Application of SREBF1 in the preparation of chemotherapeutic drugs for treating acute T-lymphocytic leukemia

Abstract

The present invention discloses the application of SREBF1 in the preparation of chemotherapeutic drugs for treating acute T-lymphoblastic leukemia, belonging to the technical field of biomedicine. The present invention clarifies the changes in the bone marrow microenvironment of T-ALL patients before and after chemotherapy and inhibits its protective effect on minimal residual disease, clarifies the role of SREBF1 in the abnormal lipidation of the bone marrow microenvironment after T-ALL chemotherapy, and inhibiting the function of SREBF1 can inhibit the generation of adipocytes induced by DEX, thereby reversing the protective effect of adipocytes on residual T-ALL cells. Therefore, inhibiting SREBF1 may be a feasible strategy to solve the recurrence dilemma of T-ALL, which is beneficial to the development of clinical therapeutic drugs for clearing minimal residual T-ALL, provides new treatment ideas and drugs for effectively clearing minimal residual disease of T-ALL and further improving the treatment of T-ALL, and has very important clinical significance.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to the application of SREBF1 in the preparation of chemotherapeutic drugs for treating acute T-lymphoblastic leukemia. Background Art

[0002] Disclosing the information of this background art section is only intended to enhance the understanding of the overall background of the present invention, and is not necessarily to be regarded as an admission or any form of implication that this information constitutes the prior art already known to those of ordinary skill in the art.

[0003] Acute T-lymphoblastic leukemia (T-ALL) is an aggressive malignant blood disease caused by the clonal expansion of variant T-cell precursor cells, accounting for about 25% of all adult cases. With the use of intensive combination chemotherapy (at least one glucocorticoid, vincristine, and an anthracycline drug) and the adjustment of treatment methods according to the patient's response, great progress has been made in the treatment of T-ALL, and the complete remission rate is very high, but relapses often occur. Most patients develop refractory and relapsed conditions, ultimately leading to treatment failure. The five-year survival rate of adults under 60 years old is only 40%-50%, and the prognosis of older patients is worse. The fundamental cause of relapse is the presence of minimal residual disease (MRD), and the bone marrow microenvironment (BMM) is the main site of MRD, which can shelter residual leukemia cells from being killed by chemotherapeutic drugs.

[0004] Bone marrow mesenchymal stem cells (BMSCs), together with adipocytes, endothelial cells, osteoblasts, fibroblasts, etc., constitute bone marrow stromal cells, which are the main components of the bone marrow microenvironment. Recent studies have shown that bone marrow adipocytes can participate in the occurrence and development of malignant tumors by providing energy to solid tumor cells; in multiple myeloma, bone marrow adipocytes protect multiple myeloma cells from chemotherapy-induced apoptosis by secreting adipokines and promote bone metastasis. Adipocytes also play an important role in the survival, proliferation, invasion, and drug resistance of leukemia.

[0005] Bone marrow mesenchymal stem cells (BMSCs) are the precursor cells of adipocytes, and some chemotherapeutic drugs can promote the differentiation of BMSCs into adipocytes. For example, in acute myeloid leukemia, the self-renewal ability of bone marrow mesenchymal stem cells is reduced, and after treatment with cytarabine (Ara-C), they are prone to differentiate into adipocytes and chondrocytes. However, the changes in adipocytes in the bone marrow microenvironment of T-ALL patients after chemotherapy have not been reported, and the molecular mechanism involved in regulating the changes in the bone marrow microenvironment of T-ALL patients before and after chemotherapy is not clear. Summary of the Invention

[0006] Aiming at the deficiencies of the existing technology, the present invention provides the application of SREBF1 in the preparation of chemotherapeutic drugs for treating acute T-lymphoblastic leukemia. The purpose of the present invention is to clarify the changes in the bone marrow microenvironment of T-ALL patients before and after chemotherapy and inhibit its protective effect on minimal residual disease, so as to provide new treatment ideas and drugs for effectively clearing minimal residual disease of T-ALL and further improving the treatment of T-ALL.

[0007] In view of the above-mentioned status quo of the existing technology and based on the above findings, the present invention provides the following technical solutions.

[0008] In the first aspect of the present invention, there is provided the application of a substance for detecting the expression level of SREBF1 in the preparation of a product for evaluating the prognosis of patients with acute T-lymphoblastic leukemia.

[0009] The present invention discovers through research that, compared with the BMSCs group without adipogenic differentiation, the expression level of SREBF1 in the BMSCs group after adipogenic differentiation induction is significantly increased.

[0010] In the second aspect of the present invention, there is provided a product for evaluating the prognosis of acute T-lymphoblastic leukemia, and the product contains a substance for detecting SREBF1.

