WO2021042321A1 - 基因修饰MSCs治疗2型糖尿病 - Google Patents

基因修饰MSCs治疗2型糖尿病 Download PDF

Info

Publication number
WO2021042321A1
WO2021042321A1 PCT/CN2019/104502 CN2019104502W WO2021042321A1 WO 2021042321 A1 WO2021042321 A1 WO 2021042321A1 CN 2019104502 W CN2019104502 W CN 2019104502W WO 2021042321 A1 WO2021042321 A1 WO 2021042321A1
Authority
WO
WIPO (PCT)
Prior art keywords
stem cells
recombinant
exendin
stem cell
cells
Prior art date
Application number
PCT/CN2019/104502
Other languages
English (en)
French (fr)
Inventor
杜亚楠
张元元
高爽
Original Assignee
清华大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 清华大学 filed Critical 清华大学
Priority to PCT/CN2019/104502 priority Critical patent/WO2021042321A1/zh
Publication of WO2021042321A1 publication Critical patent/WO2021042321A1/zh

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/26—Glucagons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C12N15/867—Retroviral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the present invention relates to the field of biotechnology, and relates to genetically modified MSCs for the treatment of type 2 diabetes, in particular to a recombinant stem cell, a preparation method and use, and a medicine for treating type 2 diabetes.
  • Diabetes is one of the most serious and critical health problems currently facing the world. In 2017, about 425 million adults worldwide were suffering from diabetes, and more than 4 million people died of diabetes and its complications each year. The total medical expenditure on diabetes reached US$727 billion. Despite the continuous efforts of people, the number of diabetic patients continues to grow. It is estimated that by 2045, the number of diabetic patients will reach 629 million.
  • the current anti-diabetic drugs can only relieve symptoms and delay the progression of the disease, but cannot be cured fundamentally, and seriously affect the quality of life of patients. Therefore, it is urgent to prevent and completely cure diabetes.
  • Some oral antidiabetic drugs mainly achieve the purpose of lowering blood sugar by stimulating the secretion of insulin from the remaining pancreatic islet cells. Insulin can only be used as a supplementary treatment or replacement treatment for the relative or absolute lack of insulin.
  • an object of the present invention is to provide a recombinant mesenchymal stem cell, a preparation method and application, and a medicine for treating type 2 diabetes.
  • GLP-1 receptor agonists and DPP4 inhibitors both regulate and enhance the GLP-1 signaling pathway to promote insulin secretion, inhibit glucagon secretion, suppress appetite, and enhance the body's insulin sensitivity. And many other functions.
  • the decrease of GLP-1 secretion leads to obesity, and the excessive secretion can lead to postprandial hypoglycemia.
  • the half-life of GLP-1 being degraded by DPP4 after synthesis in the body is only two minutes, so its analogues are often used clinically as substitutes for the treatment of diabetes.
  • Exendin-4 (abbreviated as Ex-4) is a GLP-1 analog secreted in the saliva of the Gila monster. It has 53% homology with the human GLP-1 sequence. It has the same function of activating GLP-1R, but has a half-life Longer. Ex-4 has a 39 amino acid peptide. It has been approved by the Food and Drug Administration to help control the blood sugar of T2D patients with oral metformin or/and sulfonylureas. Its main ingredient is Exendin-4.
  • Exendin-4 has a longer half-life than GLP-1, it still needs to be injected before breakfast and dinner every day, which brings great inconvenience to the work and life of patients. Therefore, it is necessary to increase the duration of action of Exendin-4 drugs and reduce the number of injections.
  • MSCs treatment aims at the key link in the onset of diabetes and fundamentally improves the hyperglycemia of diabetic patients.
  • the duration of a single injection of MSCs is about 2-4 weeks, and multiple injections are required to achieve the therapeutic effect. Therefore, the long-term effectiveness of stem cell therapy is one of the problems that need to be solved urgently.
  • Ex-4 Exendin-4
  • the survival efficiency of MSCs is enhanced, and the treatment effect of MSCs is more long-term in the treatment of diabetes.
  • MSCs In order to enhance the efficacy of MSCs in the treatment of diabetes, we genetically modified MSCs so that MSCs can stably secrete Exendin-4. After modification, MSCs and unmodified cells did not change significantly in cell proliferation, activity and function.
  • MSCs and MSCs-Exendin-4 By injecting MSCs and MSCs-Exendin-4 into diet-induced diabetic mice, we found that the MSCs-Exendin-4 group can significantly increase insulin sensitivity, lower the blood glucose and blood lipid levels of the mice, and promote the proliferation of pancreatic ⁇ -cells than the MSCs group. , Effectively increase the secretion of insulin.
  • This method obtains a genetically modified MSCs that is safe and effective for the treatment of type 2 diabetes, provides a reference for clinical trials of stem cell treatment of diabetes, and provides a basis for humans to cure diabetes, a disease that seriously harms human health.
  • the present invention provides the following technical solutions:
  • the present invention provides a recombinant stem cell capable of overexpressing Exendin-4 protein.
  • the present invention provides a recombinant stem cell, which can stably overexpress Exendin-4 protein, which can be applied to humans or animals to significantly reduce blood glucose and blood lipid levels in the body, improve insulin sensitivity, and reduce the occurrence of fatty liver ,
  • the therapeutic effect can be maintained for a month or even longer without causing side effects of damage to the body's tissues and organs. Therefore, it can be used to treat type 2 diabetes, is safe, effective, and has a long action time.
  • the above-mentioned recombinant stem cell may further include the following technical features:
  • the recombinant stem cell contains the Exendin-4 gene, and compared with natural stem cells, the expression level of the Exendin-4 gene is increased by at least 1000 times, preferably by at least 2000 times. , More preferably increased by at least 3000 times. Compared with natural stem cells, the provided recombinant stem cells have a significantly improved expression ability of Exendin-4. Under the same culture time conditions, the expression level of Exendin-4 has increased by at least 1000 times, for example, by at least 1500 times, 2000 times, 2500 times, 3000 times, or even 3500 times.
  • the recombinant stem cells can express at least 300ng Exendin-4 protein, such as at least 400ng Exendin-4 protein, and more preferably at least Express 500ng Exendin-4 protein.
  • the recombinant stem cells are modified stem cells, and the stem cells are mesenchymal stem cells or other stem cells with differentiation ability.
  • the stem cells are genetically modified to obtain modified stem cells, that is, recombinant stem cells, which can be used to lower blood sugar and blood lipid levels, and treat obesity, fatty liver, or type 2 diabetes.
  • modified stem cells can be any stem cells with self-replication and differentiation capabilities, for example, some mesenchymal stem cells or other stem cells with differentiation capabilities, including embryonic stem cells, induced pluripotent stem cells, hematopoietic stem cells, neural stem cells, liver stem cells, etc. .
  • mesenchymal stem cells have strong proliferation ability, multidirectional differentiation potential, and immune regulation ability. They are the first choice for genetically modified stem cells and show superior performance and therapeutic effects.
  • the mesenchymal stem cells are selected from bone marrow mesenchymal stem cells, placental mesenchymal stem cells, umbilical cord mesenchymal stem cells, umbilical cord blood mesenchymal stem cells, amniotic fluid mesenchymal stem cells, At least one of blood mesenchymal stem cells.
  • the other stem cells with differentiation ability are selected from at least one of embryonic stem cells, induced pluripotent stem cells, hematopoietic stem cells, neural stem cells, adult stem cells, and liver stem cells.
  • the sequence of the Exendin-4 protein is shown in SEQ ID NO:1.
  • the sequence shown in SEQ ID NO:1, as a GLP-1 analog, has the function of activating GLP-1R and has a long half-life.
  • the recombinant stem cells thus obtained maintain the therapeutic effect for a long time.
  • the present invention provides a method for preparing a recombinant stem cell, the recombinant stem cell being the recombinant stem cell according to the first aspect of the present invention, the preparation method comprising: using Exendin-4 encoding gene and a vector Connecting to obtain an expression vector containing the gene encoding Exendin-4; introducing the expression vector containing the gene encoding Exendin-4 into stem cells to obtain the recombinant stem cells.
  • mesenchymal stem cells modified by gene Exendin-4 can be rapidly prepared, that is, recombinant stem cells can be obtained.
  • the recombinant stem cells overexpress Exendin-4 protein, can be used to treat type 2 diabetes, are safe and have a long action time.
