Pharmaceutical composition

Abstract

A pharmaceutical composition comprises an oncolytic virus and odontogenic stem cells. At least a portion of the oncolytic virus is contained within the odontogenic stem cells. The pharmaceutical composition treats tumors, prevents tumor recurrence or inhibits tumor metastasis through intraperitoneal administration and intratumor injection.

Description

Pharmaceutical composition This application claims priority to the Chinese patent application filed with the State Intellectual Property Office of China on December 30, 2022, with application number 202211725797.2 and invention name “Pharmaceutical Composition”, the entire contents of which are incorporated by reference into this application. Join by reference

[0001] All publications, patents, and patent applications cited in this specification are herein incorporated by reference, as if each individual publication, patent, or patent application were specifically set forth in its entirety and were individually incorporated herein by reference. Technical Field

[0002] The present invention belongs to the field of medical technology, and relates to a pharmaceutical composition comprising an oncolytic virus and dental stem cells, and the use of dental stem cells to carry the oncolytic virus, and also relates to a method for treating tumors, inhibiting tumor metastasis and preventing tumor recurrence by intraperitoneally administering dental stem cells carrying the oncolytic virus. Background Art

[0003] In my country, cancer is the second largest cause of death, and the number of cases is still increasing at an average annual growth rate of about 3%. Therefore, it is urgent to study more effective cancer treatments. At present, the most commonly used clinical cancer treatments include traditional surgical treatment, radiotherapy, chemotherapy and new immunotherapy. Oncolytic virus therapy is one of the tumor immunotherapies. As early as the early 20th century, researchers found cases of remission or recovery in some virus-infected tumor patients, which aroused the curiosity of researchers, and then the concept of oncolytic virus and related research were born.

[0004] Oncolytic viruses are natural or recombinant viruses that can selectively infect and kill tumor cells without damaging normal cells. Compared with traditional immunotherapy, oncolytic viruses have the advantages of good targeting, few adverse reactions, multiple ways to kill tumors, and less likely to develop drug resistance. However, the administration method of oncolytic viruses is limited. Due to the limitation of intratumoral administration, systemic administration is temporarily not possible, mainly for the following reasons: on the one hand, the neutralizing antibodies existing in the human body will recognize foreign viruses and play an immune clearance role; on the other hand, intravenous or intraperitoneal administration usually results in extremely small amounts of virus reaching solid tumors, which cannot reach effective drug concentrations; and after systemic administration, adenoviruses tend to accumulate in the liver.

[0005] Mesenchymal stem cells are a type of pluripotent stem cells that have all the characteristics of stem cells, namely the ability to self-renew and multidirectional differentiation. In addition, a series of chemokine receptors, such as CXCR4 / 5 / 6, CCR1 / 4 / 7 / 9 / 10 and integrin-α4, are expressed on the cell surface. Under the stimulation of chemokines, inflammatory mediators, and specific cell secretions, they can be induced to migrate to the site of injury and tumor (Ma, X. et al., J Cell Physiol 236, 3114-3128, 2021; Wang, H. et al., Stem cells 27, 1548-1558, 2009). Therefore, mesenchymal stem cells are often used as living cell drug delivery carriers, combined with drugs in a variety of construction methods to achieve drug delivery to the target site (Chulpanova, D Set al., Front Pharmacol 9, 259, 2018; Wang, X. et al., International journal of nanomedicine 13, 5231-5248, 2018); at the same time, mesenchymal stem cells do not express MHC II, low immunogenicity (Halm, D. et al., Biol Chem 402, 693-702, 2021), express MHC I, can inhibit T cell activity, and participate in immunosuppression and immunoregulation (Hu, CD, Kosaka, Y., Marcus, P., Rashedi, I. & Keating, A., Stem cells and development 28, 933-943, 2019). In recent years, the use of mesenchymal stem cells to carry various small molecules and bioactive drugs for anti-cancer research has become one of the research hotspots.

[0006] At present, the types of stem cells used in the anti-cancer efficacy research of stem cells combined with oncolytic viruses include neural stem cells, umbilical cord blood stem cells, and mesenchymal stem cells derived from bone marrow and fat. Neural stem cells and umbilical cord blood stem cells are derived from patients undergoing cranial surgery and the umbilical cords of newborns. The sources are limited, there are ethical issues, the cost of cell culture is high, and the feasibility of mass production is low. Mesenchymal stem cells derived from human bone marrow and fat need to be obtained through bone puncture and liposuction, which is very traumatic.

[0007] In this case, it is still necessary to explore and develop different methods of administering oncolytic viruses in order to effectively exert the effects of oncolytic viruses. Summary of the invention

[0008] After a lot of research, the inventor unexpectedly discovered that the use of dental stem cells (DSCs) combined with oncolytic viruses can effectively treat tumors. The inventor also found that the use of intraperitoneal administration of dental stem cells equipped with oncolytic viruses has unexpected tumor treatment effects. The intraperitoneal administration method does not require high technical level of doctors, is easy to operate, and is easy to promote. Moreover, the oncolytic virus-dental stem cell composition administered intraperitoneally can reach various parts of the body, and can also effectively treat undiscovered micro-lesions. In addition, since the oncolytic virus-dental stem cell composition administered intraperitoneally can reach other parts of the body, it also has the effect of inhibiting tumor metastasis. On this basis, the present invention is completed.

[0009] In a first aspect, the present invention provides a pharmaceutical composition comprising an oncolytic virus and dental stem cells. In one embodiment, the oncolytic virus and dental stem cells are contained in separate containers, and the oncolytic virus and dental stem cells are mixed before use. In another embodiment, at least a portion of the oncolytic virus is contained in the dental stem cells, that is, the dental stem cells are used as carriers to carry the oncolytic virus.

[0010] The dental stem cells are mesenchymal stem cells with self-renewal ability and multi-differentiation potential derived from teeth or periodontal tissues, such as dental pulp stem cells (DPSCs), stem cells from exfoliated deciduous teeth (SHED), stem cells from apical papilla (SCAP), periodontal ligament stem cells (PDLSCs), alveolar bone-derived mesenchymal stem cells (ABMSCs), tooth germ progenitor cells (TGPCs), gingival mesenchymal stem cells (GMSCs), dental follicle precursor cells (DFPCs), etc.

[0011] In one embodiment, the dental stem cells are dental pulp stem cells. Preferably, the dental stem cells are of human origin.

[0012] In another embodiment, the oncolytic virus is a wild-type attenuated virus strain, such as reovirus and Newcastle disease virus; or, the oncolytic virus is a genetically engineered virus, such as an engineered adenovirus, herpes simplex virus, vaccinia virus, and measles virus. Preferably, the oncolytic virus is an oncolytic adenovirus.

[0013] In another embodiment, the oncolytic virus is an oncolytic virus expressing interferon, and preferably, the nucleotide sequence encoding the interferon is as shown in SEQ ID NO: 2-4. For example, an oncolytic virus prepared according to patent application publication CN111363726A.

[0014] In another embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. The pharmaceutical composition can be formulated into a dosage form suitable for intraperitoneal administration, such as an injection.

