma) diseases. In the present invention, "pharmaceutical composition" refers to a preparation of a compound of the present invention and a medium generally accepted in the art for delivering biologically active compounds to mammals (such as humans). The medium includes a pharmaceutically acceptable carrier. The purpose of the pharmaceutical composition is to promote administration of an organism, facilitate the absorption of the active ingredient, and thus exert biological activity.

[0076] The term "pharmaceutically acceptable" as used herein refers to a substance (such as a carrier or diluent) that does not affect the biological activity or properties of the compounds of the present invention and is relatively non-toxic, i.e., the substance can be administered to an individual without causing an adverse biological response or interacting in an adverse manner with any component contained in the composition.

[0077] In the present invention, "pharmaceutical excipients" include but are not limited to any adjuvant, carrier, excipient, glidant, sweetener, diluent, preservative, dye / colorant, flavoring agent, surfactant, wetting agent, dispersant, suspending agent, stabilizer, isotonic agent, solvent or emulsifier approved by the relevant government regulatory authorities as acceptable for human or livestock use.

[0078] As used herein, the term "preventing" includes reducing the likelihood of a disease or condition occurring or becoming worse in a patient.

[0079] The term "treatment" and other similar synonyms used in the present invention include the following meanings:

[0080] (i) preventing a disease or condition from occurring in a subject, particularly where such subject is susceptible to the disease or condition but has not yet been diagnosed with the disease or condition;

[0081] (ii) inhibiting a disease or condition, i.e. arresting its development;

[0082] (iii) alleviate the disease or condition, that is, cause regression of the disease or condition; or

[0083] (iv) alleviating the symptoms caused by the disease or condition.

[0084] As used herein, the terms "effective amount", "therapeutically effective amount" or "pharmaceutically effective amount" refer to an amount of at least one agent or compound sufficient to relieve to some extent one or more symptoms of the disease or condition being treated after administration. The result may be a reduction and / or alleviation of signs, symptoms or causes, or any other desired change in a biological system. For example, an "effective amount" for treatment is the amount of a composition comprising a compound disclosed herein that is required to provide a clinically significant symptom alleviation effect. Techniques such as dose escalation trials may be used to determine the effective amount appropriate for any individual case.

[0085] The terms "administering", "administering", "administering", etc. used in the present invention refer to methods that can deliver a compound or composition to a desired site for biological action. These methods include, but are not limited to, oral routes, intraduodenal routes, parenteral injections (including intravenous, subcutaneous, intraperitoneal, intramuscular, intraarterial injections or infusions), topical administration, and rectal administration. Those skilled in the art are familiar with the administration techniques that can be used for the compounds and methods described herein.

[0086] In the present invention, "in combination with" or "in conjunction with" refers to administering another treatment modality in addition to a treatment modality (such as drug therapy). Therefore, these terms refer to administering another treatment modality before, during, or after administering one treatment modality to a patient.

[0087] As used herein, the terms "drug combination", "drug combination", "combination therapy", "administration of other treatments", "administration of other therapeutic agents" and the like refer to drug treatments obtained by mixing or combining more than one active ingredient, including fixed and non-fixed combinations of active ingredients. The term "fixed combination" refers to the simultaneous administration of at least one isoquinoline alkaloid, at least one adult stem cell and optional other auxiliary ingredients to a patient in the form of a single entity or a single dosage form. The term "non-fixed combination" refers to the simultaneous administration, combined administration or sequential administration at variable intervals of at least one isoquinoline alkaloid, at least one adult stem cell and optional auxiliary ingredients to a patient in the form of separate entities.

[0088] When using a pharmaceutical composition, a safe and effective amount of the compound of the present invention is applied to a subject in need of prevention or treatment (the subject of treatment includes mammals, such as humans). Of course, the specific dosage should also take into account factors such as the route of administration, the health status of the subject in need of prevention or treatment, and these are all within the skill range of skilled physicians.

[0089] In some preferred embodiments, drugs are administered through the nose-brain route to treat glioblastoma, and drugs are directly delivered to the brain through the nasal cavity, aiming to bypass the blood-brain barrier (BBB) ​​and increase the concentration and therapeutic effect of drugs in the brain. The blood-brain barrier (BBB) ​​is composed of astrocytes, basement membranes, endothelial cells, pericytes, and extracellular matrix. It is a barrier between brain tissue and blood, which can prevent macromolecules, toxins, and drugs in the body's blood from entering the brain tissue, which is beneficial to maintaining homeostasis in the body. Due to the existence of the BBB, it is difficult for GBM-related drugs to reach the tumor and its microenvironment, thereby limiting the entry of a variety of anticancer drugs into the brain. Therefore, clinically, it is usually necessary to increase the drug dose to obtain an effective therapeutic concentration in the brain, but it will cause greater damage to the body. In order for drugs to overcome the BBB and reach GBM lesions, new methods of administration are urgently needed. Among them, the nose-brain route of administration is a non-invasive alternative that can bypass the BBB to treat central nervous system (CNS) diseases and reduce systemic adverse reactions. The nasal cavity is composed of the vestibular area, respiratory area and olfactory area, among which the olfactory area is a weak area of ​​BBB and is the main absorption site of nasal-brain drug delivery. Nasal-brain drug delivery mainly enters the central nervous system through the olfactory or trigeminal nerve, and the pathways used include intracellular and extracellular pathways. The nasal-brain drug delivery route provides a non-invasive method for delivering therapeutic agents directly to the CNS, in which a variety of therapeutic compounds or biological agents such as peptides, proteins, oligonucleotides, nanoparticles, viral vectors and stem cells can be delivered. Studies have shown that a variety of natural medicines and active ingredients have obvious inhibitory effects on a variety of malignant tumors, among which some stem cells derived from the body have a significant tendency to GBM and can inhibit tumor proliferation, so it can provide a new means to prolong the survival of GBM patients and improve the prognosis. In short, the nasal-brain drug delivery route is a drug delivery method that bypasses the BBB by utilizing the unique anatomical structure connecting the brain to the external environment. Compared with other CNS drug delivery methods, nasal-brain drug delivery, as a simple, non-invasive alternative drug delivery route, has the advantages of high bioavailability, less trauma, fewer adverse reactions, and can prevent gastrointestinal reactions and first-pass effects. The present invention administers adult stem cells for the first time via the nose-to-brain route. After entering the body, the adult stem cells will automatically aggregate to damaged organs or parts and differentiate into required specific cells to replace and repair damaged and dead cells, promote tissue repair and regeneration, and the present invention proves that the nose-to-brain route of administration has application prospects in the drug treatment of GBM and other nervous system tumors (especially the brain).

[0090] In some preferred embodiments, the dosage form for the nose-to-brain administration route includes, but is not limited to, nasal drops and sprays.

[0091] In the present invention, the administration method of the nose-brain administration route includes using nasal spray, nasal drops or nasal cannula to directly deliver the drug to the nasal mucosa, ensuring that the drug is evenly distributed in the nasal cavity and fully contacts with the nasal mucosa.