[0011] Among them, the substance for detecting SREBF1 includes but is not limited to substances for detecting the expression level of SREBF1 by RT-PCR, real-time quantitative PCR, in situ hybridization, gene chip, and gene sequencing.

[0012] The product includes but is not limited to primers, probes, chips, nucleic acid membrane strips, preparations, or kits for detecting the expression level of SREBF1 in a test sample.

[0013] The test sample can be a human sample, and more specifically, the test sample includes the spleen or bone marrow of a subject.

[0014] In the third aspect of the present invention, there is provided a system for diagnosing or assisting in diagnosing the recurrence of acute T-lymphoblastic leukemia, and the system includes:

[0015] i) An analysis unit, the analysis unit comprising: a detection substance for determining the expression level of SREBF1 in a test sample of a subject, and;

[0016] ii) An evaluation unit, the evaluation unit comprising: judging whether the acute T-lymphocytic leukemia of the subject recurs according to the SREBF1 expression level determined in i);

[0017] The specific evaluation process of the evaluation unit in step ii) includes:

[0018] Compared with the reference, if the expression level of SREBF1 in the test sample of the subject is up-regulated, then the subject is or is a candidate for a patient with recurrent acute T-lymphocytic leukemia; otherwise, the subject is not or is not a candidate for a patient with recurrent acute T-lymphocytic leukemia.

[0019] In the fourth aspect of the present invention, there is provided the use of SREBF1 as a therapeutic target for abnormal lipidation of the bone marrow microenvironment after chemotherapy for acute T-lymphocytic leukemia in the preparation of a chemotherapeutic drug for treating acute T-lymphocytic leukemia.

[0020] In the fifth aspect of the present invention, there is provided the use of a substance that inhibits the expression of SREBF1 and / or reduces its activity in the preparation of a product for treating abnormal lipidation of the bone marrow microenvironment after chemotherapy for acute T-lymphocytic leukemia.

[0021] In the sixth aspect of the present invention, there is provided the use of a substance that inhibits the expression of SREBF1 and / or reduces its activity in the preparation of a drug for inhibiting the recurrence of acute T-lymphocytic leukemia.

[0022] The beneficial effects of the present invention are:

[0023] The present invention clarifies the changes in the bone marrow microenvironment of T-ALL patients before and after chemotherapy and inhibits its protective effect on minimal residual disease, clarifies the role of SREBF1 in abnormal lipidation of the bone marrow microenvironment after T-ALL chemotherapy, and inhibiting the function of SREBF1 can inhibit the generation of adipocytes induced by DEX, thereby reversing the protective effect of adipocytes on residual T-ALL cells. Therefore, inhibiting SREBF1 may be a feasible strategy to solve the recurrence dilemma of T-ALL.

[0024] The present invention provides new insights into the survival mechanism of residual T-ALL cells after chemotherapy from the perspective of improving the bone marrow microenvironment, which is beneficial to the development of clinical treatment drugs for clearing minimal residual T-ALL, and provides new treatment ideas and drugs for effectively clearing minimal residual disease of T-ALL and further improving the treatment of T-ALL, and has very important clinical significance. Description of the Drawings

[0025] The accompanying drawings of the specification, which form a part of the present invention, are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention.

[0026] Figure 1 For detecting the proportion of adipocytes in the bone marrow microenvironment of newly diagnosed T-ALL patients before and after chemotherapy by H&E staining; among them, Diagnosis is: bone marrow biopsy specimens of newly diagnosed T-ALL patients; Post-chemo is: bone marrow biopsy specimens of T-ALL patients who achieved complete remission after chemotherapy corresponding to the newly diagnosed ones;

[0027] Figure 2 For detecting the effect of chemotherapeutic drugs on the adipogenic differentiation of BMSCs by BODIPY fluorescence staining;

[0028] Figure 3 For the effect of adipocytes on the adhesion, anti-apoptosis ability and colony formation ability of T-ALL cells;

[0029] Figure 4 For the screening of differentially expressed molecules during the adipogenic differentiation of BMSCs in Example 1 by RNA-Seq;

[0030] Figure 5 For the enrichment analysis of differentially expressed molecules during the adipogenic differentiation of BMSCs in Example 1;

[0031] Figure 6 For the change in the expression level of SREBF1 during the adipogenic differentiation of BMSCs in Example 1;

[0032] Figure 7 For verifying the effect of inhibiting the function of SREBF1 on the adipogenic differentiation of BMSCs in vitro in Example 2;

[0033] Figure 8 For verifying the effect of inhibiting the function of SREBF1 on the adipogenic differentiation of BMSCs in vivo in Example 3;

[0034] Figure 9 For verifying the effect of applying an SREBF1 inhibitor to reverse the protective effect of adipocytes in vitro in Example 4;

[0035] Figure 10 For verifying the effect of applying an SREBF1 inhibitor to reverse the protective effect of adipocytes in vivo in Example 5. Detailed implementation manners

[0036] The present invention will be further described with reference to specific examples. The following examples are only for explaining the present invention and do not limit its content. If the specific experimental conditions are not specified in the examples, they are usually in accordance with conventional conditions or the conditions recommended by the sales company; the materials, reagents, etc. used in the examples, unless otherwise specified, can be obtained through commercial channels.