[0015] In another embodiment, the oncolytic virus content carried by dental stem cells in the pharmaceutical composition is 0.1-100 vp / cell (virus particle per cell), for example 0.2-50 vp / cell, 0.3-20 vp / cell, 0.4-10 vp / cell, 0.5-5 vp / cell or 0.6-2 vp / cell. The oncolytic virus can be in the dental stem cells or adsorbed / attached to the surface of the dental stem cells. The specific content can be prepared and used according to actual clinical needs.

[0016] In a second aspect, the present invention provides the use of the pharmaceutical composition of the first aspect in the preparation of a medicament for treating a tumor. In one embodiment, the pharmaceutical composition can be formulated into a dosage form suitable for intraperitoneal administration, such as an injection. The tumor includes, but is not limited to, osteosarcoma, pancreatic cancer, bile duct cancer, vulvar cancer, prostate cancer, breast cancer, colorectal cancer, lung cancer, liver cancer, melanoma, lymphoma, gastric cancer, esophageal cancer, ovarian cancer, head and neck squamous cell carcinoma, squamous cell carcinoma, bladder cancer, glioma, cervical cancer and kidney cancer.

[0017] The present invention also provides a composition for treating a tumor, the composition comprising the pharmaceutical composition of the first aspect. In one embodiment, the composition can be formulated into a dosage form suitable for intraperitoneal administration, such as an injection. The tumor includes but is not limited to osteosarcoma, pancreatic cancer, bile duct cancer, vulvar cancer, prostate cancer, breast cancer, colorectal cancer, lung cancer, liver cancer, melanoma, lymphoma, gastric cancer, esophageal cancer, ovarian cancer, head and neck squamous cell carcinoma, squamous cell carcinoma, bladder cancer, glioma, cervical cancer and kidney cancer.

[0018] In a third aspect, the present invention provides a method for treating a tumor, comprising administering the pharmaceutical composition of the first aspect to a subject in need thereof. In one embodiment, the pharmaceutical composition is administered by intraperitoneal administration. In another embodiment, the pharmaceutical composition is administered by both intraperitoneal administration and intratumoral administration, i.e., a combination of the two. In another embodiment, the pharmaceutical composition is administered by both intraperitoneal administration and intravenous administration, i.e., a combination of the two. In yet another embodiment, the pharmaceutical composition The composition is administered in combination by intraperitoneal administration, intravenous administration and intratumoral administration. The tumors include, but are not limited to, osteosarcoma, pancreatic cancer, bile duct cancer, vulvar cancer, prostate cancer, breast cancer, colorectal cancer, lung cancer, liver cancer, melanoma, lymphoma, gastric cancer, esophageal cancer, ovarian cancer, head and neck squamous cell carcinoma, squamous cell carcinoma, bladder cancer, glioma, cervical cancer and kidney cancer.

[0019] The inventors also unexpectedly found that the use of the pharmaceutical composition described in the first aspect of the present invention to treat tumors greatly reduced the amount of virus required to achieve the therapeutic effect, reducing or avoiding the side effects that may be caused by oncolytic viruses. In certain embodiments, the content of oncolytic viruses carried by dental stem cells is 0.1-100 vp / cell, such as 0.2-50 vp / cell, 0.3-20 vp / cell, 0.4-10 vp / cell, 0.5-5 vp / cell or 0.6-2 vp / cell.

[0020] In a fourth aspect, the present invention provides a method for preventing tumor recurrence, comprising administering the pharmaceutical composition of the first aspect to a subject in need thereof. In one embodiment, the present invention also provides the use of the pharmaceutical composition of the first aspect in the preparation of a medicament for preventing tumor recurrence. In another embodiment, the present invention also provides a composition for preventing tumor recurrence, the composition comprising the pharmaceutical composition of the first aspect.

[0021] In a fifth aspect, the present invention provides a method for inhibiting tumor metastasis, comprising administering the pharmaceutical composition of the first aspect to a subject in need thereof. In one embodiment, the present invention also provides the use of the pharmaceutical composition of the first aspect in the preparation of a medicament for inhibiting tumor metastasis. In another embodiment, the present invention also provides a composition for inhibiting tumor metastasis, the composition comprising the pharmaceutical composition of the first aspect.

[0022] The technical solution provided by the present invention has at least one of the following beneficial effects: improving the limitations of intratumoral administration, achieving systemic administration, broadening the indications and improving the convenience of operation; reducing the dosage of the virus and increasing safety; having a good systemic tumor inhibition effect; and providing an adequate source of dental stem cells, which can greatly reduce the cost of cell therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the prior art description. Obviously, The drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0024] FIG1 shows the morphology of human dental pulp stem cells under a microscope (100×) (A) and the growth curve (B).

[0025] Figure 2 shows the multidirectional differentiation potential of human dental pulp stem cells. Dental pulp stem cells were induced to differentiate into adipocytes, Oil Red O staining (A); dental pulp stem cells were induced to differentiate into osteoblasts, Alizarin Red staining (B).

[0026] FIG3 shows fluorescence images of human dental pulp stem cells infected with different concentrations of oncolytic viruses (expressing EGFP) for 72 hours.

[0027] FIG. 4 shows the crystal violet staining results of human dental pulp stem cells infected with different concentrations of oncolytic virus YSCH01 (unit: vp / cell) for 72 hours.

[0028] FIG5 shows the cell growth activity curves after oncolytic virus infection of human dental pulp stem cells (A) and tumor cells SCC152 (B).

[0029] FIG. 6 shows the results of the Transwell assay, wherein (A) is a glossopharyngeal carcinoma squamous epithelial cell SCC152, and (B) is a bladder cancer epithelial cell SW780.

[0030] Figure 7 shows the fluorescence images of the distribution of different cells in the body after cell injection in mice. (A) shows the distribution of hDPSC in the tumor of tumor-bearing mice on the 10th day after intraperitoneal injection, where green fluorescence is SW780 tumor cells and red fluorescence is dental pulp stem cells; (B) shows the distribution of YSCH01 / hDPSC (red fluorescence) in the tumor on the 10th day after intraperitoneal injection.

[0031] Figure 8 shows the distribution of dental pulp stem cells in the organs of tumor-bearing mice, wherein (A) shows the distribution of dental pulp stem cells in the organs of SW780 tumor-bearing mice 48 hours after intraperitoneal injection of human dental pulp stem cells, and (B) shows the distribution of dental pulp stem cells in the organs of SW780 tumor-bearing mice 9 days after intraperitoneal injection of "oncolytic adenovirus YSCH01-human dental pulp stem cells".

[0032] FIG. 9 shows the drug administration pattern in SW780 tumor-bearing mice.

[0033] FIG. 10 shows the body weight curve of mice in Example 7.

[0034] FIG11 shows the tumor growth curve of Example 7, (A) is the tumor growth on the left, (B) The tumor growth on the right side.

[0035] FIG. 12 shows the tumor anatomy of SW780 tumor-bearing mice on day 23 of drug administration in Example 7.

[0036] FIG. 13 shows the statistical graph of tumor weights of SW780 tumor-bearing mice on day 23 of drug administration in Example 7

[0037] FIG. 14 shows the drug administration pattern in SCC152 tumor-bearing mice.