[0092] In the present invention, the administration route of the nose-brain administration route includes direct and indirect routes, wherein: the direct route includes: allowing the drug to directly enter the brain through the olfactory nerve pathway and the trigeminal nerve pathway. The olfactory nerve pathway is the shortest and most direct route, and the drug can quickly reach the olfactory bulb and be distributed to different areas of the brain. The trigeminal nerve pathway allows the drug to directly enter the tail of the brain, such as the pons and medulla oblongata, through its maxillary branch and ophthalmic branch. The indirect route includes: the drug is absorbed from the nasal mucosa into the blood circulation, and then reaches the brain through the blood-brain barrier.

[0093] In the present invention, the dosage of the nasal-cerebral administration route is determined based on the toxicity, efficacy of the drug and the specific conditions of the patient. Considering the absorption capacity of the nasal mucosa to the drug and the bioavailability of the drug, it is ensured that the dosage can effectively treat the tumor without causing serious side effects on the nasal mucosa and the brain.

[0094] In the present invention, the frequency of administration of the nose-brain route is determined according to the half-life of the drug, the duration of drug effect, and the changes in the patient's condition. Usually, it is necessary to continue to administer the drug for a period of time to maintain the effective concentration of the drug in the brain and achieve the best therapeutic effect.

[0095] In some embodiments, the dosage of isoquinoline alkaloids administered in animals is 5 mg / kg-40 mg / kg per day. In some preferred embodiments, the dosage of isoquinoline alkaloids administered in animals is 20 mg / kg per day.

[0096] In some preferred embodiments, the dosage of isoquinoline alkaloids administered in animals is 10 mg / kg-40 mg / kg per day. In some preferred embodiments, the dosage of isoquinoline alkaloids administered in animals is 20 mg / kg per day.

[0097] In some embodiments, the adult stem cells are administered to an animal at a dose of 0.5×10 5 pcs / kg-2×10 7 Pieces / kg.

[0098] In some preferred embodiments, the dosage of adult stem cells administered to an animal is 0.5×10 6 pcs / kg-1×10 7 Pieces / kg.

[0099] Example

[0100] It should be noted that, unless otherwise specified, the various materials and reagents used in the following examples are commonly used materials and reagents in the art and can be obtained through conventional commercial channels. If the experimental methods in the following examples do not specify specific conditions, they are usually implemented according to conventional conditions or the conditions adopted by the partners.

[0101] The embodiments of the present invention establish a highly stable GBM in vitro culture model, homologous orthotopic transplantation, and a subcutaneous tumor-bearing mouse in vivo model to explore the following questions (such as Figure 1 The results are as follows: ① The effect of MSCs on the occurrence and proliferation of homologous GBM, and the correlation between the deterioration and invasion of homologous GBM; ② The effect of different concentrations of Tet on the proliferation and invasion of homologous GBM cells; ③ Whether the combined treatment of GBM with Tet and MSCs can improve the therapeutic effect of Tet or MSCs alone, and compensate for the treatment risk that MSCs may promote GBM invasion.

[0102] Example 1

[0103] The overall design framework of the embodiment of the present invention is as follows Figure 2 Shown

[0104] (1) Detecting the effects of mouse mesenchymal stem cells (MSCs) on the affinity, proliferation, migration and invasion of the mouse glioblastoma (GBM) cell line G422

[0105] ① Transwell experiment was used to detect the affinity of mouse MSCs to the mouse GBM cell line G422. The lower layer of the Transwell chamber was inoculated with primary mixed cultured mouse astrocytes as the control group, and the lower layer of the Transwell chamber was inoculated with GBM cells as the experimental group. The upper layer of the Transwell chambers of the two groups was inoculated with MSCs at the same time, and the indirect co-culture was performed for 24 hours, and each well was repeated 3 times.

[0106] ② Red fluorescent marker plasmid transfection. Using pCMV-RFP plasmid as the transfection vector for red fluorescent protein, mouse MSCs were transfected with Lipo3000 for 48h and 72h, and the transfection efficiency was observed under a fluorescence microscope at two time periods, where the plasmid transfection efficiency = number of fluorescent cells per high-power field of view / total number of cells in the same field of view × 100%.

[0107] ③ In vitro fluorescence tracing of mouse MSCs. The lower layer of the Transwell chamber was inoculated with primary mixed cultured mouse astrocytes as the control group, and the lower layer of the Transwell chamber was inoculated with mouse GBM cells as the experimental group. The upper layer of the Transwell chamber in both groups was inoculated with mouse MSCs labeled with red fluorescence, and the indirect co-culture was performed for 24 hours.

[0108] ④ In vivo fluorescence tracing of mouse MSCs cells. The expression of mouse MSCs in normal brain tissue and brain orthotopic transplanted tumors was tracked through brain tissue sections. Using the successfully modeled orthotopic transplanted tumor model, the tumor area and the corresponding non-tumor area were taken to culture primary cells, and then the fluorescence expression area was observed under a fluorescence microscope.

[0109] ⑤ Use CCK-8 to detect the effect of mouse MSCs on the proliferation of mouse GBM cell line G422 cells. The experiment was divided into two groups, GBM cells cultured in complete medium for 24 hours as the control group, and GBM cells cultured in MSCs conditioned medium for 24 hours as the experimental group. The well-grown GBM cells were digested and centrifuged, and 2000 cells were inoculated in 96-well plates per 200 μL / well. Cell viability was detected by CCK-8 assay at 0h, 24h, 48h, and 72h.

[0110] ⑥ The scratch test was used to detect the effect of mouse MSCs on the migration of mouse GBM cell line G422 cells. The experiment was divided into 2 groups, GBM cells cultured in complete medium for 24 hours as the control group, and GBM cells cultured in MSCs conditioned medium for 24 hours as the experimental group. When the cells filled the 6-well plate, the cell scratch test was performed, and the control group and the experimental group were cultured for another 24 hours to calculate the cell scratch wound healing area.

[0111] ⑦qRT-PCR and Western Blot were used to detect the effect of mouse MSCs on the mRNA and protein levels of invasion-related proteins in the mouse GBM cell line G422. The experiment was divided into two groups, GBM cells cultured in complete medium for 24 hours as the control group, and GBM cells cultured in MSCs conditioned medium for 24 hours as the experimental group. In order to further explore the effect of MSCs on GBM invasion, the mRNA expression levels of EMT and invasion-related proteins such as vimentin, β-catenin, N-cadherin, and E-cadherin were analyzed by qRT-PCR experiments.

[0112] (2) Effects of combined use of mouse MSCs and tetrandrine (Tet) on the mouse GBM cell line G422

[0113] ① CCK-8 was used to detect the effect of Tet on the proliferation of the murine GBM cell line G422 cells. The experiment was divided into 5 concentration gradients according to the different drug concentrations. The GBM cells cultured in complete medium for 24 h were the control group, and the Tet drugs were divided into 5μmol / L Tet group, 10μmol / L Tet, 20μmol / L Tet and 40μmol / L Tet.

[0114] ②qRT-PCR and Western Blot were used to detect the effect of the combined use of mouse MSCs and Tet on the mRNA and protein levels of invasion proteins related to mouse GBM cell line G422 cells. In order to explore the effect of the combined use of MSCs and Tet, the cells were divided into a control group, 20μmol / L Tet, 20μmol / L Tet+MSCs group and MSCs group. The mRNA expression levels of EMT and invasion-related proteins such as vimentin, β-catenin, N-cadherin, and E-cadherin were detected by qRT-PCR.