  • the method for preparing the recombinant stem cell described above may further include the following technical features:
  • the vector is a lentiviral vector
  • using the lentiviral vector to prepare the recombinant stem cell includes: linking the Exendin-4 coding gene with the lentiviral expression vector to obtain Exendin-containing 4 Recombinant lentiviral expression vector encoding gene; Assemble the recombinant lentiviral expression vector containing Exendin-4 encoding gene in the first cell to obtain a lentivirus; Use the lentivirus to infect mesenchymal stem cells to obtain the The recombinant stem cell.
  • lentiviral vectors Compared with other vectors, for example, compared with adenovirus vectors, lentiviral vectors have strong protein expression ability after infection, which can greatly improve the transduction efficiency of Exendin-4 encoding genes, so as to achieve long-term Exendin-4 encoding genes in stem cells. Stable expression, the resulting recombinant stem cell Exendin-4 protein has a high expression level and stable expression, and it can be applied to animals or humans to exert stable therapeutic effects.
  • the first cell is a 293FT cell
  • the lentiviral expression vector is a pLV-IRES-Puro vector.
  • the lentivirus is used to infect the stem cells under the action of polybrene.
  • the present invention provides a medicament for treating obesity, fatty liver and/or type 2 diabetes and its complications.
  • the medicament includes the recombinant stem cells described in the first aspect of the present invention.
  • sexually contains a pharmaceutically usable carrier.
  • Using the recombinant stem cells provided by the present invention can lower blood sugar, blood lipids and insulin levels, and can be used to prepare drugs for treating obesity, fatty liver, or type 2 diabetes, as well as drugs for treating complications caused by these diseases. These complications can be, for example, cardiovascular disease caused by diabetes and diabetic foot.
  • the medicine is an injection.
  • the dosage of the recombinant stem cells in the medicine is 1 ⁇ 10 5 to 1 ⁇ 10 7 cells/kg of human body weight.
  • the present invention provides the use of recombinant stem cells in the preparation of drugs for the treatment of obesity, fatty liver and/or type 2 diabetes and their complications.
  • the recombinant stem cells are those described in the first aspect of the present invention. Recombinant stem cells.
  • Figure 1 is a diagram of the detection effects of recombinant mesenchymal stem cells and mesenchymal stem cells that have not been genetically modified according to an embodiment of the present invention.
  • Figure 1a shows the results of the measurement of different cells under a microscope
  • Figure 1b shows the results of different cells in different Time cell proliferation results.
  • Figure 1c shows the cell viability measurement results of different cells
  • Figure 1d shows the flow cytometry results of recombinant mesenchymal stem cells.
  • Fig. 2 is a graph showing the gene expression levels and Exendin-4 expression levels of recombinant mesenchymal stem cells and mesenchymal stem cells that have not been genetically modified according to an embodiment of the present invention, wherein Fig. 2a shows Exendin-4 of different cells Gene expression detection results.
  • Figure 2b shows the Exendin-4 expression detection results of recombinant mesenchymal stem cells at different times.
  • Figure 2c shows the injection of recombinant mesenchymal cells and tail vein into mice fed with high-fat diet. The content of Exendin-4 in mouse serum at the time point.
  • Figure 2d shows the expression results of genes related to dryness, nutritional factors, and immunity in different cells.
  • Figure 3 is the results of changes in body weight and blood glucose of mice with different treatments according to the embodiments of the present invention.
  • Figure 3a is the results of the weight changes of mice fed with conventional diet and high-fat diet
  • Figure 3b is the results of mice fed with conventional diet.
  • the blood glucose levels of mice fed with high-fat diet and starvation are shown in Figure 3c.
  • the high-fat mice were treated with tail vein injection at different times.
  • Figure 3d is the results of the body shape of the mice after different treatments.
  • Figure 3d shows the mice after different treatments. The weight result of the mouse.
  • Fig. 4 is a test result of blood glucose, blood lipid, insulin, alanine aminotransferase and aspartate aminotransferase in diabetic mice with different treatments according to an embodiment of the present invention, wherein Fig. 4a is a test result of blood glucose in diabetic mice with different treatments , Figure 4b is the test results of triglyceride in diabetic mice treated with different treatments, Figure 4c is the test results of insulin in diabetic mice treated with different treatments, and Figure 4d is the test results of alanine aminotransferase in diabetic mice treated with different treatments. Figure 4e shows the test results of aspartate aminotransferase in diabetic mice treated with different treatments.
  • Figure 5 is a test result of cell survival and distribution after injection of MSCs into mice with different treatments according to an embodiment of the present invention.
  • Figure 5a shows the cells in the cells after MSCs were injected into conventional diet and high-fat diet for 6 hours. Distribution and survival.
  • Figure 5b shows the distribution and survival of MSCs in mice after 6 days of injection of high-fat diet.
  • Fig. 6 is a test result of glucose tolerance and insulin tolerance of diabetic mice treated with different treatments according to an embodiment of the present invention, wherein Fig. 5a is a different time after the injection of glucose in diabetic mice with different treatments.
  • Figure 5b shows the test results of the area under the glucose tolerance curve of diabetic mice with different treatments after glucose injection
  • Figure 5c shows the blood glucose levels of diabetic mice with different treatments at different times after insulin injection.
  • Figure 5d shows the test results of the area under the insulin resistance curve in diabetic mice treated with different treatments after insulin injection.
  • Figure 7 is the HE staining results of the liver, fat, and pancreas tissues of the mice treated with different treatments and the detection results of triglycerides in the liver according to the embodiments of the present invention, wherein Figure 6a is the mice treated with different treatments The HE staining results of the liver and fat of the mice, Figure 6b is the HE staining results of the pancreas tissue of the mice that have undergone different treatments, and Figure 6c is the detection result of the liver triglycerides of the mice that have undergone different treatments.
  • Treatment in this article refers to the process of intervening or changing a specific health state, which is understood in a broad sense, that is, not only includes curing and eliminating diseases, but also refers to relieving, alleviating symptoms of diseases, and reducing the pain caused by diseases.
  • the term "recombinant stem cell” refers to a stem cell that has been artificially modified, which is capable of overexpressing Exendin-4 protein compared to natural stem cells.
  • the so-called “overexpression” refers to the significant increase in the expression of Exendin-4 gene and Exendin-4 protein compared with natural stem cells. Taking the expression of Exendin-4 gene as an example, this significant improvement is at least 500 times higher, which can be at least 800 times higher, at least 1000 times higher, at least 1300 times higher, or at least 1500 times higher. Increased by at least 1800 times, at least 2000 times, at least 2500 times, at least 2800 times, at least 3000 times, or even at least 3200 times. The expression of this Exendin-4 gene has been significantly increased, which can also be reflected in the significant increase in the expression of Exendin-4 protein.
  • the present invention provides a recombinant stem cell capable of overexpressing Exendin-4 protein.
  • Using the recombinant stem cells for treatment can significantly reduce blood sugar and blood lipid levels, and improve insulin sensitivity. After a course of treatment, the therapeutic effect can be maintained for more than one month. It can be used to treat obesity, fatty liver, type 2 diabetes and its complications, and is safe. Effective and long acting time.
  • the recombinant stem cell can be obtained by genetically modifying the stem cell, so that the genetically modified recombinant stem cell can overexpress the Exendin-4 protein.
  • Stem cells used for genetic modification can be purchased commercially, or obtained through personal consent, purchased through some institutions or donated.
  • the amino acid sequence of the Exendin-4 protein may be as shown in SEQ ID NO:1.
  • the amino acid sequence shown in SEQ ID NO:1 is an analog of GLP-1, which can activate GLP-1R and has a long half-life.
  • Recombinant stem cells are used to express the Exendin-4 protein, which can be used to treat type 2 diabetes, obesity, fatty liver or other metabolic diseases, is safe and effective, and has a long action time.
  • GLP-1 analogue proteins of other species or sources if they exhibit the same or similar functions as the SEQ ID NO:1 protein, can also be used for the treatment of type 2 diabetes, obesity, fatty liver or other metabolic diseases.
  • GLP-1 analogue proteins from other sources can be artificial mutations or synthetic proteins of Exendin-4, for example, compared with SEQ ID NO:1, the sequence homology is more than 80% and more than 85%. 88% or more, 90% or more, 92% or more, 95% or more, 97% or more protein.
  • these Exendin-4 proteins can be based on the sequence shown in SEQ ID NO:1 with a few amino acids added or a few amino acids reduced.