[0037] The term "expression level" refers to the amount of a gene product present in vivo or in a sample at a specific point in time. The expression level can be measured / quantified / detected, for example, by the protein or mRNA expressed by the gene. The expression level can be quantified, for example, as follows: normalizing the amount of the target gene product present in the sample by the total amount (total protein or mRNA) of the same type of gene product in the same sample or a reference sample (e.g., a sample obtained from the same individual at the same time or a portion of the same size (weight, volume) of the same sample), or determining the amount of the target gene product / defined sample size (weight, volume, etc.). The expression level can be measured or detected by any method known in the art, such as methods for the direct detection and quantification of the target gene product (e.g., mass spectrometry), or methods for the indirect detection and measurement of the target gene product that typically work by binding of the target gene product to one or more different molecules or detection devices specific for the target gene product (e.g., primers, probes, antibodies, protein scaffolds). It is also known to those skilled in the art to determine the level of gene copies, which also includes determining the absence or presence of one or more fragments (e.g., by nucleic acid probes or primers, such as quantitative PCR, multiplex ligation-dependent probe amplification (MLPA) PCR).

[0038] The terms "indicator" and "marker" are used interchangeably in the present invention and refer to a sign or signal of a disorder or for monitoring a disorder. Such a "disorder" refers to the biological state of a cell, tissue or organ, or to the health and / or disease state of an individual. An indicator can be the presence or absence of a molecule including, but not limited to, a peptide, protein and nucleic acid, or can be a change in the expression level or pattern of such a molecule in a cell, or tissue, organ or individual. An indicator can be a sign of the occurrence, development or presence of a disease in an individual or a further progression of such a disease. An indicator can also be a sign of the risk of developing a disease in an individual.

[0039] The terms "upregulation", "elevation" or "increase" in the level of an indicator refer to a decrease in the level of such an indicator in a sample compared to a reference.

[0040] The terms "downregulation", "decrease" or "drop" in the level of an indicator refer to a decrease in the level of such an indicator in a sample compared to a reference.

[0041] As used herein, the term "kit" refers to a collection of the above components, preferably provided separately or in a single container. The container also preferably contains instructions for practicing the methods of the present invention. Examples of these components of the kit and their methods of use have been given in this specification. Preferably, the kit contains the above components in a ready-to-use formulation. Preferably, the kit may additionally include instructions, such as a user manual for adjusting the components (e.g., the concentration of the detection agent) and for interpreting the results of any assays regarding the diagnosis provided by the methods of the present invention. In particular, such a manual may include information for assigning the amount of the determined gene product to a type of diagnosis. Details are found elsewhere in this specification. In addition, such a user manual may provide instructions for correctly using the components of the kit for determining the amount of the corresponding biomarker. The user manual may be provided in paper or electronic form (e.g., stored on a CD or CD ROM). The present invention also relates to the use of said kit in any method according to the present invention.

[0042] As used herein, the term "system" refers to a device system that includes at least the above-described devices that are effectively interconnected to allow for diagnosis. Preferred devices for determining the methylation status or amount of a gene product and devices for making comparisons were disclosed above in connection with the method of the present invention. How the devices are operatively connected will depend on the type of devices included in the equipment. For example, in the case of applying a device for the automated determination of the methylation status or amount of a gene product, the data obtained by the automated operating device can be processed, for example, by a computer program to establish a diagnosis. Preferably, in such a case, the devices are included in a single device. Thus, the equipment may include an analysis unit for determining the methylation status or amount of a gene product in a sample and an evaluation unit for processing the resulting data for diagnosis. Preferred detection devices were disclosed above in connection with embodiments relating to the method of the present invention. In this case, the devices are effectively connected such that the user of the system combines the result of the determination of the amount and its diagnostic value due to the instructions and explanations given in the manual. In such an embodiment, the devices may be presented as separate devices and are preferably packaged together as a kit. Those skilled in the art will understand how to connect the devices without further creative skills. Preferred equipment are those that can be applied without the specific knowledge of a professional clinician, such as test strips or electronic devices that only require loading of the sample. The results can be output as parameter diagnostic raw data, preferably given as an absolute or relative amount. It should be understood that these data will need to be interpreted by a clinician. However, expert system equipment is also envisioned, where the output includes processed diagnostic raw data that does not require a professional clinician for interpretation. Other preferred equipment includes an analysis unit / device (e.g., biosensor, array, solid support coupled to a ligand that specifically recognizes a polypeptide, surface plasmon resonance device, NMR spectrometer, mass spectrometer, etc.) or an evaluation unit / device mentioned above according to the method of the present invention.