[0038] FIG15 shows the body weight curves of mice in each group.

[0039] FIG. 16 shows the tumor volume curve of Example 8, wherein (A) is the tumor growth on the left side, which is the intratumoral injection side; and (B) is the tumor growth on the right side.

[0040] FIG. 17 shows the tumor growth curve of Example 8.

[0041] FIG. 18 shows the immunohistochemical image of bilateral tumor tissues of SCC152 tumor-bearing mice in Example 9, where the black arrows indicate typical hexon immunohistochemical positive results.

[0042] FIG. 19 shows the immunohistochemical images of bilateral tumor tissues of SW780 tumor-bearing mice in Example 9, where the black arrows indicate typical hexon immunohistochemical positive results. DETAILED DESCRIPTION

[0043] Based on the following detailed description, it is obvious that other aspects and advantages of the present invention will be easily understood by those skilled in the art. It should be noted that the following detailed description (including the accompanying drawings) is only exemplary and not limiting of the present invention. It is easy to understand that by adjusting and changing some specific details, the present invention can be implemented in other different embodiments, and these adjusted and changed embodiments do not deviate from the present invention.

[0044] Unless otherwise specified, the terms used herein have the common meanings as understood by one of ordinary skill in the art to which they belong.

[0045] The term "oncolytic virus" refers to a virus that can selectively replicate in cancer or hyperproliferative cells, thereby slowing their growth or causing their death, while having no or minimal effect on normal cells. Exemplary oncolytic viruses include vesicular stomatitis virus (VSV), Newcastle disease virus (NDV), herpes simplex virus (HSV), reovirus, measles virus, retrovirus, influenza virus, Sindbis virus, vaccinia virus and adenovirus, etc.

[0046] In the present invention, the unit of viral dose is vp (viral particle), which indicates the number of viral particles contained in 1 ml of viral solution, and is the particle titer of the virus, usually expressed as vp / ml. In some specific embodiments, it can also be expressed as vp / cell (viral particle per cell) or other suitable expressions.

[0047] The term "dental stem cells" (DSCs) refers to a type of stem cells isolated from teeth or periodontal tissues. They are mesenchymal stem cells (MSCs)-like cells with self-renewal and multi-differentiation potentials. The main DSCs reported so far include: dental pulp stem cells (DPSCs), stem cells from exfoliated deciduous teeth (SHED), stem cells from apical papilla (SCAP), periodontal ligament stem cells (PDLSCs), alveolar bone-derived mesenchymal stem cells (ABMSCs), tooth germ progenitor cells (TGPCs), gingival mesenchymal stem cells (GMSCs), and dental follicle precursor cells (DFPCs).

[0048] Dental stem cells, as a type of mesenchymal stem cells, have the advantages of rapid and simple collection, complete autologous collection, shortened treatment cycle, and small host response, and have great clinical application prospects in anti-tumor applications. Studies have shown that through genetic modification, gingival mesenchymal stem cells carrying interferon β gene expression can effectively inhibit the growth of glossopharyngeal carcinoma (Du, L., Liang, Q., Ge, S., Yang, C. & Yang, P. Stem Cell Res Ther 10, 224, 2019).

[0049] Human dental pulp stem cells come from a wide range of sources, including healthy third molars of adults, orthodontic teeth, and children's deciduous teeth. 50% of people have third molars, and most wisdom teeth themselves need to be extracted, which provides a sufficient tissue source for in vitro mass production of such stem cells. Secondly, dental pulp stem cells can be obtained by separating the ex vivo teeth obtained after tooth extraction. This type of surgery is less invasive, short in operation time, convenient, and relatively low in cost. This is the most humane sampling method for both healthy organ donors and patients who need autologous cell transplantation. In addition, compared with bone marrow mesenchymal stem cells, dental pulp stem cells have stronger proliferation ability, cloning, and multidirectional differentiation characteristics, which can greatly reduce the cost and time of stem cell culture.

[0050] The term "pharmaceutical composition" refers to at least one A combination of a drug and optionally a pharmaceutically acceptable carrier or excipient. In certain embodiments, the pharmaceutical composition includes a combination separated in time and / or space, as long as it can work together to achieve the purpose of the present invention. For example, the ingredients contained in the pharmaceutical composition can be applied to the subject as a whole, or separately. When the ingredients contained in the pharmaceutical composition are applied to the subject separately, the ingredients can be applied to the subject simultaneously or sequentially. Preferably, the pharmaceutically acceptable carrier is water, a buffered aqueous solution, an isotonic saline solution such as PBS (phosphate buffered saline), glucose, mannitol, dextrose, lactose, starch, magnesium stearate, cellulose, magnesium carbonate, 0.3% glycerol, hyaluronic acid, or a polyalkylene glycol such as polypropylene glycol, triglycerides, etc. The type of pharmaceutically acceptable carrier used depends in particular on whether the composition according to the present invention is formulated for oral, nasal, intratumoral, perfusion, intradermal, subcutaneous, intramuscular or intravenous administration. The composition according to the present invention may contain lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring substances, flavoring substances and / or aromatic substances etc. as additives.

[0051] The pharmaceutical composition comprising oncolytic virus and dental stem cells referred to herein, the oncolytic virus and dental stem cells can be contained in separate containers and mixed before use; or the oncolytic virus can be contained in the dental stem cells or attached / adsorbed to the surface of the dental stem cells, that is, the dental stem cells serve as a carrier to carry the oncolytic virus.

[0052] The term "oncolytic virus-dental stem cell composition" (sometimes referred to herein as "oncolytic virus-dental stem cell complex") refers to dental stem cells equipped with oncolytic viruses. In some embodiments, the oncolytic virus enters the dental stem cells through infection, causing the dental stem cells to carry the oncolytic virus. The oncolytic virus can be replicative or non-replicative. In some embodiments, the oncolytic virus-dental stem cell composition is an oncolytic virus-human dental pulp stem cell composition.

[0053] The terms "treatment" and "therapeutic methods" include both therapeutic and prophylactic treatments. Those in need of treatment may include those who already have a particular medical disease as well as those who may eventually develop the disease. Treating tumors includes eliminating or inhibiting tumor growth, inhibiting tumor metastasis, and removing residual tumors after / while undergoing surgery or other treatments.

[0054] The term "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, Cows, chickens, amphibians, reptiles, etc. Unless otherwise indicated, the terms "patient" or "subject" are used interchangeably.

[0055] In certain embodiments, disorders and diseases include tumors and cancers, for example, non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic cancer, leukemia, lymphoma, myeloma, mycosis fungoides, Merkel cell carcinoma and other hematological malignancies, such as classical Hodgkin lymphoma (CHL), primary mediastinal large B-cell lymphoma, T cell / histiocyte-rich B cell lymphoma, EBV-positive and EBV-negative PTLD, and EBV-associated diffuse large B-cell lymphoma. The main targets of this study are DLBCL, plasmablastic lymphoma, extranodal NK / T-cell lymphoma, nasopharyngeal carcinoma, and HHV8-related primary effusion lymphoma, Hodgkin lymphoma, central nervous system (CNS) tumors, such as primary CNS lymphoma, spinal cord tumors, and brainstem glioma.