  • the expressed Exendin-4 protein may be as shown in SEQ ID NO: 3, or as shown in SEQ ID NO: 4.
  • HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSKKKKKK (SEQ ID NO: 3),
  • the recombinant stem cells can be used to prepare medicines for the treatment of obesity, fatty liver or type 2 diabetes.
  • the present invention provides a medicine for the treatment of obesity, fatty liver and/or type 2 diabetes.
  • the medicine includes the recombinant stem cells and optionally contains pharmaceutically usable excipients.
  • pharmaceutically acceptable excipients may include any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. Specific examples may be one or more of water, saline, phosphate buffered saline, glucose, glycerol, ethanol, etc., and combinations thereof.
  • drugs include isotonic agents, such as sugars, polyalcohols (such as mannitol, sorbitol), or sodium chloride.
  • the pharmaceutically acceptable carrier may also include minor amounts of auxiliary substances, such as wetting or emulsifying agents, preservatives or buffers, to extend the shelf life or efficacy of the antibody.
  • the recombinant stem cells of the present invention can be incorporated into drugs suitable for parenteral administration (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular).
  • These drugs can be prepared in various forms, such as liquid, semi-solid and solid dosage forms, including but not limited to liquid solutions (for example, injection solutions and infusion solutions), dispersions or suspensions, tablets, pills, powders, Liposomes and suppositories.
  • liquid solutions for example, injection solutions and infusion solutions
  • dispersions or suspensions tablets, pills, powders, Liposomes and suppositories.
  • Typical drugs are in the form of injection solutions or infusion solutions.
  • the drug can be administered by intravenous infusion or injection, or intramuscular or subcutaneous injection.
  • the present invention also provides a method for treating obesity, fatty liver and/or type 2 diabetes and its complications, the method comprising administering to a patient an effective amount of recombinant stem cells, the recombinant stem cells being the aforementioned recombinant stem cells.
  • the blood glucose, blood lipid and insulin levels in the patient can be reduced, so that it can be used to treat obesity, fatty liver and/or type 2 diabetes in the patient, as well as the complications caused by these diseases.
  • cardiovascular disease and diabetic foot caused by diabetes. Diabetic foot is a complication of diabetic patients.
  • Diabetes patients have long-term chronic blood sugar increase, which leads to peripheral blood vessel and neuropathy, leading to some diseases, such as lower extremity ulcers, foot infections, neuropathic arthritis, etc., which are called diabetic feet.
  • the recombinant stem cells provided by the present invention can adjust the blood glucose level in the patient's body, and can alleviate or treat the complications caused by diabetes.
  • the so-called "effective amount of recombinant stem cells” can refer to any dose that can achieve the purpose of treating obesity, fatty liver and/or type 2 diabetes and its complications, that is, the administered recombinant stem cells are not required to meet a fixed standard. As long as the therapeutic effect is shown.
  • the dose used can be 1 ⁇ 10 5 recombinant stem cells to 1 ⁇ 10 7 recombinant stem cells/kg human body weight, one injection in 20 to 30 days, three times A total course of treatment.
  • 1 ⁇ 10 5 recombinant stem cells to 1 ⁇ 10 7 recombinant stem cells can be given, for example, 5 ⁇ 10 5 recombinant stem cells to 1 ⁇ 10 7 recombinant stem cells, which can be 8 ⁇ 10 5 recombinant stem cells ⁇ 1 ⁇ 10 7 recombinant stem cells, or 8 ⁇ 10 5 recombinant stem cells ⁇ 8 ⁇ 10 6 recombinant stem cells, 8 ⁇ 10 5 recombinant stem cells ⁇ 5 ⁇ 10 6 recombinant stem cells, etc. Wait.
  • the recombinant stem cells can be injected into the patient by intravenous injection.
  • the dosage of recombinant stem cells can be appropriately increased, and the interval of injection time can be appropriately extended; of course, the dosage of recombinant stem cells can also be appropriately reduced, while the interval of injection time can be appropriately shortened or a course of treatment can be increased. time.
  • the total course of treatment can also be appropriately extended according to the patient's condition.
  • different modes of administration can also be selected according to the manifestation of the complications.
  • the recombinant stem cells can be directly injected into the diseased part by subcutaneous injection, which can directly and quickly achieve the purpose of treatment.
  • the recombinant stem cells can also be prepared into other forms as needed, such as kits or kits.
  • Example 1 provides a method for preparing recombinant mesenchymal stem cells, including:
  • adipose-derived mesenchymal stem cells and adipose tissue were provided by the Plastic Surgery Department of Peking Union Medical College Hospital. The samples were obtained and the volunteers signed an informed consent form.
  • the fat donor is a healthy person aged 25-35, without HIV, HBV and HCV infection.
  • the fat sample is the fat discarded during liposuction. After being sucked out, it is stored in a sterile, airtight disposable drainage bag, cryopreserved and transported to the laboratory for separation of primary adipose-derived mesenchymal stem cells.
  • the peptide sequence of Exendin-4 is determined to be (SEQ ID NO:1):
  • the nucleic acid sequence encoding the above-mentioned polypeptide is (SEQ ID NO: 2):
  • the Exendin-4 gene was synthesized and then ligated to the pLV-IRES-Puro vector.
  • the lentivirus was packaged in 293FT cells, and the cell culture supernatant was concentrated.
  • the concentrated lentivirus infects P1 MSC cells with the help of polybrene. After the cells are full, they are passaged and screened with 2.5 ⁇ g/ml puromycine. Two days later, the medium is changed to complete medium, and the cells are passaged after they are full.
  • Cell lines that stably express Exendin-4 are recombinant mesenchymal stem cells (ie, recombinant MSCs, also called MSC-Exendin-4 cells).
  • the recombinant mesenchymal stem cells and the mesenchymal stem cells that have not been genetically modified are tested respectively, including:
  • the gene was cloned into a lentiviral vector, the P1 generation MSCs cells were infected with the lentivirus, and a stable cell line was constructed by passage.
  • the cell status of the P5 generation recombinant MSCs was compared with the cell status of the MSCs without genetic modification.
  • the results are as follows As shown in Figure 1a, it can be seen from Figure 1a that there is no significant difference between the status of recombinant mesenchymal stem cells and MSC cells without genetic modification. And with the increase of the culture time after the passage, for example, on the first day, the second day and the third day after the passage, the proliferation multiples of the recombinant mesenchymal stem cells and the unmodified MSC cells were recorded respectively.
  • the recombinant mesenchymal stem cells are cultured for a period of time, the cells are collected, broken, and protein is extracted, and the secretion of Exendin-4 of the recombinant mesenchymal stem cells is detected by the ELISA method.
  • different cells were injected into mice fed with high-fat diet through the tail vein, and the expression of Exendin-4 in the serum of mice at different time points was detected by the ELISA method.
  • the corresponding ELISA test results include the following steps:
  • mice-derived Exendin-4 (1:2000) monoclonal antibody (purchased from Abcam, product number: ab23407) was coated on a 96-well polystyrene plate at 4°C overnight. Wash 6 times with 200 ⁇ l/well TBST, and shake off the waste liquid. 100 ⁇ l/well 5% skimmed milk sealed, skimmed milk powder/TBST; incubate at 4°C on a shaker for 4-5h. Wash 6 times with TBST, add standard substance (purchased from Shanghai Taopu Biotechnology Co., Ltd.) and 100 ⁇ l/well of the sample to be tested, and incubate at 37°C for 2h.
  • mice were fed high-fat for 16 weeks to establish an obese diabetic mouse model, and the blood glucose concentration in the tail vein blood was measured with a blood glucose meter.
  • Blood samples were collected from the heart and placed in an EDTA anticoagulation tube, and centrifuged at 2000 rpm at 4°C for 15 minutes. Take the supernatant for the detection of blood indicators.
  • kit Sigma, TR0100
  • mice with high fat By feeding the mice with high fat for 16 weeks, an obese diabetic mouse model was established.
  • the weight of the mice increased significantly and reached obesity, as shown in Figure 3a.
  • the blood glucose level of mice fed with high-fat diet was significantly higher than that of mice fed with conventional diet, reaching 120mg/dl, as shown in Figure 3b.
  • PBS, MSC and MSC-Exendin-4 were divided into three groups and injected into the high-fat C57B6 mice through the tail vein.
  • the number of cells was 1 ⁇ 10 6 .
  • multiple injections are used to enhance the therapeutic effect, so the injections are divided into three times, which are carried out on the 16th, 18th and 20th weeks of high-fat feed, as shown in Figure 3c.