[0043] As introduced in the background art, in view of the phenomenon that minimal residual disease in current T-ALL leads to the recurrence of acute T-lymphoblastic leukemia, the object of the present invention is to provide the application of SREBF1 in the preparation of chemotherapeutic drugs for treating acute T-lymphoblastic leukemia, to clarify the mechanism by which the bone marrow microenvironment changes after chemotherapy and then shelters residual T-ALL cells, and to provide a new therapeutic target for the treatment of minimal residual disease.

[0044] In a typical embodiment of the present invention, the application of a substance for detecting the expression level of SREBF1 in the preparation of a product for evaluating the prognosis of patients with acute T-lymphoblastic leukemia.

[0045] Wherein, SREBF1 is up-regulated in patients with relapsed acute T-lymphoblastic leukemia after chemotherapy.

[0046] The present invention uses the bone marrow biopsy tissues of adult T-ALL patients before and after treatment in a one-to-one correspondence, compares the changes in the bone marrow biopsy pathology of T-ALL patients before and after chemotherapy, and finds that after chemotherapy in T-ALL patients, the adipocytes in the bone marrow increase significantly; further research finds that after adding the chemotherapeutic drug dexamethasone (DEX) to the BMSCs of T-ALL patients cultured in vitro, it can promote their differentiation into adipocytes. And it is further confirmed that the adipocytes derived from BMSCs have a supporting and protective effect on the residual T-ALL cells. At the same time, through RNA-Seq, the key molecule SREBF1 for DEX-induced abnormal adipogenic differentiation of BMSCs is screened out, and it is first confirmed by subsequent in vitro cell experiments and in vivo animal experiments that inhibiting SREBF1 can reduce the promoting effect of DEX on the adipogenic differentiation of BMSCs and inhibit the protective effect of adipocytes on residual T-ALL cells. Therefore, SREBF1 can be used as one of the targets for minimal residual disease of T-ALL. The present invention finds through research that compared with the BMSCs group without adipogenic differentiation, the expression level of SREBF1 in the BMSCs group after adipogenic differentiation induction is significantly increased.

[0047] In another specific embodiment of the present invention, a product is provided, and the product contains the above-mentioned substance for detecting SREBF1, and the product is used to evaluate the prognosis of acute T-lymphoblastic leukemia.

[0048] Among them, the substance for detecting SREBF1 includes but is not limited to substances for detecting the expression level of SREBF1 by RT-PCR, real-time quantitative PCR, in situ hybridization, gene chip, and gene sequencing.

[0049] The product includes but is not limited to primers, probes, chips, nucleic acid membrane strips, preparations, or kits for detecting the expression level of SREBF1 in a test sample.

[0050] The test sample is a human-derived sample and a non-human-derived sample. More specifically, the test sample includes the cells, tissues, organs, and body fluids of a subject.

[0051] Among them, the tissue can be the spleen, bone marrow;

[0052] Further, in addition to the above-mentioned SREBF1 biomarker, the product further comprises substances suitable for detecting other biomarkers for the recurrence of acute T-lymphocytic leukemia, and the other biomarkers include, but are not limited to, biomarkers related to endocrine factors, biomarkers related to infectious factors (such as any severe infection that can cause bacteremia or viremia), biomarkers related to immune function (such as antiphospholipid antibody, antinuclear antibody, anti-DNA antibody, anti-sperm antibody, anti-thyroid antibody, increased number and activity of natural killer (NK) cells, abnormal macrophage function, abnormal dendritic cell function, abnormal complement system, lack of blocking antibody, abnormal T and B lymphocytes, abnormal Th1 / Th2 cytokines of helper T lymphocytes, etc.), biomarkers related to prethrombotic state (such as gene mutations of factor V and factor II (prothrombin), protein S deficiency), and other systemic diseases of the subject. By using in combination with other biomarkers, the interference of other physiological and pathological states can be further excluded, thereby improving the sensitivity and specificity of detection.