[0115] ③Construction of subcutaneous tumor-bearing mouse model. In order to further explore the effects of the combined use of MSCs and Tet on the proliferation and invasion of GBM in vivo, a subcutaneous tumor-bearing model was established in BALB / c mice, which were divided into control group, 20 mg / Kg Tet, 20 mg / Kg Tet+MSCs group and MSCs group.

[0116] ④Construction of intracranial orthotopic transplant tumor model in mice. In order to further explore the effect of combined use of mouse MSCs and Tet on the proliferation and invasion of mouse GBM in situ tumors in the intracranial orthotopic transplant tumor model, an orthotopic transplant tumor model was established in the brain of BALB / c mice, and the experiment was divided into control group, 20 mg / Kg Tet, 20 mg / Kg Tet+MSCs group and MSCs group.

[0117] (1) Main experimental materials, working fluid preparation and equipment

[0118] a. Main experimental materials

[0119]

[0120]

[0121]

[0122] b. Preparation method of experimental working solution

[0123] (b-1) Sodium citrate buffer

[0124] Dissolve and mix according to the instructions for use of sodium citrate buffer (ready-to-use dry powder), add distilled water to make up to 1000 mL and use immediately.

[0125] (b-2) 10% fetal bovine serum complete medium

[0126] Add 50 mL of fetal bovine serum, 5 mL of 1% double antibody, dilute to 500 mL with DMEM medium, and store at 4°C for later use.

[0127] (b-3) Cell freezing medium

[0128] 92% volume of complete medium and 8% DMSO were prepared as needed and used immediately.

[0129] (b-4) PBS solution

[0130] PBS buffer (dry powder) was prepared according to the instructions for use. One packet of dry powder was diluted to 2 L with double distilled water, dissolved and mixed, and sterilized by high pressure and stored at 4°C for later use.

[0131] (b-5) TBST solution

[0132] TBS buffer (powder) was prepared according to the instructions, with double distilled water added to 2 L, and then 2 mL of Tween-20 was added at a ratio of 1000:1. After fully dissolved, it was stored at 4°C for later use.

[0133] (b-6) 0.1% DEPC solution

[0134] Measure 0.5 mL of DEPC and dissolve it in 500 mL of double distilled water. After it is fully dissolved, store it at 4°C for later use.

[0135] (b-7)RNase-Free ddH 2 O

[0136] Dispense 0.1% DEPC solution into centrifuge tubes, sterilize by high pressure at 121°C for 30 min, and then dispense for use.

[0137] (b-8) 10% ammonium persulfate (APS)

[0138] Weigh 0.5 g of ammonium persulfate, add 5 mL of double distilled water to completely dissolve, and store at 4 °C.

[0139] (b-9) Electrophoresis fluid

[0140] Take 15.1g of Tris, 94g of glycine, and 5.0g of SDS, add double distilled water to 1000mL, mix thoroughly to make 5× electrophoresis buffer, and store at 4°C. During electrophoresis, dilute electrophoresis buffer (5×) and double distilled water in a ratio of 1:4 to make electrophoresis buffer (1×).

[0141] (b-10) Transfer buffer

[0142] Weigh 30.3 g of Tris and 151.1 g of glycine respectively, add double distilled water to 1000 mL, stir thoroughly until clear, and make 10× transfer buffer, and store at 4°C. When transferring, add 10× transfer buffer, methanol and double distilled water in a ratio of 1:2:7 to make 1× transfer buffer.

[0143] (b-11) 5% BSA blocking solution

[0144] Weigh 5 g of BSA powder, add 100 mL of 1×TBST solution, fully dissolve, prepare 5% BSA blocking solution, and store at 4°C.

[0145] (b-12) Immunoblotting Chemiluminescent Developer

[0146] Reagent A and reagent B were diluted in a 1:1 ratio, prepared and used as needed, and stored away from light.

[0147] (b-13) 1% sodium pentobarbital

[0148] Weigh 0.5 g of sodium pentobarbital and dissolve it in 50 mL of distilled water, and store it at room temperature for later use. According to the body weight of BALB / c mice, the intraperitoneal injection dose is 0.1 mL / 20 g.

[0149] (b-14) Preparation of Tetrandrine (Tet)

[0150] Cell experiment: Dissolve 5g of tetrandrine in DMSO to a concentration of 20mmol / L and store in a -80℃ refrigerator for later use;

[0151] In vivo experiment: Tetrandrine was diluted with PEG 300 reagent and normal saline in a ratio of 1:1 to a concentration of 20 mg / Kg, and ultrasonicated for 10 min. The mixture was used immediately after preparation.

[0152] c. Experimental instruments, equipment and consumables

[0153]

[0154]

[0155]

[0156] (2) Experimental methods

[0157] a. Experimental Grouping

[0158] (a-1) In vitro experiment: divided into four groups: control group (0.1% DMSO), 20 μmol / L Tet, 20 μmol / L Te+MSCs group and MSCs group.

[0159] (a-2) In vivo experiment: Subcutaneous tumor-bearing mice and intracranial orthotopic transplanted tumors were divided into four groups: control group (PEG 300: normal saline = 1:1), 20 mg / Kg Tet group, 20 mg / Kg Tet+MSCs group and MSCs group.

[0160] b. Cell culture

[0161] Mouse G422 GBM cells and mouse MSCs were purchased from Shanghai Xuanya Biotechnology Co., Ltd., and mouse normal brain glial cells BV2 were purchased from Xiamen Yimo Biotechnology Co., Ltd. The cells were cultured in 5% CO 2 , 37 ℃ incubator, using high-glucose DMEM medium containing 10% fetal bovine serum (FBS), 100μg / mL streptomycin and 100U / mL penicillin. According to the cell status, the medium was changed every 1-2 days. When the cell growth reached 85%-90%, 1mL 0.25% trypsin-EDTA was added to digest the cells for cell passage and cell cryopreservation.

[0162] (b-1) Cell passaging

[0163] Preheat the prepared culture medium, autoclaved PBS and trypsin in a 37°C water bath in advance, observe the cells under a microscope, discard the original culture medium when the cell growth reaches 85%-90% of the culture bottle, wash 3 times with PBS, add 1mL trypsin to digest the cells, observe the cells become brighter and shrink under the microscope for about 1min, add 2mL culture medium to stop digesting the cells, gently blow the culture bottle into a 15mL centrifuge tube, centrifuge at 1000rpg for 10min, discard the supernatant, add 2mL complete culture medium to resuspend the cells, and inoculate them into the culture bottle at a ratio of 1:2. Place in an incubator at 37°C, 5% CO 2 The cells were cultured under the following conditions. The next day, the cells were observed under a microscope, the original culture medium was discarded, the cells were washed with PBS three times, and fresh culture medium was replaced to continue the culture.

[0164] (b-2) Cell recovery

[0165] Add 4-5 mL of new culture medium to the culture flask, take out the cell cryopreservation tube from the liquid nitrogen tank, and quickly put it into a 37°C water bath to thaw for about 1 minute. Use a pipette to transfer the cell suspension to a new culture flask, and then put it into a 5% CO 2 , culture in a 37°C incubator, observe the cell growth status under a microscope every other day, and replace the culture medium with new one under aseptic operation in a clean bench.