  • Four weeks after the third injection (4 weeks of treatment, that is, the 24th week of high-fat feed feeding), the mice of different treatments were photographed, and the mice were weighed. It was found that there was no significant difference between the MSC group and the PBS control group. , While the body weight of mice in the MSC-Exendin-4 group was significantly lower than that in the PBS and MSC groups (as shown in Figure 3d and Figure 3e).
  • mice treated with different treatments during starvation found that the MSC and MSC-Exendin-4 groups were significantly reduced, and the MSC-Exendin-4 group decreased more significantly than the MSC group (as shown in Figure 4a).
  • the blood lipids and insulin levels of mice treated with different treatments were tested and found that the MSC and MSC-Exendin-4 groups were significantly lower, and the MSCs-Ex-4 group was significantly lower than the MSC The group more significantly reduced blood lipids and insulin levels in mice (as shown in Figure 4b and Figure 4c).
  • liver function will also be destroyed.
  • the liver function of each group of mice treated with different treatments was tested, and it was found that alanine aminotransferase and aspartate aminotransferase in the blood of mice fed high-fat diet were in MSC.
  • MSC-Exendin-4 significantly decreased after treatment, and the MSC-Exendin-4 group decreased more significantly (as shown in Figure 4d and Figure 4e).
  • MSCs cells The survival and distribution of MSCs in the body are very important for their therapeutic effects. Therefore, using Akaluc luciferase and luciferin to react to generate bioluminescence signals, it is possible to detect the location of cells in the body, their distribution in the body, and the number of cells.
  • MSCs cells by constructing a lentivirus containing the gene encoding Akaluc luciferase, and infecting MSCs cells with the lentivirus, MSC-Akaluc cells (that is, MSC cells capable of expressing Akaluc luciferase) can be obtained, and the cells can be stabilized.
  • MSC-Akaluc cells that is, MSC cells capable of expressing Akaluc luciferase
  • Lumina II captures the strength of the signal, which reflects the amount of cells.
  • Insulin plays an important role in regulating glucose and lipid metabolism. It can inhibit gluconeogenesis, promote glycogen and fatty acid synthesis, and ultimately stabilize the balance of glucose and lipid metabolism. Intraperitoneal injection of glucose was used to test the glucose tolerance of the mice that had undergone different treatments (treatment for 4 weeks) in Example 2 above, and insulin was injected to test the insulin tolerance of the mice that had undergone different treatments (treatment for 4 weeks) in Example 2 above. , To detect the overall insulin sensitivity of mice. The mice were starved overnight, up to 5 mice per cage to prevent bites from fighting. Prepare 20% glucose or insulin with PBS (1:500 dilution).
  • HE hematoxylin-eosin staining
  • the lipid droplets in the liver of the MSC group were slightly reduced, while the lipid droplets in the liver of the MSC-Exendin-4 group were significantly reduced.
  • the reduction in the MSC-Exendin-4 group was more significant than that in the MSC group, indicating that MSC -Exendin-4 is more effective than MSC in relieving fatty liver induced by high-fat diet.
  • the adipocytes in the MSC-Exendin-4 group were significantly smaller than those in the PBS and MSC groups, indicating that MSC-Exendin-4 can also reduce fat accumulation caused by high-fat diet.
  • liver tissue weigh 40mg liver tissue, add 1ml 5% NP40 (prepared in water), sonicate it and place it on a metal bath at 100°C for 5 minutes and let it stand for 15 minutes. Use a vortex mixer to vigorously shake, then continue to heat and stand at 100°C. And shaking process, heating for a total of three times, and then centrifuging at 15000 rpm at room temperature for 10 min. Take the supernatant for testing. Take 10 ⁇ l of the supernatant and perform detection according to the instructions of the triglyceride detection kit (Sigma, TR0100).