[0053] In another specific embodiment of the present invention, there is provided a system for diagnosing or assisting in the diagnosis of the recurrence of acute T-lymphocytic leukemia, and the system comprises:

[0054] i) An analysis unit, the analysis unit comprising: a detection substance for determining the expression level of SREBF1 in a sample to be tested of a subject, and;

[0055] ii) An evaluation unit, the evaluation unit comprising: judging whether the subject has recurrence of acute T-lymphocytic leukemia according to the expression level of SREBF1 determined in i).

[0056] In another specific embodiment of the present invention, the above-mentioned sample to be tested includes cells, tissues, organs and body fluids of the subject;

[0057] Wherein, the tissue may be the spleen, bone marrow.

[0058] In another specific embodiment of the present invention, the above-mentioned detection substances include, but are not limited to, substances for detecting the expression level of SREBF1 by RT-PCR, real-time quantitative PCR, in situ hybridization, gene chip and gene sequencing.

[0059] In yet another specific embodiment of the present invention, in the analysis unit, there is further included a substance for detecting other biomarkers currently suitable for detecting the recurrence of acute T-lymphocytic leukemia. The other biomarkers include, but are not limited to, biomarkers related to endocrine factors, biomarkers related to infectious factors (such as any severe infection that can cause bacteremia or viremia), biomarkers related to immune function (such as antiphospholipid antibody, antinuclear antibody, anti-DNA antibody, anti-sperm antibody, anti-thyroid antibody, increased number and activity of natural killer (NK) cells, abnormal macrophage function, abnormal dendritic cell function, abnormal complement system, lack of blocking antibody, abnormal T and B lymphocytes, abnormal Th1 / Th2 cytokines of helper T lymphocytes, etc.), biomarkers related to prethrombotic state (such as gene mutations of factor V and factor II (prothrombin), protein S deficiency), and other systemic diseases of the subject. By using in combination with other biomarkers, interference from other physiological and pathological states can be further excluded, thereby improving the sensitivity and specificity of the detection.

[0060] In yet another specific embodiment of the present invention, the specific evaluation process of the evaluation unit includes:

[0061] Compared with the reference, if the expression level of SREBF1 in the test sample of the subject is up-regulated, then the subject is or is a candidate for a patient with recurrent acute T-lymphocytic leukemia; otherwise, the subject is not or is not a candidate for a patient with recurrent acute T-lymphocytic leukemia.

[0062] Among them, the "reference" can be a suitable control sample, such as a sample from a normal healthy subject who has no symptoms related to recurrent miscarriage and no abnormal physiological and pathological findings. The reference can also be a sample from the same subject before showing symptoms of the disease or before being diagnosed with recurrent miscarriage. The reference can be a standardized sample. For example, a sample containing materials or data from samples of several healthy subjects who have no symptoms of recurrent miscarriage and no related physiological and pathological findings.

[0063] The system for diagnosing or assisting in the diagnosis of recurrent miscarriage of the present invention can be a virtual device, as long as it can implement the functions of the analysis unit and the evaluation unit. The analysis unit can include various detection reagent materials and / or detection instrument devices, etc.; the evaluation unit can be any computing instrument, module or virtual device that can analyze and process the detection results of the analysis unit to obtain the risk assessment status of recurrent miscarriage. For example, various possible detection results and corresponding disease risk situations can be pre-formulated into a corresponding data chart, and the detection results of the detection unit are compared with this data chart to obtain the risk assessment result of recurrent miscarriage.

[0064] In another specific embodiment of the present invention, there is provided the use of SREBF1 as a therapeutic target for abnormal lipidation of the bone marrow microenvironment after chemotherapy for acute T-lymphoblastic leukemia in the preparation of a chemotherapeutic drug for treating acute T-lymphoblastic leukemia.

[0065] In another specific embodiment of the present invention, the use includes substances that inhibit SREBF1 expression and / or reduce its activity for the preparation of a chemotherapeutic drug for treating acute T-lymphoblastic leukemia.

[0066] In another specific embodiment of the present invention, the substances that inhibit SREBF1 expression and / or reduce its activity can be interfering molecules that target SREBF1 sequences and inhibit SREBF1 expression, specifically including shRNA (small hairpin RNA), small interfering RNA (siRNA), dsRNA, microRNA, antisense nucleic acids, or constructs that can express or form the shRNA, small interfering RNA, dsRNA, microRNA, antisense nucleic acids; it can also include compound inhibitors. Preferably, the substances include, but are not limited to, Fatostatin HBr, shRNA that knocks down SREBF1.