[0166] (b-3) Cell cryopreservation

[0167] Prepare a cryopreservation solution containing 92% complete medium + 8% DMSO in advance, and precool it in a 4°C refrigerator for later use. When the cells grow to about 90%, wash them with PBS three times, add trypsin to digest for about 1 minute, observe under a microscope until the cells are detached, add 2mL of new culture medium to stop digestion, collect the cell fluid into a 15mL centrifuge tube, centrifuge at 1000rpg for 10min, remove the supernatant, add 1mL of cryopreservation solution to mix the cells and then transfer them to cryopreservation tubes. Mark the cryopreservation tubes (freezing date, cell name, operator name), put the cells into a gradient cryopreservation box, store them in a -80°C refrigerator, and move them to a liquid nitrogen tank to store the cells the next day.

[0168] (b-4) Cell counting

[0169] Digest the cells with trypsin, discard the supernatant after centrifugation, add 1 mL of culture medium to resuspend the cells, take 10 μL of the resuspended solution and add it to the counting plate, and count the sum of the number of cells in the four large grids under a microscope. (The sum of the number of cells in the four large grids / 4)×10 4 = number of cells per mL

[0170] c.CCK-8 experiment

[0171] When the cells were growing well, the cells were collected and counted, and the cell density was diluted to 2 × 10 4 200 μL of cell suspension was inoculated into each well of a 96-well plate, and PBS was dripped around each well. Three replicate wells were made for each group and placed in a 37°C, 5% CO 2 Culture overnight in a constant temperature incubator. Remove the original culture medium, wash 3 times with PBS, add MSC-conditioned medium or different concentrations of Tet medium according to the purpose of the experiment and continue to culture. The control group is 0.1% DMSO. Take out the 96-well plate at different time points, add 10 μL of CCK-8 reagent to each well and continue to culture in the incubator for 2 hours. Measure the absorbance (OD) value at a wavelength of 450nm with an enzyme reader. Repeat the experimental results for each group 3 times. Calculate the survival rate of each group of cells: according to the cell survival rate formula = (OD value of the treatment group - OD value of the blank group) / (OD value of the control group - OD value of the blank group) × 100%.

[0172] d. Cell scratch assay

[0173] Before using the items required for the experiment, the clean bench was ultraviolet disinfected for 30 minutes. A vertical line was drawn from top to bottom with a marker pen on the bottom of the 6-well plate, with a distance of about 0.5 cm.

[0174] The cells were divided into 1×10 5The cells were inoculated into a six-well plate at a density of 100 cells / well. According to the experimental requirements, when the cells filled the six-well plate, a 200 μL sterile pipette tip was used to vertically scratch the cells along the drawn line. The cells were gently rinsed 3 times with PBS to remove the cells remaining in the scratches, and cultured with stem cell culture medium or 0.1% DMSO serum-free culture medium, with DMSO as the control group. Photos were taken at 0h and 24h, and the wound healing rate was finally analyzed using image J software. The experiment was repeated three times each time.

[0175] e. Real-time quantitative PCR (qRT-PCR)

[0176] (e-1) Extraction of total cell RNA:

[0177] Take out the 6-well plate, wash it evenly with PBS 3 times, add 1mL Trizol to each well, lyse it on ice for 2 minutes, blow it with a pipette tip and transfer it to a 1.5mL enzyme-free EP tube to collect the liquid, mark the tube. Immediately add 200μL chloroform, turn it upside down several times to mix it thoroughly, and let it stand on ice for 5 minutes.

[0178] Centrifuge (4°C, 12000 rpm, 12 min) and carefully collect 300 μL-400 μL of the supernatant in a new 1.5 mL EP tube that has been labeled. Add an equal volume of isopropanol to the EP tube, mix well, and place on ice for 10 min. Centrifuge (4°C, 12000 rpm) for 10 min.

[0179] Remove the supernatant, add 1 mL of 75% ethanol (anhydrous ethanol diluted with DEPC water, prepared immediately before use) to each EP tube for washing, and centrifuge at 4°C, 7500 rpm for 10 min. Use a pipette to carefully remove the liquid, and place at room temperature for 3-5 min to allow the 75% ethanol to fully evaporate (RNA can be seen to gradually change from milky white to transparent). Add 30-50 μl of DEPC water to fully dissolve the RNA.

[0180] (e-2) After agarose gel electrophoresis, RNA concentration and purity were determined, the concentration, purity and integrity of the total RNA were determined. Subsequent experiments could be performed if the OD 260 / OD 280 ratio was between 1.8 and 2.0 as measured by a microplate reader.

[0181] (e-3) cDNA synthesis:

[0182] RNA was reverse transcribed into cDNA according to the instruction manual. The reaction system (20 μl) and reaction conditions were: 37°C for 15 min, 85°C for 5 s, and 4°C for 1 h.

[0183]

[0184] (e-4) Real-time fluorescence quantitative PCR amplification and analysis:

[0185] Then the synthesized cDNA was diluted for fluorescence quantitative PCR amplification, the reaction system (25 μl), the reaction conditions: 95°C 30s, 95°C 5s, 60°C 30s, for a total of 40 cycles.

[0186]

[0187] The test results were compared with β-actin as an internal reference, and the mRNA relative quantification of the target gene was All PCR primer sequences were designed and synthesized by Shanghai Bioengineering Co., Ltd.

[0188] PCR primer sequences

[0189]

[0190] f. Western Blot experiment

[0191] (f-1) Extraction of total protein:

[0192] Extraction of adherent cell protein: Before the experiment, turn on the low-temperature centrifuge to 4°C in advance, take out the 6-well plate, put it on ice, wash it 3 times with 4°C pre-cooled PBS, and add 100μL lysis solution containing PMSF (RIPA:PMSF=100:1) to each well. After lysis on ice for 30 minutes, use a cell scraper to take the cells and use a pipette to suck them into a 1.5mL sterile EP tube. Pay attention to changing the pipette tip for different groups. Centrifuge (12000rpm, 4°C, 20min), transfer the supernatant to a new EP tube, and record the extraction date and name.

[0193] Extraction of tissue protein: Take about 20 mg of brain tissue and add it to a sterile 1.5 mL EP tube, add 200 μL of lysis buffer (the same ratio as for adherent cells), grind the tissue on ice until it is no longer visible, lyse on ice for 30 minutes, mix twice in a vortex mixer to fully lyse, centrifuge (12000 rpm, 4°C, 20 min), transfer the supernatant to a new EP tube, and record the extraction date and name.

[0194] (f-2) Protein concentration determination:

[0195] First, dilute the BSA with an initial concentration of 5 mg / mL with PBS to a concentration of 0.5 mg / mL, and dispense into 1.5 mL EP tubes for later use. Prepare the BCA working solution before use, with BCA reagent: Cu reagent = 50:1. Take the diluted protein standard and operate according to the instructions of the BCA protein concentration determination kit. Use an enzyme-labeled instrument to determine the concentration of each group in A. 562nm wavelength absorbance value, and draw a standard curve (y = 0.8795x + 0.0916, R 2 ≥0.995 was qualified), and finally the protein concentration of each sample was calculated according to the formula, and the corresponding volume of RIPA lysis buffer was added to adjust the protein concentration of each group to the same level.