  • liver triglyceride levels were significantly reduced in the MSC and MSC-Exendin-4 groups, and the reduction was more significant in the MSC-Exendin-4 group. It shows that MSC and MSC-Exendin-4 treatment can not only reduce blood lipids, but also alleviate fatty liver induced by high-fat diet, and MSC-Exendin-4 treatment shows a more superior effect on lowering blood lipids and alleviating fatty liver induced by high-fat diet.
  • MSCs Mesenchymal stem cell MSCs have self-renewal, clone formation and multiple differentiation potentials. They can play a therapeutic role through two forms of nutritional support and immune regulation. They have been used to treat a variety of diseases with good results.
  • Existing MSCs treatment of diabetes has the problem of too short treatment time.
  • Exendin-4 polypeptide can be effectively used as an adjuvant drug to treat type 2 diabetes, but its retention time in the body is short, and the need for multiple injections is also a major problem.
  • MSCs are modified with Exendin-4 gene, so that they can secrete long-acting GLP-1 analogs in the body without changing the proliferation, activity and cell characteristics of MSCs.
  • MSCs stably overexpressing Exendin-4 into mice, and observed that genetically modified MSCs can significantly reduce blood glucose, blood lipids and insulin levels in mice than unmodified MSCs, improve insulin sensitivity, and reduce fatty liver. happened.
  • modified MSCs that are safer, more effective and longer-acting have been obtained. According to the research results provided, it is reasonable to believe that mesenchymal stem cells overexpressing Exendin-4 can be used to treat obesity, fatty liver and type 2 diabetes.
  • first and second are only used for descriptive purposes, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of indicated technical features. Therefore, the features defined with “first” and “second” may explicitly or implicitly include at least one of the features. In the description of the present invention, “plurality” means at least two, such as two, three, etc., unless otherwise specifically defined.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Zoology (AREA)
  • Biomedical Technology (AREA)
  • Wood Science & Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Diabetes (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • Epidemiology (AREA)
  • Endocrinology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Immunology (AREA)
  • Virology (AREA)
  • Cell Biology (AREA)
  • Plant Pathology (AREA)
  • Emergency Medicine (AREA)
  • Hematology (AREA)
  • Obesity (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

一种基因修饰MSCs治疗2型糖尿病的方法及其重组干细胞，该重组干细胞中能够过表达Exendin-4蛋白。利用该重组干细胞可以制备治疗肥胖、脂肪肝和2型糖尿病药物。该重组干细胞能够降低血糖和血脂水平，提升胰岛素敏感性，一个疗程后治疗效果可以维持一个月以上，可用于治疗肥胖、脂肪肝和2型糖尿病等代谢疾病。

Description

基因修饰MSCs治疗2型糖尿病 技术领域
本发明涉及生物技术领域，涉及基因修饰MSCs治疗2型糖尿病，具体涉及一种重组干细胞、制备方法和用途以及治疗2型糖尿病的药物。
背景技术
糖尿病是目前全球面临的最严重和危急的健康问题之一。2017年，全球约有4.25亿成年人患有糖尿病，每年有400多万人因为糖尿病及其并发症而死亡，糖尿病总医疗支出达7270亿美元。尽管人们不断努力，但糖尿病人数依然持续增长，预计到2045年，糖尿病患病人数将达到6.29亿。目前的抗糖尿病药物只能缓解症状，延缓疾病进展，不能从根本上治愈，严重影响患者的生活质量。因此预防和彻底根治糖尿病刻不容缓。一些口服抗糖尿病药物主要通过刺激残存胰岛细胞分泌胰岛素来实现降糖的目的，而胰岛素仅能作为胰岛素相对或绝对缺乏的补充治疗或替代治疗，两者均不能针对发病机制从源头上治疗糖尿病。
糖尿病治疗药物的开发还需要进一步改进。
发明内容
本发明旨在至少在一定程度上解决相关技术中的技术问题之一。为此，本发明的一个目的在于提出一种重组间充质干细胞、制备方法和用途以及治疗2型糖尿病的药物。
发明人在研究过程中发现：
目前治疗糖尿病的药物中，GLP-1受体激动剂和DPP4抑制剂，均采用调节增强GLP-1信号通路，起到促进胰岛素分泌，抑制胰高血糖素分泌，抑制食欲，增强机体胰岛素敏感性等多种功能。GLP-1分泌量的降低导致肥胖，过量的分泌又会导致餐后低血糖。然而GLP-1在体内合成后被DPP4降解半衰期只有两分钟，因此临床上常使用其类似物作为替代物用于治疗糖尿病。
Exendin-4(缩写为Ex-4)是希拉毒蜥唾液中分泌的一种GLP-1类似物，与人的GLP-1序列同源性53％，具有同样激活GLP-1R的功能，但半衰期更长。Ex-4有39个氨基酸多肽。
Figure PCTCN2019104502-appb-000001
是己被食药监局批准上市的辅助控制口服二甲双胍或/和磺脲类药物的T2D病人血糖的药品，其主要成分为Exendin-4。多项研究报道了Exendin-4抗糖尿病的作用，例如依赖于葡萄糖增加胰岛素分泌，抑制胰高血糖素分泌，延迟胃排空，减少食物摄取并降低体重，增加β细胞重量。Exendin-4虽然较GLP-1有更长的半衰期，但依然需要在每日早晚餐前注射，给患者工作生活带来极大不便，因此提高Exendin-4类药物的作用时长，减少注射次数是亟待解决的问题之一。
干细胞治疗糖尿病的基础和临床研究的开展为糖尿病患者点燃了新的希望，尤其是间充质干细胞为未来彻底治愈糖尿病提供了一种新模式、新思路，也为研究人员及医生提供 了糖尿病治疗的新的突破口。MSCs治疗针对糖尿病发病的关键环节，从根本上改善糖尿病患者的高血糖。同时在小鼠和人体试验中都发现，一次注射MSCs持续治疗时间约为2-4周，需持续多次注射才能起到治疗效果。因此干细胞治疗的长效性是目前亟待解决的问题之一。为增强治疗效果，我们对MSCs过表达Exendin-4(Ex-4)后用于治疗糖尿病，检测修饰后MSC对糖尿病的治疗的靶向性、有效性和安全性，目的是提高MSCs在治疗中的存活效率，增强MSCs治疗效果，更加长效的治疗糖尿病。并寻找MSCs治疗糖尿病的分子机制，为MSCs治疗糖尿病的临床试验提供参考，为人类彻底治愈糖尿病这一严重危害人类健康疾病提供依据。