[0056] The term "administering" or "administering" means providing a substance, such as a pharmaceutical composition, to a subject in a pharmacologically acceptable manner.

[0057] The term "intraperitoneal administration" also known as intraperitoneal administration refers to the injection of drugs into the abdominal cavity. This method is easy to control, fast, suitable for chronic treatment, and less stressful to the person being administered.

[0058] The dosage of a pharmaceutical composition provided to a subject is a dosage sufficient to show benefit to the subject to which it is administered, and may also be referred to herein as a "pharmaceutically effective amount" or "effective amount". The actual amount administered, as well as the rate and time course of administration, will depend on the individual condition and severity of the subject being treated. The prescription of treatment (e.g., determination of dosage, etc.) is ultimately the responsibility of and is relied upon by physicians and other medical practitioners, typically taking into account the disease being treated, the individual patient's condition, the delivery site, the method of administration, and other factors known to physicians.

[0059] As used herein, unless otherwise explicitly stated, a noun is not preceded by an article or is modified by "the" to indicate that the term can be one or more. It should also be noted that as used in this specification, a singular form includes a plural form of its referent unless it is clearly and unambiguously limited to one referent.

[0060] The term "about" or "approximately" generally means within a range of error for a particular value determined by a person of ordinary skill, which depends in part on the manner of measurement, i.e., the limitations of the measurement system. For example, "about" may refer to one or more standard deviations, depending on the practice of a particular field. In particular, "about" may refer to values ​​within 20%, 10%, 5%, or 1% of a given value.

[0061] Terms such as "comprises," "comprising," "including," and "including" used herein are not intended to be limiting. In addition, "or," "or" means "and / or" unless otherwise stated.

[0062] The technical solution will be described clearly and completely below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are merely exemplary and not exhaustive of all implementation methods of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention. Example Example 1: Extraction and identification of human dental pulp stem cells

[0063] 1. Primary extraction of dental pulp stem cells

[0064] Healthy third molars and orthodontic teeth aged 18-30 years were extracted, and the isolated teeth were placed and rinsed in a culture medium containing 1% double antibiotics (penicillin and streptomycin); a small groove was ground along the neck of the tooth with a turbine to separate the crown and root of the tooth, expose the pulp cavity, and the pulp was removed with a pulp needle, and the apical 1 / 3 was discarded; the pulp tissue was rinsed 3 times with PBS containing 1% double antibiotics, then the tissue was cut into minced meat, and a mixed enzyme solution (3 mg / ml type I collagenase and 4 mg / ml type II protease) was added, and cultured at 37°C and 5% CO2.

[0065] 2. Screening and culture of dental pulp stem cells

[0066] Fibronectin is used to screen out dental pulp stem cells with good growth conditions. The screening method is as follows:

[0067] a. One day before screening, prepare fibronectin solution with PBS to make a 10 mg / ml working solution, add 1 ml of the working solution to each well of a six-well plate, and incubate at 4°C overnight.

[0068] b. On the day of screening, P1 dental pulp stem cells were obtained and prepared into a cell suspension using serum-free a-MEM.

[0069] c. Take out the pre-laid six-well plate, carefully aspirate and discard the supernatant, and culture the P1 cells at 4000 cells / cm 2 The density is spread in the perforated plate.

[0070] d. Incubate at 37°C for 20 min, wash twice with PBS, discard the non-adherent cells, add α-MEM (Hyclone, SH30265.01) culture medium containing 10% FBS and continue culturing. Change the medium every 2-3 days and pass the culture medium when the cell density reaches 80-90%.

[0071] e. Passage at a density of 1:3 to reserve enough dental pulp stem cells as subsequent cell carriers. P3-P6 cells are used for in vitro experiments, and P6-P10 cells are used for in vivo experiments.

[0072] Dental pulp stem cell growth culture medium: α-MEM (Hyclone, catalog number: SH30256.01) containing 10% FBS (Sciencell, catalog number: 0500) and 1% double antibody (Sigma, catalog number: V900929).

[0073] The morphology of the human dental pulp stem cells obtained by culture is shown in FIG1A , where the cells are spindle-shaped and fibrous.

[0074] The growth curve is shown in Figure 1B. It can be seen that the hDPSC cell growth curve is basically "S"-shaped. Within 1 day after subculturing, the cells grow slowly; on the 2nd to 4th day, they enter the logarithmic growth phase and the cells grow rapidly; on the 5th to 7th day, the cells grow slowly and tend to be flat.

[0075] 3. Identification of Dental Pulp Stem Cells

[0076] Flow cytometry was used to identify the cell surface markers of stem cells (CD146, CD90, CD44, STRO-1), and it was found that more than 99% of the cells positively expressed Stro-1, CD90, and CD44, which are the dental pulp mesenchymal stem cells we need.

[0077] 4. Identification of multidirectional differentiation of dental pulp stem cells

[0078] Human dental pulp stem cells were induced into adipogenesis and osteogenetically using adipogenic induction solution (α-MEM (Hyclone, SH30265.01), containing 10% FBS, 1 μM dexamethasone, 10 μg / ml insulin, 200 μM indomethacin and 0.5 mM IBMX) and osteogenic induction solution (α-MEM (Hyclone, SH30265.01), containing 10% FBS, 10 mmol / L sodium β-glycerophosphate, 0.05 mmol / L vitamin C and 100 mmol / L dexamethasone), respectively, and the solution was changed every 2-3 days. Osteogenic induction lasted for 14-21 days, and adipogenic induction lasted for 21-28 days. Then, mineralized nodules and oil granules were identified by alizarin red and oil red staining, respectively. Alizarin red can stain mineralized nodules, and oil red can stain oil granules. The results are shown in Figure 2.

[0079] Figure 2A is an Oil Red O staining image of hDPSCs after 28 days of culture in adipogenic induction medium. Red plump fat particles can be seen, indicating that the stem cells can differentiate into adipocytes; in addition, hDPSCs were cultured in mineralization induction medium for 18 days, and mineralized nodules were formed under the microscope. After Alizarin Red staining, the mineralized nodules became heterogeneous red blocks, as shown in Figure 2B, indicating that hDPSCs can differentiate into osteoblasts / odontogenics. The results showed that human dental pulp stem cells can be induced to differentiate into adipocytes and osteoblasts, and have multi-differentiation potential. Example 2 Preparation of oncolytic virus-human dental pulp stem cell composition

[0080] 1. Oncolytic virus infection of human dental pulp stem cells (hDPSC)

[0081] 1) Plating: P5 hDPSCs were plated in 24-well plates, with 1E+5 cells per well and 1 ml of culture medium;

[0082] 2) Infection: 18 hours after plating, dilute the OncoMul-V2-EGFP virus stock solution (the control empty virus of YSCH01, carrying the EGFP gene, for the preparation method, see patent application publication CN111363726A) with serum-free α-MEM medium. The stock solution concentration is 6.9E+9 vp / ml. Add the virus dilution solution into the 24-well plate according to the preset dose;

[0083] 3) Photography: 72 hours after infection, the expression of green fluorescence in hDPSCs was observed under an inverted fluorescence microscope and the images were recorded (Figure 3). The results showed that as the amount of virus added increased, the number of human dental pulp stem cells infected by the virus also increased.