[0196] (f-3) Protein treatment and sample loading:

[0197] The protein sample adjusted to the same concentration and the loading buffer (5×) were mixed in a ratio of 4:1, and placed in a 100°C boiling water bath for 10 min to denature the protein, mixed by centrifuge, and stored at -20°C.

[0198] (f-4) Preparation of separation gel and stacking gel:

[0199] Prepare relevant reagents and supplies, clean the glass plate in advance and dry it for later use.

[0200] Prepare SDS-PAGE gel (see table below)

[0201]

[0202] First pour the lower layer of separation gel, then add anhydrous ethanol to the upper liquid surface of the separation gel, let it stand at room temperature for about 30-40 minutes until a horizontal separation line appears, absorb the excess anhydrous ethanol with filter paper, prepare the concentrated gel, after pouring the concentrated gel, do not generate bubbles, quickly insert the comb, let it stand at room temperature for 35 minutes before loading the sample.

[0203] (f-5) SDS-PAGE electrophoresis:

[0204] First add 3 μL of rainbow marker, then add protein samples in sequence, with 30 μg per well. The voltage is 80 V for about 25-30 min. When the marker is separated, the voltage is changed to 120 V and the electrophoresis is terminated when bromophenol blue reaches the bottom of the separation gel.

[0205] (f-6) Transfer:

[0206] Prepare 10 minutes in advance, cut the PVDF membrane suitable for the target protein (mark it well), and soak it in methanol for 3 minutes to activate it. Soak the PVDF membrane and transfer clip in the transfer solution (1×). Make a "sandwich", and put the sponge-filter paper-peeled gel-PVDF membrane-filter paper-sponge from the black negative electrode to the white positive electrode one by one, and use clean tweezers to remove bubbles.

[0207] Place it in the electroporation tank filled with transfer solution, paying attention to the direction of the positive and negative electrodes. Place the electroporation box in a basin filled with ice and operate at low temperature. The transfer conditions are constant current 300mA, 90min.

[0208] (f-7) Closed:

[0209] After the transfer, the PVDF membrane was placed in a blocking box containing 5% BSA blocking solution and blocked at room temperature for 2 h.

[0210] (f-8) Primary antibody incubation:

[0211] After blocking, wash the membrane three times with 1×TPST solution, each time for 8 min, add rabbit primary antibodies vimentin (1:2000), β-catenin (1:5000), N-cadherin (1:5000), E-cadherin (1:1000), β-actin (1:5000), and incubate in a shaker at 4°C overnight.

[0212] (f-9) Secondary antibody incubation:

[0213] After taking out the incubation box from the 4°C refrigerator, let it stand at room temperature for 30 minutes to recover, recover the primary antibody, wash the membrane three times with TPST solution, each time for 8 minutes, add goat anti-rabbit IgG secondary antibody (1:10000), and incubate on a shaker at room temperature for 1.5 hours.

[0214] (f-10) Development and analysis:

[0215] Recover the secondary antibody and wash the membrane 4 times with TPST solution, 6 minutes each time. Mix the luminescent solution A and solution B in a ratio of 1:1 and prepare them immediately before use. When the temperature of the luminescent instrument drops to -30°C, put the PVDF membrane into the luminescent instrument and add an appropriate amount of the mixed solution with a pipette according to the size of the cut PVDF membrane, and take pictures. The grayscale value of the bands was analyzed using Image J software for Western Blot image results.

[0216] g. Transwell experiment

[0217] Matrigel was thawed at 4°C overnight, and the cooled serum-free DMEM medium and thawed Matrigel were mixed in a 4:1 ratio. 45 μL of the mixture was added to each Transwell chamber in a 24-well plate, and the inner membrane of the Transwell chamber was evenly spread. The plate was placed in a cell culture incubator and allowed to stand for 1 hour before the next experiment.

[0218] Remove the cells from the incubator, wash them twice with autoclaved PBS to remove the residual culture medium, add 1 mL of trypsin, digest for about 1 min, add 2 mL of complete culture medium to terminate the digestion, centrifuge (1000 rpm, 5 min), remove the supernatant, wash once with PBS, resuspend the cells and count the cells to make the cell suspension concentration 1×10 4 Pieces / mL.

[0219] The upper chamber was inoculated with homologous brain glial cells BV2 of the control group and homologous MSCs of the experimental group, and 200 μL of cell suspension was added to each Transwell chamber. 500 μL of homologous GBM cells of the same concentration were added to the outer chamber and then placed in a constant temperature cell culture incubator (37°C, 5% CO 2 ) were cultivated.

[0220] After 24 hours, remove the culture medium inside and outside the chamber and wash three times with PBS. Move the chamber to 800 μL of 4% paraformaldehyde solution and fix the cells for 30 minutes. Then remove the chamber, wash three times with PBS, use tweezers to put it in 800 μL of crystal violet staining solution for 25 minutes, and wash three times with PBS. Gently wipe the cells at the bottom of the chamber with a cotton swab, then observe the stained cells under an inverted microscope, take pictures and save them, and repeat the experiment 3 times.

[0221] h. Subcutaneous tumor-bearing mouse experiment

[0222] (h-1) Twelve 4-week-old female BALB / c mice were purchased, with a weight difference of about 1 g, and were randomly divided into four groups and raised in an animal room.

[0223] (h-2) After BALB / c mice were raised for about a week and their growth was good, subsequent experiments were performed. The cells were digested with trypsin and then resuspended in PBS to a density of 5×10 7 Each BALB / c mouse was injected with 1×10 7 A subcutaneous tumor-bearing mouse model was established.

[0224] (h-3) After anesthetizing BALB / c mice with pentobarbital, 200 μL of cells were injected into the axilla. After the injection, the mice were kept in the animal room.

[0225] (h-4) When the tumor grows to 150cm 3 The control group was injected with 200 μL of drug-dissolving reagent, and the experimental group was injected with 200 μL (20 mg / Kg) Tet and 10 μL of 1×10 Tet+MSCs into the nasal cavity according to the weight of BALB / c mice. 5 MSCs were injected intraperitoneally and dripped into the nose once every 1 day (using a 10 μL pipette tip to drip into the nose, alternating between the left and right nasal cavities, 5 μL in each nasal cavity, and the state of the BALB / c mice was observed during the operation). The mice were allowed to leave the hospital only after they recovered.

[0226] (h-5) The growth status of BALB / c mice was observed. When the BALB / c mice were about to die, they were immediately killed by cervical dislocation and the brains were removed. The time of death was recorded and pictures were collected.

[0227] i. Orthotopic transplantation tumor experiments in mice

[0228] (i-1) Twelve 4-week-old female BALB / c mice were purchased and randomly divided into four groups and raised in an animal room.

[0229] (i-2) After BALB / c mice were raised for about a week and their growth was good, subsequent experiments were performed. The cells were digested with trypsin and then resuspended in PBS to a density of 2×10 7 Pieces / mL.