为了增强MSCs治疗糖尿病的疗效，我们对MSCs进行基因修饰，使MSCs能够稳定分泌Exendin-4。修饰后MSCs与未修饰细胞在细胞增殖、活性和功能上并未发生明显改变。通过对对饮食诱导的糖尿病小鼠注射MSCs和MSCs-Exendin-4，我们发现，MSCs-Exendin-4组比MSCs组能够更显著提高胰岛素敏感性，降低小鼠血糖血脂水平，促进胰岛β细胞增殖，有效提高胰岛素的分泌量。该方法获得了一种治疗2型糖尿病安全有效的基因修饰MSCs，为干细胞治疗糖尿病的临床试验提供参考，为人类治愈糖尿病这一严重危害人类健康疾病提供依据。
具体而言，本发明提供了如下技术方案：
根据本发明的第一方面，本发明提供了一种重组干细胞，所述重组干细胞能够过表达Exendin-4蛋白。本发明提供了一种重组干细胞，该重组干细胞能够稳定过表达Exendin-4蛋白，将其应用于人体或者动物体内，能够明显降低体内血糖和血脂水平，提升胰岛素的敏感性，降低脂肪肝的发生，该治疗效果能够维持一个月甚至更长的时间，而不引起机体组织器官的损伤副作用。因此可以将其用于治疗2型糖尿病，安全，有效，作用时间长。
根据本发明的实施例，以上所述的重组干细胞可以进一步包括如下技术特征：
在本发明的一些实施例中，所述重组干细胞含有Exendin-4基因，所述重组干细胞相较于天然干细胞，所述Exendin-4基因的表达量至少提高了1000倍，优选至少提高了2000倍，更优选至少提高了3000倍。所提供的重组干细胞相较于天然的干细胞而言，Exendin-4的表达能力有了明显的提高，在相同的培养时间条件下，Exendin-4的表达量至少提高了1000倍，例如至少提高了1500倍，2000倍，2500倍，3000倍，甚至是3500倍。
在本发明的一些实施例中，以5×10 5个重组干细胞培养24小时为例，所述重组干细胞至少能够表达300ng Exendin-4蛋白，例如至少能够表达400ng Exendin-4蛋白，更优选至少能够表达500ng Exendin-4蛋白。
在本发明的一些实施例中，所述重组干细胞为经过改造的干细胞，所述干细胞为间充质干细胞或者其他具有分化能力的干细胞。将干细胞进行基因修饰，获得经过改造的干细胞，即重组干细胞，可以用于降低血糖和血脂水平，治疗肥胖、脂肪肝或者2型糖尿病。这些可用的干细胞可以是任何具备自我复制和分化能力的干细胞，例如可以是一些间充质干细胞或者其他具有分化能力的干细胞，包括胚胎干细胞、诱导多能干细胞、造血干细胞、神 经干细胞、肝脏干细胞等。其中间充质干细胞具有强大的增殖能力和多向分化潜能，以及免疫调节能力，其作为该进行基因修饰的干细胞的首选，表现出优越的性能和治疗效果。
在本发明的一些实施例中，所述间充质干细胞为选自骨髓间充质干细胞，胎盘间充质干细胞，脐带间充质干细胞，脐带血间充质干细胞，羊水间充质干细胞，宫血间充质干细胞中的至少一种。
在本发明的一些实施例中，所述其他具有分化能力的干细胞选自胚胎干细胞、诱导多能干细胞、造血干细胞、神经干细胞、成体干细胞、肝脏干细胞中的至少一种。
在本发明的一些实施例中，所述Exendin-4蛋白的序列如SEQ ID NO:1所示。该SEQ ID NO:1所示序列作为一种GLP-1类似物，具有激活GLP-1R的功能，半衰期长。由此获得的重组干细胞，治疗效果维持时间长。
根据本发明的第二方面，本发明提供了一种重组干细胞的制备方法，所述重组干细胞为本发明第一方面所述的重组干细胞，所述制备方法包括：利用Exendin-4编码基因与载体连接，以便获得含有Exendin-4编码基因的表达载体；将所述含有Exendin-4编码基因的表达载体导入到干细胞中，以便获得所述重组干细胞。由此可以快速制备获得经基因Exendin-4修饰的间充质干细胞，即获得重组干细胞，该重组干细胞过表达Exendin-4蛋白，能够用于治疗2型糖尿病，而且安全、作用时间长。
根据本发明的实施例，以上所述重组干细胞的制备方法可以进一步包括如下技术特征：
在本发明的一些实施例中，所述载体为慢病毒载体，利用所述慢病毒载体制备所述重组干细胞包括：利用Exendin-4编码基因与所述慢病毒表达载体连接，以便获得含有Exendin-4编码基因重组慢病毒表达载体；将所述含有Exendin-4编码基因的重组慢病毒表达载体在第一细胞中组装，以便获得慢病毒；利用所述慢病毒感染间充质干细胞，以便获得所述重组干细胞。相较于其他载体，例如相较于腺病毒载体，慢病毒载体感染后蛋白表达能力强，能够大大提高Exendin-4编码基因的转导效率，从而可以实现Exendin-4编码基因在干细胞内的长期稳定表达，由此所获得的重组干细胞Exendin-4蛋白的表达量高，而且表达稳定，应用到动物或者人体内，可以发挥稳定的治疗效果。
在本发明的一些实施例中，所述第一细胞为293FT细胞，所述慢病毒表达载体为pLV-IRES-Puro载体。
在本发明的一些实施例中，利用所述慢病毒在聚凝胺的作用下感染所述干细胞。
根据本发明的第三方面，本发明提供了一种治疗肥胖、脂肪肝和/或2型糖尿病及其并发症的药物，所述药物包括本发明第一方面所述的重组干细胞，还可以选择性地含有药学上可用的载体。应用本发明所提供的重组干细胞可以降低血糖、血脂以及胰岛素水平，能够用于制备治疗肥胖、脂肪肝、或者2型糖尿病药物，以及制备治疗由这些疾病所引起的并发症的药物。这些并发症例如可以是糖尿病所引起的心血管疾病和糖尿病足。在本发明的一些实施例中，所述药物为注射剂。
在本发明的一些实施例中，所述药物中所述重组干细胞的用量为1×10 5～1×10 7个/kg 人的体重。
根据本发明的第四方面，本发明提供了重组干细胞在制备治疗肥胖、脂肪肝和/或2型糖尿病及其并发症的药物中的应用，所述重组干细胞为本发明第一方面所述的重组干细胞。
附图说明
图1是根据本发明的实施例提供的重组间充质干细胞以及未进行基因修饰的间充质干细胞的检测影响图，其中图1a为不同细胞在显微镜下测定结果，图1b为不同细胞在不同时间细胞增殖结果，图1c为不同细胞的细胞活性测定结果，图1d为重组间充质干细胞的流式检测结果。
图2是根据本发明的实施例提供的重组间充质干细胞以及未进行基因修饰的间充质干细胞的基因表达量以及Exendin-4表达量检测结果图，其中图2a为不同细胞的Exendin-4基因表达量检测结果，图2b为重组间充质干细胞培养不同时间Exendin-4的表达量检测结果，图2c为将重组间充质细胞以及尾静脉注射到高脂饲料喂养小鼠体内，在不同时间点小鼠血清中Exendin-4的含量，图2d为不同细胞中与干性相关、营养因子、免疫相关等基因的表达结果。
图3是根据本发明的实施例提供的不同处理的小鼠体重血糖变化结果，其中图3a为采用常规饲料和高脂饲料喂养小鼠，小鼠体重变化结果，图3b为常规饲料喂养小鼠和高脂饲料喂养小鼠饥饿时血糖水平，图3c为在不同时间对高脂小鼠进行尾静脉注射治疗，图3d为经过不同治疗后小鼠的体型结果，图3d为经过不同治疗后小鼠的体重结果。
图4是根据本发明的实施例提供的不同治疗处理的糖尿病小鼠的血糖、血脂、胰岛素、谷丙转氨酶以及谷草转氨酶的检测结果，其中图4a为不同治疗处理的糖尿病小鼠的血糖检测结果，图4b为不同治疗处理的糖尿病小鼠的甘油三酯检测结果，图4c为不同治疗处理的糖尿病小鼠的胰岛素检测结果，图4d为不同治疗处理的糖尿病小鼠的谷丙转氨酶检测结果，图4e为不同治疗处理的糖尿病小鼠的谷草转氨酶检测结果。
图5是根据本发明的实施例提供的MSCs注射到不同处理的小鼠体内后细胞存活和分布的检测结果，其中图5a为MSCs注射到常规饲料和高脂饲料喂养小鼠6小时后细胞体内分布和存活情况，图5b为MSCs注射到高脂饲料喂养小鼠6天后细胞体内分布和存活情况。
图6是根据本发明的实施例提供的经过不同治疗处理的糖尿病小鼠的葡萄糖耐受性和胰岛素耐受性检测结果，其中图5a为不同治疗处理的糖尿病小鼠在注射葡萄糖后的不同时间的血糖水平检测结果，图5b为不同治疗处理的糖尿病小鼠在注射葡萄糖后，葡萄糖耐受曲线下面积检测结果，图5c为不同治疗处理的糖尿病小鼠在注射胰岛素后的不同时间的血糖水平检测结果，图5d为不同治疗处理的糖尿病小鼠在注射胰岛素后，胰岛素耐受曲线下面积检测结果。
图7是根据本发明的实施例提供的经过不同治疗处理的小鼠的肝脏、脂肪、胰腺组织的HE染色结果以及肝脏中甘油三酯的检测结果，其中图6a为经过不同治疗处理的小鼠的肝脏 和脂肪的HE染色结果，图6b为经过不同治疗处理的小鼠的胰腺组织HE染色结果，图6c为经过不同治疗处理的小鼠的肝脏甘油三酯的检测结果。
具体实施方式
下面参考附图详细描述本发明的实施例，所描述的实施例是示例性的，旨在用于解释本发明，而不能理解为对本发明的限制。
下面对本文中的一些术语进行解释和说明，本领域技术人员应该理解的是，这些解释和说明仅用来帮助理解，而不能看做是对本申请保护范围的限制。
本文中“治疗”是指干预或者改变特定健康状态的过程，其作广义上理解，即不单单包括治愈，消除疾病，还可以指缓解，减轻疾病症状，降低疾病所带来的痛苦。
本文中，术语“重组干细胞”是指经过人工改造的干细胞，其相较于天然干细胞而言，能够过表达Exendin-4蛋白。所谓“过表达”是指相较于天然干细胞而言，Exendin-4基因和Exendin-4蛋白的表达量有了显著的提升。以Exendin-4基因的表达为例，这种显著的提升至少表现为提高了500倍，其可以是至少提高了800倍，至少提高了1000倍，至少提高了1300倍，至少提高了1500倍，至少提高了1800倍，至少提高了2000倍，至少提高了2500倍，至少提高了2800倍，至少提高了3000倍，甚至是至少提高了3200倍。这种Exendin-4基因的表达量有了显著的提升，也可以相应地反映在Exendin-4蛋白的表达量有了显著的提升。