[0067] In the present invention, T-ALL patient BMSCs were treated with lentivirus for downregulating SREBF1 (shSREBF1) or with an SREBF1 inhibitor (Fatostatin HBr, FH). DEX was used to induce the differentiation of BMSCs towards the adipose direction. As the induction time extended, the number of lipidated cells and lipid droplets in each group increased significantly; when lentivirus was used to downregulate SREBF1 or an SREBF1 inhibitor was used for intervention, the number of lipidated cells and lipid droplets induced by DEX decreased significantly. Among them, the nucleotide sequence of the lentivirus for downregulating SREBF1 (shSREBF1) is: TCTCCATCAGTTCCAGCAT (SEQ ID No.1).

[0068] In another specific embodiment of the present invention, the chemotherapeutic drug for treating acute T-lymphoblastic leukemia includes a drug for inhibiting minimal residual disease of T-ALL.

[0069] In another specific embodiment of the present invention, there is provided the use of substances that inhibit SREBF1 expression and / or reduce its activity in the preparation of a product for treating abnormal lipidation of the bone marrow microenvironment after chemotherapy for acute T-lymphoblastic leukemia.

[0070] Among them, the substances include Fatostatin HBr, shRNA that knocks down SREBF1. The nucleotide sequence of the shRNA that knocks down SREBF1 is: TCTCCATCAGTTCCAGCAT (SEQ ID No.1).

[0071] In another specific embodiment of the present invention, the functions of the product are any one or more of the following:

[0072] a. Inhibiting the generation of adipocytes in the bone marrow microenvironment induced by glucocorticoids;

[0073] b. Inhibiting the protective effect of adipocytes in the bone marrow microenvironment on residual T-ALL cells.

[0074] In another specific embodiment of the present invention, the glucocorticoid includes dexamethasone DEX.

[0075] In another specific embodiment of the present invention, the product is composed of a substance that inhibits the expression of SREBF1 and / or reduces its activity and pharmaceutically necessary excipients.

[0076] In another specific embodiment of the present invention, the above product can be a drug.

[0077] The drug may further include a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be a buffer, an emulsifier, a suspending agent, a stabilizer, a preservative, an excipient, a filler, a coagulant and a conditioner, a surfactant, a dispersant or an antifoaming agent.

[0078] The drug may further include a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be a virus, a microcapsule, a liposome, a nanoparticle or a polymer and any combination thereof. The delivery carrier of the pharmaceutically acceptable carrier may be a liposome, a biocompatible polymer (including natural polymers and synthetic polymers), a lipoprotein, a polypeptide, a polysaccharide, a lipopolysaccharide, an artificial virus envelope, an inorganic (including metal) particle, and a bacterium or a virus (such as a baculovirus, an adenovirus and a retrovirus), a phage, a cosmid or a plasmid vector.

[0079] The drug may also be used in combination with other drugs for preventing and / or treating recurrent miscarriage. Other preventive and / or therapeutic compounds may be administered simultaneously with the main active ingredient, even in the same composition.

[0080] The drug may also be administered to other preventive and / or therapeutic compounds in a separate composition or in a dosage form different from that of the main active ingredient. A partial dose of the main ingredient may be administered simultaneously with other therapeutic compounds, while other doses may be administered separately. During the treatment process, the dose of the drug of the present invention may be adjusted according to the severity of the symptoms, the frequency of recurrence and the physiological response of the treatment regimen.

[0081] The medicament of the present invention can be administered into the body by known methods. For example, it can be systemically delivered via intravenous injection or locally injected into the tissue of interest. Optionally, it can be administered via intravenous, percutaneous, intranasal, mucosal or other delivery methods. Such administration can be carried out via a single dose or multiple doses. Those skilled in the art understand that the actual dose to be administered in the present invention can vary to a large extent depending on various factors, such as the target cells, the biological type or its tissue, the general condition of the subject to be treated, the administration route, the administration method, and so on.

[0082] In another specific embodiment of the present invention, there is provided the use of a substance that inhibits the expression of SREBF1 and / or reduces its activity in the preparation of a medicament for inhibiting the recurrence of acute T-lymphoblastic leukemia.

[0083] Among them, the substance includes Fatostatin HBr and shRNA that knocks down SREBF1. The nucleotide sequence of the shRNA that knocks down SREBF1 is: TCTCCATCAGTTCCAGCAT (SEQ ID No.1).