[0230] (i-3) BALB / c mice were anesthetized with pentobarbital and disinfected with iodine. The scalp was cut with a disposable blade. The distance from the middle of the two ears to the middle of the two eyes was about 1 cm. The bregma of the BALB / c mouse was taken as the origin, and then 1.5 mm to the right and 1.5 mm to the back as the injection point. A small hole was drilled from the injection point in the skull with a 1mL syringe. A 10μL microsyringe was used to drill vertically 3mm from the injection point and then lifted 0.5mm. Then 5μL cells (1×10 per point) were injected. 5 After the injection, the scalp was sutured with needle and thread and the animals were kept in the animal room.

[0231] (i-4) The mice were divided into four groups: control group, 20 mg / Kg Tet group, 20 mg / Kg Tet+MSCs group and MSCs group. When the mice grew to 3 days old, they were intraperitoneally injected with drugs and nasally dripped with MSCs using the same method as the subcutaneous tumor model.

[0232] (i-5) Observe the growth status of BALB / c mice. When the mice are about to die, they are immediately killed by cervical dislocation and the brain is removed. The time of death is recorded and pictures are collected.

[0233] j. Tissue embedding and sectioning

[0234] (j-1) Take out the tissue sample and immerse it in 4% paraformaldehyde solution for 48 hours.

[0235] (j-2) The fixed tissue samples were rinsed with running water overnight and placed in a dehydrator with a programmed program for dehydration. The program settings are as follows:

[0236]

[0237]

[0238] (j-3) After dehydration, the tissue samples were taken out, embedded in paraffin using an embedding machine, and marked.

[0239] (j-4) The embedded paraffin blocks were cut into paraffin sections with a thickness of 3-5 μm using a microtome. The sections were first unfolded in cold water, then flattened in a 42°C slide spreader, and the wax sections were picked up with a slide (lysine treated), and stored at 4°C after natural air drying.

[0240] kH&E staining

[0241] (k-1) Set the oven to 65°C. When the temperature reaches 65°C, place the paraffin sections in the oven and bake for 2 hours.

[0242] (k-2) Dewaxing and hydration are performed according to the following steps:

[0243]

[0244]

[0245] (k-3) Stain the sections with hematoxylin for 10-15 minutes, then rinse with running water until the color does not change. Follow the steps below:

[0246] Ethanol hydrochloride 2s Running water flushing 5min 1% ammonia water 15s Running water flushing 5min 75% ethanol 2min 85% ethanol 2min 95% ethanol 2min 100% Ethanol I 2min 100% Ethanol II 2min 1 / 2 xylene 2min Xylene I 2min Xylene II 2min

[0247] After completing the above operations, seal the slides with neutral resin and air dry them naturally. Collect images under a microscope and save them for analysis.

[0248] 1. Immunohistochemical staining

[0249] (l-1) Set the oven to 65°C. When the temperature reaches 65°C, place the paraffin sections in the oven and bake for 2 hours.

[0250] (1-2) Dewaxing and hydration according to the following steps:

[0251]

[0252]

[0253] (l-3) Antigen retrieval: Place the slices in a box containing citric acid buffer and place in a microwave oven on medium-high heat for 10 min each time, replacing the buffer each time, for a total of three times. Remove from the dehydration box and cool naturally to room temperature.

[0254] (1-4) Add an appropriate amount of endogenous peroxidase blocker and incubate at room temperature for 10 min. Then wash three times with PBS, each time for 3 min.

[0255] (l-5) Add the primary antibody and dilute it according to the instructions. According to the size of the tissue, add an appropriate amount of primary antibody to cover the paraffin section tissue. Place the sections in a humid incubation box and incubate at 4°C. Wash with PBS three times, 3 minutes each time.

[0256] (l-6) Add appropriate amount of enhancement solution, incubate at room temperature for 20 min, and wash three times with PBS, each time for 3 min.

[0257] (l-7) Add an appropriate amount of enhanced enzyme-labeled goat anti-mouse / rabbit IgG polymer, incubate at room temperature for 20 minutes, and wash three times with PBS, each time for 3 minutes.

[0258] (1-8) Prepare DAB colorimetric reagent in a 1:1 ratio according to the instructions, and drop it onto the paraffin section tissue for color development for about 5-8 minutes (the time depends on the actual situation). Then wash with PBS three times, 3 minutes each time.

[0259] (l-9) Stain the sections with hematoxylin for about 10 minutes, then rinse with running water until the color does not change, and proceed as follows:

[0260] Ethanol hydrochloride 2s Running water flushing 5min 1% ammonia water 15s Running water flushing 5min 75% ethanol 2min 85% ethanol 2min 95% ethanol 2min 100% Ethanol I 2min 100% Ethanol II 2min 1 / 2 xylene 2min Xylene I 2min Xylene II 2min

[0261] After completing the above steps, seal the slide with neutral resin. After air drying, collect pictures under a microscope and save them for analysis.

[0262] The experimental results of Example 1 are as follows:

[0263] (1) Results of testing the effects of mouse mesenchymal stem cells (MSCs) on the affinity, proliferation, migration and invasion of the mouse glioblastoma (GBM) cell line G422

[0264] ① The results of the Transwell experiment showed the number of cells that migrated and passed through the bottom membrane of the chamber. The number of cells in the control group and the experimental group was (45±3.512 and 77±2.517), respectively. The results showed that compared with the control group, the affinity of MSCs in the experimental group to GBM cells increased, and the difference was statistically significant (P<0.05). Figure 3 shown).

[0265] ② The results of red fluorescent marker plasmid transfection showed that the transfection efficiency was low at 48h and 72h (e.g. Figure 4 There was no significant difference in transfection efficiency between 48h and 72h (P>0.05).

[0266] ③ The results of the Transwell experiment showed that the number of MSCs expressing red fluorescence in the experimental group was greater than that in the control group when the cells migrated and passed through the bottom membrane of the chamber were observed using a fluorescence microscope. The difference was statistically significant (P<0.05). This indicates that in vitro, mouse MSCs have an affinity for mouse GBM cells and can migrate in the direction of the latter (e.g. Figure 5 shown).

[0267] Brain tissue sections were observed using a fluorescence microscope. Compared with non-tumor areas, the fluorescence in tumor areas was significantly higher, and the difference was statistically significant (P<0.05). This indicates that mouse MSCs have affinity for mouse GBM cells in vivo, and the former can specifically target the latter and achieve selective localization (e.g. Figure 6 shown).

[0268] ⑤ At 0h, 24h, 48h, and 72h, the cell viability was detected by CCK-8 assay. The results showed that the experimental group was significantly different from the control group (P<0.05). The 48h and 72h experimental groups significantly inhibited the proliferation of GBM cells (P<0.05). Figure 7 shown).

[0269] ⑥ The experiment showed that the wound healing rates of the control group and the experimental group were 11.97% ± 1.7% and 35.66% ± 2.45%, respectively, and the difference was statistically significant (P < 0.05) (e.g. Figure 8 The results showed that the MSCs-conditioned medium in the experimental group promoted the migration of GBM cells.

[0270] ⑦ The results showed that compared with the control group, the MSCs-conditioned medium in the experimental group significantly promoted the mRNA expression of vimentin, β-catenin, N-cadherin and other proteins, and reduced the mRNA expression level of E-cadherin (such as Fig. 9 This suggests that MSCs may promote the invasion of GBM cells.