为此，根据本发明的一个方面，本发明提供了一种重组干细胞，该所述重组干细胞能够过表达Exendin-4蛋白。利用该重组干细胞进行治疗，能够明显降低血糖和血脂水平，提升胰岛素敏感性，一个疗程后治疗效果可以维持一个月以上，可用于治疗肥胖、脂肪肝和2型糖尿病及其并发症，并且安全，有效，作用时间长。该重组干细胞可以通过对干细胞进行基因修饰获得，使得经过基因修饰的重组干细胞能够过表达Exendin-4蛋白。用于进行基因修饰的干细胞可以通过商购获得，也可以经由个人同意，经由一些机构购买或者受捐赠获得。
在本发明的至少一些实施方式中，该Exendin-4蛋白的氨基酸序列可以如SEQ ID NO:1所示。SEQ ID NO:1所示的氨基酸序列，其是GLP-1类似物，其能够激活GLP-1R，而且半衰期长。利用重组干细胞表达该Exendin-4蛋白，能够用于治疗2型糖尿病、肥胖、脂肪肝或者其他代谢类疾病，安全有效，作用时间长。其它物种或者其它来源的GLP-1类似物蛋白，如果与SEQ ID NO:1蛋白表现出相同或者相似的功能，也可以用作2型糖尿病、肥胖、脂肪肝或者其他代谢类疾病的治疗中。这些其他来源的GLP-1类似物蛋白可以是一些Exendin-4的人工突变或者人工合成蛋白，例如可以是与SEQ ID NO:1相比，序列同源性在80％以上，在85％以上，88％以上，在90％以上，92％以上，95％以上，97％以上的蛋白。在一些实施方式中，这些Exendin-4蛋白可以是在SEQ ID NO:1所示序列的基础上，增加几个氨基酸，或者减少几个氨基酸。例如，所表达的Exendin-4蛋白可以如SEQ ID NO:3，或 者如SEQ ID NO:4所示。
HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSKKKKKK(SEQ ID NO:3)，
TSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS(SEQ ID NO:4)。
利用该重组干细胞可以制备药物，用来治疗肥胖、脂肪肝或者2型糖尿病。为此，在本发明的另一个方面，本发明提供了一种治疗肥胖、脂肪肝和/或2型糖尿病的药物，所述药物包括该重组干细胞，还可以选择性地含有药学上可用的辅料。这些“药学上可接受的辅料”可以包括生理学上相容的任何和所有溶剂、分散介质、包衣、抗细菌剂和抗真菌剂、等渗剂和延迟吸收剂等等。具体实例可以是水、盐水、磷酸盐缓冲盐水、葡萄糖、甘油、乙醇等以及它们的组合物中的一种或多种。有许多情况下，药物中包括等渗剂，例如糖类、多元醇(如甘露醇、山梨醇)或氯化钠等。当然药学上可接受的载体还可包括微量的辅助物质，例如润湿剂或乳化剂、防腐剂或缓冲剂，用来延长抗体的保存限期或效力。例如，本发明的重组干细胞可掺入适用于胃肠外施用(例如静脉内、皮下、腹膜内、肌肉内)的药物中。这些药物可以被制备成各种形式，例如液体、半固体和固体剂型等，包括但不限于液体溶液(例如，注射溶液和输注溶液)、分散剂或悬浮剂、片剂、丸剂、粉末、脂质体和栓剂。典型的药物为注射溶液或输注溶液形式。所述药物可通过静脉输注或注射或肌肉内或皮下注射来施用。
本发明还提供了一种治疗肥胖、脂肪肝和/或2型糖尿病及其并发症的方法，所述方法包括给予患者有效量的重组干细胞，所述重组干细胞为上述重组干细胞。通过给予患者有效量的重组干细胞，能够降低患者体内血糖、血脂以及胰岛素水平，从而能够用于治疗患者的肥胖、脂肪肝和/或2型糖尿病，以及由这些疾病所引起的并发症。例如由糖尿病所引起的心血管疾病和糖尿病足。糖尿病足是糖尿病患者的一种并发症。糖尿病患者由于长期的慢性血糖升高，导致周围血管和神经病变，导致一些病症，例如出现下肢溃疡、足部感染、神经性关节炎等，称为糖尿病足。通过本发明所提供的重组干细胞，调整患者体内血糖水平，可以缓解或者治疗由糖尿病所引起的并发症。
所称的“有效量的重组干细胞”可以指任何能够达到治疗肥胖、脂肪肝和/或2型糖尿病及其并发症目的的剂量，即所给予的重组干细胞不要求必须要达到一个固定的标准，只要表现出治疗效果即可。为了获得良好的治疗效果，在给予患者重组干细胞时，采用的剂量可以为1×10 5个重组干细胞～1×10 7个重组干细胞/kg人的体重，20天～30天注射一次，三次为一总疗程。即按照每kg人的体重来计算，可以给予1×10 5个重组干细胞～1×10 7个重组干细胞，例如可以是5×10 5个重组干细胞～1×10 7个重组干细胞，可以是8×10 5个重组干细胞～1×10 7个重组干细胞，也可以是8×10 5个重组干细胞～8×10 6个重组干细胞，8×10 5个重组干细胞～5×10 6个重组干细胞等等。通常可以采用静脉注射的方式，将重组干细胞注射到患者体内。当然，也可以根据患者的患病情况，参考上述剂量进行相应的调整。例如，可以适当性的加大重组干细胞的剂量，间隔的注射时间可以适当性的延长；当然也可以适当性地减少重组干细胞的剂量，同时适当性地缩短间隔的注射时间或者增加一疗程 的治疗时间。也可以根据患者的患病情况，治疗的总疗程可以适当的延长。针对一些并发症情况，根据并发症的表现形式不同，也可以选择不同的给药方式。例如，针对糖尿病足，可以直接采用皮下注射的方式将重组干细胞注射到患病部位，能够直接地快速发挥治疗目的。该重组干细胞也可以根据需要被制备成其他形式，例如试剂盒或者药盒等形式。
下面将结合实施例对本发明的方案进行解释。本领域技术人员将会理解，下面的实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体技术或条件的，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
实施例1
实施例1提供了一种制备重组间充质干细胞的方法，包括：
1、脂肪间充质干细胞分离鉴定
人类脂肪来源的间充质干细胞，脂肪组织由北京协和医院整形外科提供，样品获取与志愿者签订知情同意书。脂肪供体是25-35岁健康人，无HIV、HBV和HCV感染。脂肪样品为做抽脂手术时废弃的脂肪，吸出后储存在无菌、密闭一次性引流袋中，低温保存运输到实验室进行原代脂肪间充质干细胞的分离。准备collagenase type II 0.2％,大量HBSS+2％P/S，用与脂肪等体积HBSS彻底清洗脂肪5次，洗去血液成分。每管中加与脂肪体积等量的collagenase溶液，37℃，摇动，每5min观察消化情况(剩结缔组织时即可停止，即无肉眼可见固体)。10％collagenase溶液体积的FBS终止消化后100目筛网过滤，1500rpm离心20min，HBSS重悬，吹打冲洗，1500rpm离心15min后，培养基重悬，每15-20ml脂肪的细胞量种进1个T75，隔天换液，约5-7day可以长满(80-90％密度)。
2、Exendin-4稳定细胞株建立
确定Exendin-4多肽序列为(SEQ ID NO:1)：
HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS
编码上述多肽的核酸序列为(SEQ ID NO:2):
Figure PCTCN2019104502-appb-000002
合成Exendin-4基因，然后连接到pLV-IRES-Puro载体上。在293FT细胞中包装成慢病毒，浓缩细胞培养上清。浓缩后慢病毒在polybrene的辅助下感染P1代的MSC细胞，待细胞长满后传代并使用2.5μg/ml的puromycine进行筛选，两天后换液为完全培养基，待细胞长满后传代，获得稳定表达Exendin-4的细胞株，即重组间充质干细胞(即重组MSCs，也称MSC-Exendin-4细胞)。
然后分别对重组间充质干细胞和未进行基因修饰的间充质干细胞进行检测，具体包括：
将基因克隆到慢病毒载体，用慢病毒感染P1代次MSCs细胞，并将传代构建稳定细胞株，将P5代次重组MSCs细胞状态与未经基因修饰的MSCs细胞状态进行比对，其结果如图1a所示，从图1a可以看出，重组间充质干细胞与未经基因修饰的MSC细胞状态无明显 差别。且随着传代后培养时间的增加，例如在传代后的第一天，第二天和第三天，分别记录下重组间充质干细胞与未经基因修饰的MSC细胞的增殖倍数，实验结果如图1b所示，重组间充质干细胞与未经基因修饰的MSC细胞的增殖状态良好。而且采用MTT法测定两种细胞活性，结果表明，两种细胞活性也无明显差异(如图1c所示)。
同时，采用流式细胞术分析重组MSCs细胞的表面标志物，实验结果如图1d所示，实验结果表面重组MSCs细胞的表面标志物相比较于未经基因修饰的MSC细胞并未发生明显变化。
另外，用
Figure PCTCN2019104502-appb-000003
法提取细胞或组织总RNA，按照试剂盒说明进行反转录(购自于Vazyme，产品货号为：R223-01)，SYBR green进行实时荧光定量PCR反应，检测重组间充质干细胞与未经基因修饰的间充质干细胞中Exendin-4的基因表达量，实验结果如图2a所示，实验结果表明，经过基因修饰的重组间充质干细胞相较于未经基因修饰的间充质干细胞，其Exendin-4基因过表达3289倍。
同时将重组间充质干细胞培养一段时间，收集细胞，破碎，提取蛋白质，采用ELISA的方法检测重组间充质干细胞的Exendin-4的分泌量。另外，将不同的细胞通过尾静脉注射到高脂饲料喂养小鼠体内，采用ELISA方法检测小鼠体内不同时间点血清中Exendin-4的表达量。相应的ELISA检测结果包括如下步骤：