[0084] In order to verify the feasibility of using a substance that inhibits the expression of SREBF1 and / or reduces its activity as a medicament for inhibiting the residual active ingredients after chemotherapy for acute T-lymphoblastic leukemia, the present invention conducted in-vivo experiments using an NPG xenograft mouse model. At the end of the 3rd treatment cycle, compared with the Control group, the proportion in the DEX group was significantly reduced, and the DEX group showed a statistically significant therapeutic effect. However, compared with the DEX group, there was no significant difference in the therapeutic effect in the DEX + SREBF1 inhibitor group. At the end of the 4th and 5th treatment cycles, compared with the Control group, the proportion in the DEX group was significantly reduced, and the DEX group had a more obvious therapeutic effect; compared with the DEX group, the proportion of T-ALL cells in the combined treatment group (DEX + SREBF1 inhibitor group) was significantly reduced. After the administration at the end of the 5th cycle, the mice were euthanized. Compared with the Control group, the size and weight of the spleen in the DEX group were significantly reduced, and the proportion of T-ALL cells in the spleen and bone marrow was also significantly reduced; compared with the DEX group, the size and weight of the spleen in the combined treatment group (DEX + SREBF1 inhibitor group) were significantly reduced, and the proportion of T-ALL cells in the spleen and bone marrow was also significantly reduced. This indicates that a substance that inhibits the expression of SREBF1 and / or reduces its activity can be used as a medicament for inhibiting the residual active ingredients after chemotherapy for acute T-lymphoblastic leukemia.

[0085] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the following will specifically describe the technical solution of the present invention in detail with reference to specific examples.

[0086] Changes in the expression of SREBF1 during DEX-induced adipogenic differentiation of BMSCs in Example 1

[0087] RNA-seq analysis was performed using T-ALL specimens to systematically explore the differential changes in BMSCs during adipogenic differentiation.

[0088] BMSCs from 3 T-ALL patients were isolated and cultured in vitro and divided into 2 groups for treatment:

[0089] ① BMSC group (BMSCs): BMSCs were cultured in α-MEM containing 10% fetal bovine serum;

[0090] ② BMSC group induced for 14 days of adipogenic differentiation (Induced-BMSCs): Added to 10% fetal bovine serum α-MEM containing a final concentration of 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 10 μg / ml insulin, and 1 μM dexamethasone (DEX) to culture and induce adipogenic differentiation of BMSCs.

[0091] Cells were collected on the 14th day, RNA was extracted, and RNA-Seq was performed to screen for genes with differential expression before and after adipogenic differentiation of BMSCs.

[0092] The detection results showed that there were obvious molecular differences between the BMSC group and the BMSC group after adipogenic differentiation induction. A total of 3886 genes had significant differential expression between the two groups (q < 0.05,

[0093] |log2fold - chang| > 0, Figure 4 ), including 1813 up-regulated genes and 2073 down-regulated genes.

[0094] Then, KEGG functional enrichment analysis was performed on the 1813 up-regulated genes in the BMSC group after adipogenic differentiation induction ( Figure 5 ), and the results showed that these up-regulated genes were significantly involved in oxidative phosphorylation, fatty acid metabolism, biosynthesis of unsaturated fatty acids, etc., all of which are related to fat formation.

[0095] According to the functional enrichment results, 209 genes related to fat formation were further screened out. Among them, the expression of SREBF1 was significantly up-regulated in the induced BMSC group. Therefore, SREBF1 may be a key target for abnormal adipogenic differentiation of bone marrow BMSCs in T-ALL patients after chemotherapy.

[0096] The expression of SREBF1 in BMSCs after adipogenic differentiation induction was verified by qRT-PCR and Western-Blot, and the results were as Figure 6As shown, compared with the BMSCs group, the expression level of SREBF1 in the BMSCs group after adipogenic differentiation induction was significantly increased, which was consistent with the RNA-Seq results, suggesting that DEX may promote the differentiation of BMSCs into adipocytes by regulating the expression of SREBF1.

[0097] Example 2 Detection of the effect of inhibiting the function of SREBF1 on the adipogenic differentiation of BMSCs in vitro

[0098] In vitro experiments, BMSCs from T-ALL patients were treated with lentivirus for downregulating SREBF1 (shSREBF1) or with an SREBF1 inhibitor (Fatostatin HBr, FH) respectively, and DEX was used to induce the adipogenic differentiation of BMSCs. On the 7th, 14th, and 21st days of induction, Oil Red O staining was used to detect the effect of intervening SREBF1 on the adipogenic differentiation of BMSCs induced by DEX. Among them, the nucleotide sequence of the lentivirus shSREBF1 for downregulating SREBF1 is: TCTCCATCAGTTCCAGCAT (SEQ ID No.1).

[0099] The results were as Figure 7 shown: As the induction time extended, the number of lipidized cells and lipid droplets in each group increased significantly; when SREBF1 was downregulated by lentivirus or intervened with an SREBF1 inhibitor, the number of lipidized cells and lipid droplets induced by DEX decreased significantly. It is suggested that inhibiting the expression or function of SREBF1 can reverse the promoting effect of DEX on the differentiation of BMSCs into adipocytes, indicating that SREBF1 plays a key role in the process of DEX-induced adipogenic differentiation of BMSCs.