[0271] The expression of EMT and invasion-related proteins such as vimentin, β-catenin, N-cadherin, and E-cadherin was analyzed by Western Blot experiments. The results showed that compared with the control group, the MSCs-conditioned medium in the experimental group significantly promoted the expression of vimentin, β-catenin, and N-cadherin-related invasion proteins, and reduced the expression level of E-cadherin (P<0.05), suggesting that MSCs may promote the occurrence of GBM cell invasion, which is consistent with the results of qRT-PCR experiments (such as Fig.10 shown).

[0272] (2) Effects of combined use of mouse MSCs and tetrandrine (Tet) on the mouse GBM cell line G422

[0273] ① The CCK-8 experiment was used to detect the cell viability at 0h, 24h, 48h, and 72h. Compared with the control group, the inhibitory effect of Tet on the proliferation of the mouse GBM cell line G422 cells in the experimental groups of 5μmol / L Tet, 10μmol / L Tet, 20μmol / L Tet, and 40μmol / L Tet was not only dose-dependent, but also time-dependent, and the differences were statistically significant (P<0.05) (e.g. Fig.11 According to the above effects, there was no significant difference between 20μmol / L Tet and 40μmol / L Tet (P>0.05), so 20μmol / L Tet was selected for subsequent experiments.

[0274] ②The results showed that compared with the MSCs group, the 20μmol / L Tet group and the 20μmol / L Tet+MSCs group could significantly reduce the mRNA expression levels of vimentin, β-catenin, and N-cadherin (e.g. Figure 12-14 ), increasing the mRNA expression level of E-cadherin (as shown Fig.15 (P<0.05), indicating that both Tet alone and MSCs combined therapy can inhibit the GBM invasion induced by MSCs in vitro.

[0275] Western Blot was used to detect the expression levels of EMT and invasion-related proteins such as vimentin, β-catenin, N-cadherin, and E-cadherin. The results showed that compared with the MSCs group, the 20μmol / L Tet and 20μmol / L Tet+MSCs groups could reduce the expression levels of vimentin, β-catenin, and N-cadherin proteins, and increase the protein expression level of E-cadherin (e.g. Fig.16 The results showed that Tet alone or in combination with MSCs could inhibit the mRNA and protein expression levels of EMT and invasion-related proteins in GBM, and the combined effect of Tet and MSCs was better than that of Tet or MSCs alone, which was consistent with the results of qRT-PCR experiments.

[0276] ③ The experimental results showed that the weight of GBM tumors in the 20mg / Kg Tet, 20mg / Kg Tet+MSCs and MSCs groups decreased significantly, but the effect of the Tet and MSCs combination group was significantly better than that of the other groups (such as Fig.17 shown).

[0277] The results of immunohistochemical staining showed that compared with the control group, the expression levels of EMT and invasion-related proteins N-cadherin and vimentin in the MSCs group were significantly increased, while the expression levels of N-cadherin and vimentin in the 20mg / Kg Tet and 20mg / Kg Tet+MSCs groups were significantly decreased, among which the 20mg / Kg Tet+MSCs group had the best effect (e.g. Fig.18 This suggests that MSCs still have the risk of promoting GBM tumor invasion in vivo, and the combined use of Tet can reduce this risk to a certain extent.

[0278] ④H&E staining revealed that the GBM tumor area in the 20mg / Kg Tet+MSCs group was smaller than that in the 20mg / Kg Tet and MSCs group, suggesting that the combination of Tet and MSCs can inhibit the proliferation and invasion of GBM in situ tumors (e.g. Fig.19 shown).

[0279] The mRNA expression levels of EMT and invasion-related proteins such as vimentin, β-catenin, N-cadherin, and E-cadherin in GBM orthotopic transplanted tumors in vivo were detected by qRT-PCR. Compared with the MSCs group, the 20 mg / Kg Tet and 20 mg / KgTet+MSCs groups could significantly reduce the mRNA expression levels of vimentin, β-catenin, and N-cadherin (e.g. Figure 20-Figure 22 ), increasing the mRNA expression level of E-cadherin (as shown Fig.23 (P<0.05), indicating that both Tet alone and MSCs combined therapy can inhibit MSCs-induced GBM metastasis in vivo.

[0280] The expression levels of EMT and invasion-related proteins such as vimentin, β-catenin, N-cadherin, and E-cadherin were analyzed by Western Blot experiments. The results showed that compared with the MSCs group, the expression levels of vimentin, β-catenin, and N-cadherin in the 20 mg / Kg Tet group and the 20 mg / Kg Tet+MSCs group were significantly reduced, and the protein expression level of E-cadherin was significantly increased (e.g. Fig.23 The results showed that the combined use of Tet and MSCs can inhibit the EMT and invasion of GBM in orthotopic transplanted tumors in vivo (P<0.05), and the combined use of Tet and MSCs is better than that of Tet or MSCs alone, which is consistent with the trend of qRT-PCR experimental results.

[0281] Immunohistochemistry experiments showed that compared with the control group, the expression levels of EMT and invasion-related proteins vimentin and N-cadherin in the MSCs group were increased (P < 0.05), suggesting that MSCs can promote GBM invasion in orthotopic transplanted tumors (e.g. Fig.24 Compared with the MSCs group, the 20mg / Kg Tet and 20mg / Kg Tet+MSCs groups were able to significantly reduce the expression of vimentin and N-cadherin (P < 0.05), inhibiting the invasion of GBM in situ (as shown in Figure 2A). Fig.25 shown).

[0282] Summary of the implementation effect of the technical solution of the present invention:

[0283] Glioblastoma (GBM) is one of the major malignant tumors in the central nervous system and one of the most common and lethal solid tumors. Typical treatments of surgery, chemotherapy, and radiotherapy are often limited by recurrence or severe treatment-related complications. Currently, in most basic or clinical studies related to GBM, GBM tumor models are mainly in vitro (cell experiments) and in vivo (animal experiments). Although the former is easy to operate and easy to conduct molecular biology-related research, in vitro cells cannot be in the tumor microenvironment, resulting in differences from the actual tumor occurrence and development process, and there are limitations and defects in the study of deterioration processes such as tumor invasion. In contrast, in vivo experiments mainly use nude mice or immunodeficient mice as in vivo experimental animal carriers. Although the establishment of in vivo tumor models in immunodeficient animals can simulate the in vivo environment of human-derived GBM tumors to a certain extent, the animal models used are rodents that do not have normal immune capabilities, which cannot truly reflect the changes in GBM-related tumor biological processes under homologous conditions, and cannot conduct studies related to tumor microenvironment and immune regulation. In addition, human-derived mesenchymal stem cells (MSCs) are generally considered to be non-carcinogenic, but studies have shown that in rodents, GBM tumor cells and their microenvironment still affect the tumor suppressor effect of MSCs and cause malignant transformation, and the relevant mechanism research is not in-depth.

[0284] Therefore, in view of the important role of animal immunity and microenvironment in the development and growth of tumors, and in order to be closer to clinical samples, the present invention uses BALB / c mice with normal immunity and homologous GBM cell line G422 tumor strain as in vivo animal model carriers, and uses homologous GBM cells of mice for in vivo and in vitro experimental studies, aiming to simulate the real environment of tumor occurrence and development to study the therapeutic effect and biosafety of MSCs on GBM. Due to the low survival rate of GBM patients and limited clinical treatment methods, it is particularly important to develop targeted and precise treatment methods or drug combinations based on the biological characteristics of GBM tumors.