[0084] 2. Tolerance of human dental pulp stem cells to oncolytic viruses

[0085] a. Crystal violet staining

[0086] 1) Wash the 24-well plate with α-MEM serum-free medium in advance;

[0087] 2) digesting P4 hDPSCs to prepare a cell suspension containing P5 hDPSCs;

[0088] 3) Plate the cells at a density of 1E+5 cells per well, add 1 ml of culture medium to each well, let stand for 30 minutes, and place in an incubator overnight;

[0089] 4) Add OncoMul-V2-EGFP and YSCH01 virus solutions of different concentrations and volumes (for preparation methods, see patent application publication CN111363726A) to a 24-well plate and mix well. Continue to cultivate;

[0090] 5) Crystal violet staining after 72 hours: After discarding the cell supernatant, the cells were fixed with 4% paraformaldehyde (PFA) for 20 minutes, washed 3 times with PBS, stained with 2% crystal violet stain (0.5g crystal violet powder dissolved in 25ml 20% methanol) for 15 minutes, rinsed with running water, and photographed after drying, as shown in Figure 4. The results showed that hDPSCs had good tolerance to the oncolytic adenovirus YSCH01, and there was no significant difference in the number of live cells of hDPSCs within the infection dose range of 2000vp / cell, indicating that the infection dose of 2000vp / cell would not affect the normal proliferation ability of hDPSCs.

[0091] b. CCK 8 assay to detect cell viability

[0092] 1) Digest well-growing P4 hDPSCs with trypsin (sigma-Adrich T4049) at 37°C. After 3 minutes, add growth medium (a-MEM containing 10% FBS and 1% double antibody) to terminate digestion. Prepare a cell suspension of 5E+4 cells / ml and plate it in a 96-well plate with 100 μl per well.

[0093] 2) 6 hours after plating, take a 96-well plate, add 10 μl CCK8 reagent (company: Yisheng; catalog number: 40203ES80) to each well, incubate at 37°C for 3.5 hours, read the OD value at 450nm with a spectrophotometer, and conduct subsequent experiments after verifying that there is no significant difference in cell viability between wells, otherwise replate;

[0094] 3) 24 hours after plating, the original culture medium was discarded, 100 μl of freshly prepared culture medium containing different concentrations of virus particles (YSCH01) was added, and the culture was continued;

[0095] 4) Take out a 96-well plate 24 hours, 48 ​​hours, 72 hours, 96 hours, and 120 hours after adding the virus, add 10 μl of CCK8 reagent to each well, incubate for 3.5 hours, and read the OD value at 450 nm using a spectrophotometer.

[0096] Similar experiments were performed using tumor cells SCC152.

[0097] The cell growth activity curve was drawn based on the data obtained, see Figure 5, where Figure 5A is human dental pulp stem cells and Figure 5B is tumor cells SCC152. From the curve in Figure 5A, it can be seen that within 24 hours of infection, the activity of hDPSC dental pulp stem cells infected with a dose of less than 1000vp / cell is not affected; it will only be affected after 48 hours of infection. This shows that dental pulp stem cells infected with the virus can still maintain normal cell activity for a sufficient period of time and can be used as a carrier to carry oncolytic viruses. For tumor cells, the results of Figure 5B show that a YSCH01 concentration of 50 vp / cell can play a significant role in killing tumor cells within 24 hours, and tumor cells are significantly more sensitive to oncolytic viruses than human dental pulp stem cells.

[0098] 3. Construction of oncolytic virus-human dental pulp stem cell (OV-hDPSC) composition