[0100] Example 3 Detection of the effect of inhibiting the function of SREBF1 on the adipogenic differentiation of BMSCs in mice in vivo

[0101] In in vivo experiments, first, the human T-ALL cell line Jurkat cells were transfected with a lentivirus carrying green fluorescent protein (GFP). After sorting GFP+ cells by flow cytometry and amplifying and culturing them, severe immunodeficient mice aged 6 - 8 weeks were given 1.5 Gy of radiation. The GFP+ Jurkat cells were inoculated into each mouse through tail vein injection to establish a T-ALL human xenograft model. Flow cytometry was continuously used to monitor the content of GFP+ cells in the peripheral blood of mice. When the proportion of GFP+ cells in the peripheral blood could be monitored, the mice were divided into 3 groups and started to be administered drugs.

[0102] The first group: Control group, treated with a control solvent;

[0103] The second group, DEX group, treated with 1.5 mg / kg / d DEX;

[0104] Group 3: DEX + SREBF1 inhibitor group, treated with 1.5 mg / kg / d DEX combined with 15 mg / kg / d SREBF1 inhibitor (Fatostatin HBr (FH)).

[0105] Taking continuous administration for 5 days and discontinuing for 2 days per week as a cycle, a total of 5 cycles were administered, for a total of 35 days.

[0106] Mice were selected after the end of the 3rd and 5th cycles, and the femurs of the mice were extracted, sectioned and stained to observe the content of adipocytes.

[0107] The results are as Figure 8 shown. Compared with the Control group, the fat content in the DEX group was significantly higher than that in the Control group and the DEX + SREBF1 inhibitor group. The bone marrow fat content in the DEX group at the end of the 5th cycle was significantly higher than that in the DEX group at the end of the 3rd cycle. It shows that during the treatment, DEX promotes the adipogenic differentiation of bone marrow BMSCs in T-ALL mice, and the application of SREBF1 inhibitor can block the adipogenic differentiation of bone marrow BMSCs in mice induced by DEX.

[0108] Example 4 In vitro verification of the effect of applying SREBF1 inhibitor to reverse the protective effect of adipocytes

[0109] The adipogenic differentiation process of BMSCs was continuously intervened with the SREBF1 inhibitor (Fatostatin HBr), and BMSCs were induced to differentiate into adipocytes for 21 days. Using the adipocyte group without inhibitor treatment as a control, the above SREBF1-inhibited adipocytes or adipocytes were directly co-cultured with T-ALL cells. It was found that after the application of the SREBF1 inhibitor, with the decrease in the lipidation ratio of BMSCs, the adhesion to T-ALL cells was significantly reduced; the apoptosis rate of T-ALL cells induced by drugs was significantly increased, and the proportion of surviving cells was significantly reduced; the colony formation ability of T-ALL cells was significantly reduced. ( Figure 9 )

[0110] Example 5 Effect of applying SREBF1 inhibitor to reverse the protective effect of adipocytes after chemotherapy in T-ALL mice

[0111] According to the mouse model in Example 3, at the end of the 3rd cycle of treatment, compared with the Control group, the proportion of GFP+ cells in the DEX group was significantly reduced. The DEX group showed a statistically significant therapeutic effect, but there was no significant difference in the therapeutic effect between the DEX + SREBF1 inhibitor group and the DEX group.

[0112] At the end of the 4th and 5th treatment cycles, compared with the Control group, the proportion of GFP+ cells in the DEX group was significantly reduced, and the DEX group had a more obvious therapeutic effect; compared with the DEX group, the proportion of GFP+ cells in the combined drug group was significantly reduced in the DEX+SREBF1 inhibitor group.

[0113] After the administration in the 5th cycle was completed, the mice were euthanized. Compared with the Control group, the size and weight of the spleen in the DEX group were significantly reduced, and the proportion of T-ALL cells in the spleen and bone marrow was also significantly reduced.

[0114] Compared with the DEX group, the size and weight of the spleen in the combined drug group were significantly reduced in the DEX+SREBF1 inhibitor group, and the proportion of T-ALL cells in the spleen and bone marrow was also significantly reduced. ( Figure 10 )

[0115] The above are only the preferred embodiments of the present invention and are not used to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Use of dexamethasone combined with SREBF1 inhibitor in the preparation of a chemotherapeutic drug for treating acute T-lymphoblastic leukemia; The SREBF1 inhibitor is Fatostatin HBr.

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