[0285] However, there are still difficulties in the spatiotemporal targeting and dosage control of drug delivery via MSCs. Although many existing studies have shown that human-derived MSCs have a low risk of promoting tumorigenesis, some researchers still believe that MSCs may stimulate the growth of tumor cells. Existing studies have shown that the interaction between GBM and MSCs has a pro-tumor effect. MSCs promote GBM neovascularization in vivo by fusing with tumor stem cells. In addition, co-culture of MSCs and GBM increases the migration and invasiveness of tumor cells, accompanied by low expression of E-cadherin, a negatively correlated protein for invasion, and high expression of vimentin, a positively correlated protein. The molecular biological characteristics induced by MSCs are consistent with the tumor-induced EMT-like phenotype, which, as an important biological process, is closely related to the progression and invasion of tumors. It not only enhances the invasive ability of tumor cells, but also induces drug resistance in tumor cells as the disease worsens.

[0286] In the present invention, the Transwell experiment verifies that MSCs have tropism and homing to GBM. The proliferation level of GBM cells treated with MSCs conditioned medium was significantly inhibited both in vitro and in vivo. The in vitro and in vivo experiments of the present invention also show that the expression of invasive proteins vimentin, β-catenin, and N-cadherin in the MSCs intervention group was increased compared with the control group, and it is clear that GBM cells treated with MSCs conditioned medium showed EMT phenotype both in vitro and in vivo, and had a certain risk of invasion. Therefore, when MSCs are selected alone as a tumor treatment tool or targeted carrier to deliver therapeutic drugs to the tumor area, there is a potential risk of promoting cancer.

[0287] In order to verify whether Tet can inhibit the proliferation and invasion of homologous GBM cells in mice, the present invention observes the changes in the proliferation level of GBM cells and the expression level of invasion-related proteins after Tet treatment through a variety of in vivo and in vitro experiments. The results show that Tet can inhibit the proliferation of homologous GBM cells in a time- and dose-dependent manner, and promote the decrease in the expression of invasion-positive related proteins such as vimentin, β-catenin, and N-cadherin, and the increase in the expression of negative related proteins such as E-cadherin, indicating that Tet can significantly inhibit the proliferation and invasion of GBM. In order to further explore whether the combined use of Tet and MSCs can reduce the related effects of MSCs on GBM tumor invasion and improve the therapeutic effect of Tet, the present invention designs a control group, a Tet monotherapy group, an MSCs intervention group, and a Tet+MSCs combined medication group in vivo and in vitro for relevant research. The results of the study showed that the use of Tet combined with MSCs can not only improve the inhibitory effect of Tet or MSCs alone on the proliferation of GBM in vitro and in vivo, but also promote the reduction of vimentin, β-catenin, and N-cadherin expression and the increase of E-cadherin expression in GBM in vitro and in vivo, indicating that the addition of Tet can alleviate the occurrence of MSCs-promoted GBM tumor EMT and invasion to a certain extent, and inhibit the deterioration of the disease caused by tumor invasion. In short, the in vitro and in vivo experiments of the present invention confirmed that the markers of invasion were significantly reduced, and the tumor area was significantly reduced.

[0288] The present invention designs the effect of combined use of Tet and MSCs to treat GBM in BALB / c mice with normal immune function, which can provide real and effective basic research data and theoretical basis for subsequent research on the participation of MSCs and Tet in or improvement of tumor microenvironment and immunotherapy effect.

[0289] In summary, the above results show that MSCs have homing to mouse homologous GBM, MSCs and Tet inhibit the proliferation of mouse homologous GBM cells, but MSCs intervention alone promotes the expression of EMT-related invasion proteins, Tet inhibits the proliferation and invasion of tumor cells in GBM, and the combined use of MSCs and Tet has a better inhibitory effect on tumor cell proliferation, and EMT-related invasion proteins are reduced. The specific molecular mechanism of combined use on GBM needs to be further verified by subsequent experiments. In addition, RNA-seq sequencing results show that the combined use of Tet and MSCs has a large impact on the gene expression level in GBM, and the signaling pathways that may be involved are cytokine-cytokine receptor interaction, chemokine signaling pathway, extracellular matrix-receptor interaction, etc. At the same time, it has a significant effect on EMT and invasion-related tumor biological processes including chemotaxis, inflammatory response and muscle contraction formation, which provides a clearer target and research idea for the next step of the combined use of Tet and MSCs to inhibit GBM related molecular mechanism research and the introduction of immunotherapy.

[0290] In summary, the present invention takes into full consideration the side effects of conventional chemotherapy drugs, the BBB hindering drug entry into the brain, MSCs promoting tumor invasion, the influence of tumor microenvironment and the development of natural lipophilic drugs, and proposes to use mice with normal immune capacity as carriers for GBM treatment, establish a tumor model that simulates the real in vivo tumor microenvironment, and explore the optimization of the efficacy of various treatment combinations (including MSCs treatment and chemotherapy), especially the use of the nose-to-brain administration route to promote MSCs to enter the GBM tumor area in the brain and use the lipophilic Tet to eliminate the risk of the former promoting GBM invasion, thereby improving and improving the GBM cure rate and prognosis.

[0291] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention and do not constitute a limitation on the content of the present invention. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims

1. A pharmaceutical composition for preventing or treating nervous system tumors, characterized in that: The invention comprises a pharmaceutically effective amount of adult stem cells and isoquinoline alkaloids.

2. The pharmaceutical composition for preventing or treating nervous system tumors according to claim 1, characterized in that: The adult stem cells are adult stem cells of human or animal origin.

3. The pharmaceutical composition for preventing or treating nervous system tumors according to claim 1, characterized in that: The adult stem cells include at least one of mesenchymal stem cells, neural stem cells, hematopoietic stem cells, and vascular endothelial stem cells originating from human or animal tissues.

4. The pharmaceutical composition for preventing or treating nervous system tumors according to claim 1, characterized in that: The isoquinoline alkaloids include bisbenzylisoquinoline and / or benzylisoquinoline.

5. The pharmaceutical composition for preventing or treating nervous system tumors according to claim 1, characterized in that: The isoquinoline alkaloids are at least one of tertrine, nortertrine, thalidomide, thalidomide, chelidonine, berberine, lycorine, vincristine, liesinine, neferine and sinomenine.

6. The pharmaceutical composition for preventing or treating nervous system tumors according to claim 1, characterized in that: The nervous system tumors include central nervous system tumors.

7. The pharmaceutical composition for preventing or treating nervous system tumors according to claim 1, characterized in that: The central nervous system tumors include gliomas.

8. The pharmaceutical composition for preventing or treating nervous system tumors according to claim 7, characterized in that: The glioma includes glioblastoma.

9. The pharmaceutical composition for preventing or treating nervous system tumors according to any one of claims 1 to 8, characterized in that: The pharmaceutical composition is administered via at least one of a nose-brain administration route, an eye administration route, an oral administration route, and an injection administration route.

10. Use of the pharmaceutical composition for preventing or treating nervous system tumors according to any one of claims 1 to 8 in the preparation of drugs for preventing or treating nervous system tumors.

Images