[0099] a. Take dental pulp stem cells from the logarithmic growth phase of P6-P10 and calculate the density of the stem cells according to 4.5E+5 cells / cm 2 18 hours after plating, oncolytic virus YSCH01 was used to infect dental pulp stem cells at a dose of 1000 vp / cell and mixed; b. Infect for 4 hours at 37°C and 5% CO2 to obtain an oncolytic virus-dental pulp stem cell composition. The construction and production of oncolytic virus YSCH01 can be found in patent application publication CN111363726A, which is an oncolytic adenovirus expressing interferon. The amino acid sequence of interferon carried by oncolytic virus YSCH01 is: Preferably, the nucleotide sequence encoding interferon carried by the oncolytic virus YSCH01 is selected from SEQ ID NO: 2, 3 and 4. In this embodiment, the oncolytic virus carries SEQ ID NO: 2. Example 3: Tumor tropism of oncolytic virus-human dental pulp stem cell composition 1. Transwell experiment a. Take P5 dental pulp stem cells and plate them at a density of 4E+5 hDPSCs per 6 cm culture dish; b. Tumor cells were inoculated in the lower chamber of the Transwell plate, with 1.5E+6 cells in each well, and resuspended in 650 μl of culture medium; the tumor cells used in this experiment were: SCC152 cells and SW780 cells; c. Digest hDPSCs and count them, seed 2E+4 hDPSCs per well in the upper chamber, and resuspend in 200 μl of serum-free / serum-containing medium; d. After 16 hours of inoculation on the Transwell plate, the supernatant was discarded, the cells in the upper chamber were fixed with 4% paraformaldehyde for 20 minutes, washed with PBS three times, stained with 2% crystal violet for 15 minutes, and then the excess dye was washed away with running water; e. Allow to dry for 24 hours, photograph under a microscope (objective lens 10x), take 5 fields of view for each well (top, bottom, left, right, and center), count, and perform statistical analysis. The results are shown in Figure 6. There was no serum concentration gradient between the upper and lower chambers in Group A, which was the negative control of the experiment; there was a serum concentration gradient between the upper and lower chambers in Group B. Compared with Group A, a large number of hDPSC dental pulp stem cells migrated to the lower chamber, which verified the establishment of the transwell experimental system. Tumor cells were cultured in the lower chamber of Group C. The results of the Transwell in vitro experiment showed that hDPSC dental pulp stem cells could migrate toward tumor cells, whether it was glossopharyngeal carcinoma squamous epithelial cells SCC152 (Figure 6A) or bladder cancer epithelial cells SW780 (Figure 6B). Example 4: Distribution of dental pulp stem cells in tissues after intraperitoneal injection Dental pulp stem cells expressing fluorescent protein were constructed, and in vivo tracing experiments were performed to test whether dental pulp stem cells could reach the tumor site after intraperitoneal administration. 1. Construction of RFP-hDPSC stable strain Take the P4 generation hDPSCs, plate them at a density of 1E+5 cells / cm2, and grow them overnight; infect hDPSCs with 20MOI of lentivirus Lenti-RFP, and replace with fresh dental pulp stem cell culture medium 24 hours later; 72 hours later, use a medium containing 2μg / ml puromycin to screen out uninfected cells and obtain the first generation of cell lines carrying the RFP fluorescent gene; later, use a growth medium containing 1μg / ml puromycin to culture, passage, and verify the infection efficiency by flow cytometry to obtain a stable cell line. Lenti-RFP is a lentivirus expressing RFP prepared according to conventional molecular biology methods, carrying a gene encoding the red fluorescent protein RFP. 2. Construction of EGFP-SCC152 and EGFP-SW780 Stable Transgenic Lines The construction method is basically the same as above, and tumor cells SCC152 and SW780 cells are infected with the lentivirus Lenti-EGFP expressing EGFP, and stable cell lines are screened and obtained. Lenti-EGFP is a lentivirus expressing EGFP prepared according to conventional molecular biological methods, and carries the gene encoding green fluorescent protein EGFP. 3. Animal Experiments Tumor formation: 5E+6 cells / 100μl of EGFP-SW780 cells or SW780 cells were injected subcutaneously on both sides of nude mice. 3 ; Injection: RFP-hDPSC or oncolytic virus-infected RFP-hDPSC (OV / RFP-hDPSC) were injected intraperitoneally, with an injection number of 2E+6 cells; Isolation: Tumor tissues were isolated on day 10 after injection. 4. Detection Sample processing: The isolated tumors were embedded in OCT (optimal cutting temperature compound) and frozen at -80 degrees Celsius; Frozen sections: sliced ​​with a cryostat, with a slice thickness of 10 μm, added with a mounting medium containing DAPI, and covered with a coverslip; Shooting: The slices were photographed with excitation wavelengths / absorption wavelengths of 488 / 507 mm and 558 / 583 mm, respectively. The results were analyzed after panoramic scanning. The image is shown in Figure 7. In Figure 7A, green fluorescence indicates SW780 tumor cells, and red indicates dental pulp stem cells. Dental pulp stem cells with red fluorescence are distributed in both tumor sites, with more distributed outside the tumor, but hDPSCs can also be observed inside the right tumor (box), indicating that human dental pulp stem cells can reach the tumor site under the administration method of intraperitoneal injection. Ten days after intraperitoneal injection of the OV / RFP-hDPSC composition, bilateral tumor tissues were treated in the same manner, and frozen sections revealed that red fluorescent protein was expressed in both tumors, not only around the tumor but also inside the tumor (Figure 7B). The above results show that dental pulp stem cells and dental pulp stem cells carrying oncolytic viruses can reach the tumor site through intraperitoneal injection. Example 5: Distribution of oncolytic virus-dental pulp stem cell composition in tissues after intraperitoneal injection 1. Preparation of dental pulp stem cells and oncolytic virus-dental pulp stem cell composition 1) The 8th generation dental pulp stem cells (hDPSC) and YSCH01 / hDPSC (hDPSC infected with oncolytic virus YSCH01) were taken and counted after normal digestion; 2) Centrifugation at 1000 rpm for 5 min; 3) Add DiR dye (Fubaike, Cat. No. 22070) to stain the desired cells, make a cell suspension of 1E+7 cells / ml, blow evenly, and stain for 30 minutes; 4) Centrifuge at 1000 rpm for 5 min and discard excess dye; 5) Wash twice with PBS; 6) Resuspend the cells in serum-free a-MEM medium to prepare a cell suspension of 1E+7 cells / ml, namely the stained dental pulp stem cells (hDPSC-dir) and the stained oncolytic virus-dental pulp stem cell composition (OV / hDPSC-dir, dir-stained oncolytic virus YSCH01 infected hDPSC). 2. Animal Experiments and Results Analysis 1) Tumor-bearing mice prepared in advance were injected with hDPSC-dir intraperitoneally, intravenously, and intraperitoneally with OV / hDPSC-dir (n=3), and a non-treated group was set as a control (all cell suspensions were filtered through a 0.70 μm filter membrane before injection). Each mouse was injected with 100 μl of cell suspension, i.e., 1E+6 cells; 2) Before shooting, mice were anesthetized by intraperitoneal injection of tribromoethane (Aibei Biotech M2920) at a concentration of 17.5 μl / g. Live imaging was performed after complete anesthesia. The shooting parameters were as follows: focus 112, aperture 1.8, channel ICG, exposure 800 ms; 3) Same parameters as above, taking photos at 48h, 96h, 168h, and 216h after administration; 4) Analyze the data, use the "Cleve" analysis software, adjust the light intensity, select "Radiance efficiency" as the unit, adjust the threshold, circle the target area, obtain ROI information, and export the image and fluorescence value data. Figure 8A shows the distribution of dental pulp stem cells in the organs of SW780 tumor-bearing mice at 48 hours after intraperitoneal injection of human dental pulp stem cells, indicating that human dental pulp stem cells can reach different tissues and organs within 2 days. Figure 8B shows the distribution of dental pulp stem cells in the organs of SW780 tumor-bearing mice at 9 days after intraperitoneal injection of OV / hDPSC-dir (oncolytic adenovirus YSCH01-human dental pulp stem cells), indicating that the oncolytic virus carried by dental pulp stem cells can stay in different tissues and organs for a long enough time. Example 6: Determination of viral load Determination of actual viral load a. Extraction of intracellular viruses from YSCH01-hDPSCs 1) Take a six-well plate and plate it at a density of 4E+5 cells / well. 18 hours after plating, add YSCH01 virus to infect hDPSCs; 2) 4 hours after infection, discard the supernatant, wash twice with PBS, add 2 ml of serum-free medium to each well, freeze and thaw three times to lyse the cells and release the virus, take the supernatant after centrifugation, make up to 2 ml, and store it at -80°C as the original solution of the sample to be tested. b. Immunochemical cell staining Hexon is the largest and most abundant structural protein in the adenovirus capsid. Hexon staining can be used to detect the presence and titer of adenovirus. TM The virus titer was detected by using the Adenovirus Titer Immunoassay Kit (CELL BIOLABS, Catalog No. VPK-109). 1) 293A cell plating: 24-well plate, 2.5E+5 cells per well; 2) 100 μl of the sample solution to be tested was added 24 hours after plating, and the cells were infected for 48 hours; 3) Fixation: Fix with methanol precooled at -20°C for 20 min, and wash with PBS three times; 4) Blocking: Block with 1% BSA for 1 hour; 5) Primary antibody: dilute hexon primary antibody (Santa Cruz, sc-58651) at a concentration of 1:500, add 200 μl to each well, dilute with 1% BSA, and incubate at 37°C for 1 hour; 6) Wash 3 times with PBS; 7) Secondary antibody: dilute HRP-labeled secondary antibody at a concentration of 1:1000, add 200 μl to each well, dilute with 1% BSA, and incubate at 37°C for 1 hour; 8) Wash 3 times with PBS; 9) Staining: Prepare fresh DAB staining solution (Solution A and Solution B are prepared at a ratio of 1:1), add 250 μl to each well, observe the color development under a microscope, discard the staining solution after 10-15 minutes of good color development, wash twice with water, and add PBS; 10) Photography: Take photos under a 10x objective lens, take photos of 9 fields of view in each well, including the upper left, upper, upper right, left, center, right, lower left, lower, and lower right, count the number of positive virus particles, and calculate the titer. The titer calculation formula is as follows: (ifu / m) = 135.2 × average field of view × dilution factor, according to the kit manual. The results are shown in Table 1. Table 1 As can be seen from Table 1, the starting dose of virus-infected stem cells is 1000 viruses / cell. After 4 hours of infection, the average number of active viruses carried by the cells was detected to be 0.77 viruses / cell, which is only about 7 / 10,000 of the initial infection dose. This result shows that the use of human dental pulp stem cells as a carrier greatly reduces the number of oncolytic viruses that directly enter the body. The following examples show that the oncolytic virus-human dental pulp stem cell composition obtained has a significant anti-tumor effect. Example 7: Anti-tumor effect of YSCH01-hDPSC in nude SW780 tumor-bearing mice 1. Construction of nude SW780 tumor-bearing mice: 6-8 week-old Balb / c mice were implanted with human bladder cancer cells SW780 in the axillae of both sides. Other operations were the same as in Example 5. The tumor volume of the mice was 80-100 mm 3 There were 5 mice in each group, and the tumor formation period was about 7-14 days. 2. Preparation of cells for treatment a. One day before administration: replace the P6-P9 generation hDPSC dental pulp stem cells for plating at a density of 6E+6 cells / T175 cell flask; b. On the day of administration: 18 hours after plating, take 3 T175 cell culture flasks, add oncolytic adenovirus YSCH01 at an infection dose of 1000 vp / cell, and mix the supernatant culture medium; the remaining T175 cell culture flasks are left untreated as the pure stem cell experimental group; c. In vivo administration: 4 hours after infection, trypsinize and resuspend in PBS phosphate buffer solution. A cell suspension of 2E+7 cells / ml was prepared. To avoid animal death caused by injection of aggregated cell clumps, the cell suspension was filtered through a 70 μm cell filter before injection. According to the experimental design, hDPSCs and YSCH01 / hDPSCs were injected in different ways, with 100 μl injected per animal, and the cell number was 2E+6 cells. The drug was administered once every 4 days, marked as Q4D, for a total of 4 times ( FIG. 9 ). During the above experiment, the mice were weighed every 3 days to monitor their growth and the tumor volume was monitored at the same time. The tumor volume was calculated as follows: volume (mm 3 )=Length(mm)×Width 2 (mm 2 ) / 2, body weight was in g, and “Graphpad Prism” software was used to plot tumor growth curves and body weight curves, and statistical analysis was performed. *, p<0.05 indicated a significant difference, **, p<0.01 indicated an extremely significant difference. The weight of mice is shown in Figure 10, indicating that both intravenous and intraperitoneal injections are safe in the SW780 model. In terms of anti-cancer effects, Figures 11A (left tumor) and 11B (right tumor) show that simple injection of hDPSC has no tumor-suppressing effect, and intraperitoneal injection (IP) or intravenous injection (IV) of "YSCH01 / hDPSC" can inhibit tumor growth to a certain extent, among which the effect of intraperitoneal injection is significantly better than that of intravenous injection. It can also be more intuitively seen from Figures 12 and 13 that the tumor volume and weight of the YSCH01 / hDPSC-IP group are the smallest. Example 8: Antitumor effect of YSCH01-hDPSC in human glossopharyngeal squamous cell carcinoma SCC152 tumor-bearing mice Construction of SCC152 bilateral tumor-bearing mouse model: As in Example 6, the subcutaneously implanted tumor cell line was human glossopharyngeal carcinoma cell line SCC152. According to the experimental design, hDPSC and YSCH01 / hDPSC were injected in different ways, 100 μl per mouse, the number of cells was 2E+6 cells, and the drug was administered once every 4 days, marked as Q4D, for a total of 5 times (see Figure 14). The animal dosing experiment was basically the same as in Example 7. In addition, a positive control group was set up, that is, YSCH01 oncolytic adenovirus was injected intratumorally, 2E+9 vp / injection, and a total of 5 times. The left tumor was the injection site, and the right tumor was not injected with oncolytic virus. The results in Figure 15 show that the body weight of mice in each group increased significantly, regardless of whether they were injected with simple hDPSC dental pulp stem cells or the “YSCH01 / hDPSC” dental pulp stem cell experimental group carrying oncolytic adenovirus. There was no obvious change, indicating that both dental pulp stem cells themselves and dental pulp stem cells carrying viruses were very safe under this model. From the perspective of tumor inhibition (Figure 16), the positive control group, i.e., the group with intratumoral injection of YSCH01 oncolytic adenovirus (marked as YSCH01-IT in the figure), still had the best effect. The group with intravenous injection of YSCH01 / hDPSC (marked as YSCH01 / hDPSC-IV in the figure) also had a certain tumor inhibition effect. The growth rate of the left tumor in this group was significantly lower than that of the dental pulp stem cell experimental group with simple injection and the control PBS experimental group without any treatment (Figure 16A), but the overall anti-cancer effect was weaker than that of the intraperitoneal injection of YSCH01 / hDPSC group (marked as YSCH01 / hDPSC-IP in the figure) and the intratumoral injection of IT experimental group; however, there was no statistical difference between its anti-cancer effect on the other side of the tumor and the control group (Figure 16B). Surprisingly, intraperitoneal injection of "YSCH01 / hDPSC" (labeled as YSCH01 / hDPSC-IP in the figure) showed a good effect of inhibiting tumor growth, and this effect was more obvious than that of the intravenous injection group, with a very significant statistical difference. The growth rate of bilateral tumors in this experimental group was significantly inhibited, and even the size of the right tumor was similar to the remote anti-cancer effect of the non-administered tumor in the intratumoral administration group (Figure 16B), indicating that this administration method has great potential, suggesting that intraperitoneal administration can play a good anti-cancer effect on multiple tumors in the body. However, after simple intravenous injection (abbreviated as hDPSC-IV in the figure), the left tumor did not shrink significantly, and there was no statistical difference with the control group. The right tumor showed the same growth characteristics as the left tumor between 25 days after tumor transplantation, and there was no statistical difference with the control group. It is worth noting that, based on the data measured in Example 6, the actual intracellular viral load of 0.77 VP / cell in the YSCH01 / hDPSC-IP group was much lower than the initial viral infection dose used to infect dental pulp stem cells. The actual viral load per administration was: 0.77 vp / cell×2×10 6 cells=1.54x10 6 VP. See Table 2. Table 2. Analysis of actual viral load of dental pulp stem cells carrying YSCH01 virus injected intraperitoneally / intravenously in vivo In order to further compare with different virus doses injected intratumorally, an experiment was designed to directly intraperitoneally administer oncolytic virus YSCH01 to three groups of SCC152 bilateral tumor-bearing mice under low-dose conditions, with doses of 2×10 5 VP / dosage, 2×10 6 VP / dosage and 2×10 7 VP / dosage, 2×10 6 VP / dosage is equivalent to the actual virus loading of dental pulp stem cells injected intraperitoneally / intravenously calculated in Table 2. The results showed that the three doses of oncolytic virus injected intratumorally had no anticancer effect and were no different from the control group (Figure 17). Based on the above analysis, it can be seen that the YSCH01 / hDPSC-IP group achieved a very significant anticancer effect under the condition of carrying a very low dose of virus. Based on the results of Example 7 and Example 8, it can be seen that YSCH01 / hDPSC has significant anticancer effects on human glossopharyngeal squamous cell carcinoma and bladder cancer under the intraperitoneal administration route, indicating that its anticancer effect does not depend on the tissue type of the cancer source and may have a good broad-spectrum anticancer effect. Example 9: Distribution of viruses in tumor tissues Hexon immunohistochemistry was used to detect the distribution of viral particles in tumor tissues. The experimental steps are as follows: Tumor tissues were isolated from both sides of the mice (samples from SW780 nude mice were taken 23 days after injection, and samples from SCC152 nude mice were taken 29 days after injection), fixed with 4% PFA for 24 h, embedded in paraffin, cut into 6 μm thick slices, and spread on positively charged glass slides; dried at 58°C for 1 h, and the wax on the tissue surface was observed to melt; dewaxing: xylene × 3, anhydrous ethanol × 2, 95% ethanol, 90% ethanol, 80% ethanol, 70% ethanol, distilled water × 3, xylene 10min each time, the rest 5min each time; Antigen repair (high pressure repair method): Add antigen repair solution (pH 6.0 citrate or citrate antigen repair solution) to the pressure cooker and heat it to boiling on the induction cooker. Put the dewaxed and hydrated tissue sections into the boiling buffer, cover the lid tightly, wait for a while until the air nozzle is lifted, add the pressure limiting valve, count for 2min after the pressure cooker sprays, and stop the fire; After antigen repair, the slides are naturally cooled to room temperature (25-30℃, about 1h). Remove the slides and wash with PBS for 5 min × 3 times; soak in 3% H2O2 for 20 min to remove endogenous oxidase, PBS × 3, 5 min each time; serum blocking: incubate with goat serum at room temperature for 10 min; primary antibody: add hexon antibody diluted 1:100, incubate at 37℃ for 1 h, PBS × 3; spin dry PBS on the sections, incubate with secondary antibody for 1 h, PBS × 3; wipe off the water around the tissue, add DAB color developer, observe under the microscope, the color development time is about 5 min; counterstaining: hematoxylin staining for 1 min, rinse with running water; dehydration: 70% ethanol, 80% ethanol, 90% ethanol, 95% ethanol, anhydrous ethanol × 2, xylene × 2, 5 min each time; seal the slides, scan, and analyze after taking pictures. In the SCC152 nude mouse tumor model (Figure 18), there were large areas of positive virus particles in both tumor sites after intraperitoneal injection, while there were no positively stained virus particles in the control experimental group. The tumor on the administration side of the intratumoral administration group regressed and could not be stained for observation; the tumor tissue on the non-administration side also had a certain tumor inhibition efficiency, but no obvious virus particles were found in the tumor tissue sections of this experimental group. The results in the SW780 nude mouse tumor model (Figure 19) also showed that there were large areas of positive virus particles in the bilateral tumor sites after intraperitoneal injection of YSCH01 / hDPSC, but not in the control group, which was consistent with the trend of the tumor inhibition curve results. The above experimental results show that dental pulp stem cells carrying oncolytic viruses can reach different tumor sites after intraperitoneal administration and exert anti-cancer effects without being restricted by the site of administration. Compared with intratumoral administration, the administration method of the present invention can better exert systemic anti-cancer effects and has obvious advantages in the treatment of metastatic tumors. The above description is only part of the embodiments of the present invention and is not intended to limit the protection scope of the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention are included in the protection scope of the present invention.

Claims

1. A pharmaceutical composition comprising an oncolytic virus and dental stem cells. 2 . The pharmaceutical composition according to claim 1 , wherein the oncolytic virus and dental stem cells are contained in separate containers, respectively.

3. The pharmaceutical composition according to claim 1, wherein at least a portion of the oncolytic virus is contained in the dental stem cells.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the dental stem cells are mesenchymal stem cells with self-renewal ability and multidifferentiation potential derived from tooth or periodontal tissue, such as dental pulp stem cells, exfoliated deciduous tooth stem cells, apical papilla stem cells, periodontal ligament stem cells, alveolar bone mesenchymal stem cells, tooth germ progenitor cells, gingival mesenchymal stem cells and dental follicle precursor cells. The pharmaceutical composition according to claim 4 , wherein the dental stem cells are dental pulp stem cells.

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein the dental stem cells are of human origin.

7. A pharmaceutical composition according to any one of claims 1-6, wherein the oncolytic virus is a attenuated wild-type virus strain, such as reovirus and Newcastle disease virus; or, the oncolytic virus is a genetically engineered virus, such as an engineered adenovirus, herpes simplex virus, vaccinia virus and measles virus.

8. The pharmaceutical composition according to any one of claims 1-7, wherein the oncolytic virus is an oncolytic adenovirus.

9. The pharmaceutical composition according to any one of claims 1-8, wherein the oncolytic virus is an oncolytic virus expressing interferon, preferably, the nucleotide sequence encoding the interferon is as shown in SEQ ID NO: 2-4.

10. The pharmaceutical composition according to any one of claims 1 to 9, further comprising a pharmaceutically acceptable carrier. The composition according to claim 10 , which is formulated for intraperitoneal administration.

12. The pharmaceutical composition according to any one of claims 1-11, wherein the content of the oncolytic virus carried by the dental stem cells is 0.1-100 vp / cell, for example, 0.2-50 vp / cell, 0.3-20 vp / cell, 0.4-10 vp / cell, 0.5-5 vp / cell or 0.6-2 vp / cell.

13. Use of the pharmaceutical composition according to any one of claims 1 to 12 in the preparation of a medicament for treating tumors.

14. The use according to claim 13, wherein the pharmaceutical composition is formulated as a dosage form for intraperitoneal administration.

15. The method of claim 13 or 14, wherein the tumor is selected from osteosarcoma, pancreatic cancer, bile duct cancer, vulvar cancer, prostate cancer, breast cancer, colorectal cancer, lung cancer, liver cancer, melanoma, lymphoma, gastric cancer, esophageal cancer, ovarian cancer, head and neck squamous cell carcinoma, squamous cell carcinoma, bladder cancer, glioma, cervical cancer and renal cancer.

16. A method for treating a tumor, comprising administering the pharmaceutical composition according to any one of claims 1 to 12 to a subject in need thereof.

17. The method of claim 16, wherein the pharmaceutical composition is administered by intraperitoneal administration.

18. The method of claim 17, further comprising intratumoral injection of the pharmaceutical composition.

19. The method of claims 16-18, wherein the tumor is selected from osteosarcoma, pancreatic cancer, bile duct cancer, vulvar cancer, prostate cancer, breast cancer, colorectal cancer, lung cancer, liver cancer, melanoma, lymphoma, gastric cancer, esophageal cancer, ovarian cancer, head and neck squamous cell carcinoma, squamous cell carcinoma, bladder cancer, glioma, cervical cancer and renal cancer.

20. A method for preventing tumor recurrence, comprising administering the pharmaceutical composition of any one of claims 1 to 12 to a subject in need thereof.

21. A method for inhibiting tumor metastasis, comprising administering the pharmaceutical composition according to any one of claims 1 to 12 to a subject in need thereof.