包被鼠来源Exendin-4(1：2000)单抗(购自于Abcam，产品货号为：ab23407)在聚苯乙烯材质的96孔板上，4℃过夜。200μl/well TBST洗6次，甩干废液。100μl/well 5％的脱脂牛奶封闭，脱脂奶粉/TBST；4℃摇床孵育4-5h。TBST洗6次，加入标准品(购于上海淘普生物科技有限公司)和待测样品100μl/well，37℃孵育2h。TBST洗6次，加入兔来源一抗(1：2000)(购自于Cusabio，产品货号为：PA853477)，37℃孵育2h。TBST洗6次，加入兔二抗(1：5000)，37℃孵育1h。TBST洗6次，TMB底物显色液100μl/well，观察颜色反应，一般需要5-10分钟显色。加100μl 2M的H 2SO 4(或NaOH)终止反应，酶标仪读450nm OD值。
实验结果发现：将起始量为5×10 5个的重组间充质干细胞(即MSCs-Exendin-4)培养24h、48和72小时后分泌Exendin-4的总量分别为532.15ng、1118.78ng和2150.01ng(如图2b所示)。将起始量为10 6个的MSCs-Exendin-4尾静脉注射到小鼠体内24h后，检测到小鼠血浆中Exendin-4的浓度为166.4ng/ml，在注射第5天达到最高值1180.7ng/ml(如图2c所示)。
另外，利用
Figure PCTCN2019104502-appb-000004
法提取细胞或组织总RNA，按照试剂盒说明进行反转录，SYBR green进行实时定量PCR反应检测重组间充质干细胞以及未进行基因修饰的间充质干细胞中干性相关、营养因子相关基因和免疫相关基因表达量，如图2d所示，实验结果相较于未进行基因修饰的间充质干细胞，重组间充质干细胞中与干性相关、营养因子相关和免疫相关的基因表达也未发生明显改变。
实施例2生化指标测定
通过高脂喂养小鼠16周，建立肥胖糖尿病小鼠模型，用血糖仪检测尾静脉血液中血糖浓度。通过心脏采血收集血液样品后放入EDTA抗凝管中，4℃2000rpm离心15min。取上清用于血液指标的检测。取血浆4μl用于试剂盒(Mercodia，10-1247-01)检测胰岛素含量。取10μl血浆用试剂盒(Sigma，TR0100)检测甘油三酯含量。取20微升送医院进行肝功能检测。
通过高脂喂养小鼠16周，建立肥胖糖尿病小鼠模型，小鼠体重明显增加，达到肥胖，如图3a所示。检测高脂饲料喂养小鼠饥饿时血糖水平明显高于常规饲料喂养小鼠，达到120mg/dl，如图3b所示。
然后将PBS、MSC和MSC-Exendin-4分三组尾静脉注射到高脂喂养C57B6小鼠体内，细胞数量为1×10 6。同时采用多次注射的方法来加强治疗效果，因此将注射分为三次，分别于高脂饲料喂养第16、18和20周进行，如图3c所示。在第三次注射完4周后(治疗4周，即高脂饲料喂养第24周)对不同治疗处理的小鼠进行拍照，并称量小鼠体重，发现MSC组与PBS对照组无明显差异，而MSC-Exendin-4组小鼠体重比PBS和MSC组明显降低(如图3d和图3e所示)。
检测经过不同治疗处理的小鼠饥饿时的血糖发现MSC和MSC-Exendin-4组均有明显降低，MSC-Exendin-4组比MSC组降低更显著(如图4a所示)。考虑到糖尿病人同时伴有高血脂和高胰岛素血症，因此检测了不同治疗处理的小鼠血脂和胰岛素水平发现MSC和MSC-Exendin-4组均有明显降低，MSCs-Ex-4组比MSC组更明显降低小鼠血脂和胰岛素水平降低(如图4b和图4c所示)。
另外，考虑到肥胖小鼠伴有脂肪肝发生，肝功能也会被破坏，检测不同治疗处理的各组小鼠肝功能情况，发现高脂饮食喂养的小鼠血液中谷丙转氨酶和谷草转氨酶在MSC和MSC-Exendin-4治疗后明显降低，且MSC-Exendin-4组降低更加明显(如图4d和图4e所示)。
实施例3 MSCs体内存活和分布检测
MSCs在体内的存活和分布对于其发挥治疗作用非常重要。因此利用Akaluc荧光素酶和荧光素反应，产生生物发光信号，可以检测细胞在体内的位置、在体内的分布以及细胞数量。以MSCs细胞为例，通过构建含有Akaluc荧光素酶编码基因的慢病毒，用该慢病毒感染MSCs细胞，获得MSC-Akaluc细胞(即能够表达Akaluc荧光素酶的MSC细胞)，该细胞能够稳定过表达荧光素酶，当加入Akaluc底物时产生生物发光，通过
Figure PCTCN2019104502-appb-000005
Lumina II拍摄信号强弱，反应细胞量的多少。
因此我们对常规饲料和高脂饲料喂养的小鼠注射MSC-Akaluc细胞，腹腔注射Akaluc底物，观察注射6小时后生物发光信号，发现高脂饲料喂养小鼠体内信号显著高于常规饲料喂养小鼠，且信号多集中在肺(如图5a所示)。注射6天后，高脂饲料喂养组小鼠体内生 物发光信号主要集中在肝部(如图5b所示)。
实施例4胰岛素敏感性检测
胰岛素在调节糖脂代谢方面发挥着重要作用，它可以抑制糖异生，促进糖原和脂肪酸合成，最终起到稳定糖脂代谢平衡的作用。用腹腔注射葡萄糖检测上述实施例2中经过不同治疗处理(治疗4周)的小鼠对葡萄糖耐受能力，注射胰岛素检测上述实施例2中经过不同治疗处理(治疗4周)的胰岛素耐受能力，以检测小鼠的整体胰岛素敏感性情况。饥饿小鼠过夜，每笼小鼠最多5只防止打架咬伤。用PBS配制20％葡萄糖或胰岛素(1:500稀释)。对小鼠进行尾部标记，称量体重，测定注射前的血糖作为0min的值。根据体重计算小鼠葡萄糖或胰岛素注射量，计算方法Volume(μl)＝Body Weight(g)×5μl，注射剂量为1g/kg。腹腔注射葡萄糖或胰岛素以后测量15min，30min，60min和90min时的血糖值并记录。
其实验结果如下：
在注射同等剂量葡萄糖后MSC和MSC-Exendin-4组小鼠比PBS组小鼠的血糖浓度更低，说明两组葡萄糖耐受能力均有提高，且MSC-Exendin-4组比MSC组小鼠葡萄糖耐受能力更强(如图6a和图6b所示)。
在注射同等剂量胰岛素后MSC和MSC-Exendin-4组小鼠比PBS组血糖浓度更低，说明两组胰岛素敏感性均有提高，且MSC-Exendin-4组比MSC组小鼠对胰岛素更敏感(如图6c和图6d所示)。由此说明经过MSC-Exendin-4治疗比单纯的MSC治疗能够显著改善高脂饮食诱导的小鼠胰岛素抵抗问题。
实施例5苏木精-伊红染色
为观察经过治疗后各组中肝、脂肪、胰腺和肌肉等代谢组织的生理结构是否发生改变，对于实施例2治疗4周的小鼠，获取相应的组织。使用石蜡切片技术里常用的苏木精-伊红染色法(hematoxylin-eosin staining,HE)进行组织染色。利用苏木精染液使细胞核内核酸着紫蓝色，伊红使细胞质和细胞外基质着红色。
其实验结果如下：
如图7a所示，与PBS组相比，MSC组肝脏中脂滴略有减少而MSC-Exendin-4组肝脏中脂滴明显减少，MSC-Exendin-4组比MSC组降低更明显，说明MSC-Exendin-4比MSC具有更强的缓解高脂饮食诱导的脂肪肝。MSC-Exendin-4组脂肪细胞比PBS和MSC组明显减小，说明MSC-Exendin-4还能减少高脂饮食引起的脂肪堆积。
另外，如图7b所示，MSC-Exendin-4组胰腺组织中胰岛比PBS组和MSC组有增多，说明MSC-Exendin-4促进了胰岛再生。
实施例5肝脏中甘油三酯检测
本实施例对于上述实施例2经过不同治疗处理的小鼠(治疗4周)的肝脏组织中的甘油三酯进行了检测。
称取40mg肝脏组织，加入1ml 5％NP40(水中配制)，超声破碎后放于100℃金属浴上加热5分钟，静置15min，使用旋涡混匀器剧烈震荡，随后继续100℃加热、静置和震荡过程，总共加热三次，随后室温15000rpm离心10min。取上清进行检测。取10μl上清，按甘油三酯检测试剂盒(Sigma，TR0100)说明进行检测。
其实验结果如下：
如图7c所示，肝脏甘油三酯水平在MSC和MSC-Exendin-4组明显降低，且MSC-Exendin-4组降低更明显。说明MSC和MSC-Exendin-4治疗不仅能降低血脂，还可以缓解高脂饮食诱导的脂肪肝，而且MSC-Exendin-4治疗表现出更加优越的降血脂以及缓解高脂饮食诱导的脂肪肝效果。
本申请首次地对MSCs进行基因Exendin-4修饰。间充质干细胞MSCs具有自我更新，形成克隆以及多项分化潜能，可以通过营养支持和免疫调节两种形式发挥治疗作用，目前已用于治疗多种疾病，均具有良好效果。已有的MSCs治疗糖尿病研究存在治疗时间过于短的问题。Exendin-4多肽能够有效的作为辅助药物治疗2型糖尿病，但是其在体内留存时间短，需要多次注射也是其目前存在的一个主要问题。因此本申请首次对MSCs进行Exendin-4基因修饰，在不改变MSCs增殖、活性以及细胞特性情况下，使其在体内分泌长效GLP-1类似物。我们用稳定过表达Exendin-4的MSC注射到小鼠体内，观察到经过基因修饰的MSCs比未经基因修饰的MSC能够明显降低小鼠血糖、血脂和胰岛素水平，提升胰岛素敏感性，降低脂肪肝的发生。最终获得了更加安全、有效和作用时间更长的修饰后MSCs，所提供的研究结果，有理由相信过表达Exendin-4的间充质干细胞可以用来治疗肥胖、脂肪肝和2型糖尿病。
此外，术语“第一”、“第二”仅用于描述目的，而不能理解为指示或暗示相对重要性或者隐含指明所指示的技术特征的数量。由此，限定有“第一”、“第二”的特征可以明示或者隐含地包括至少一个该特征。在本发明的描述中，“多个”的含义是至少两个，例如两个，三个等，除非另有明确具体的限定。
在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不必针对的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外，在不相互矛盾的情况下，本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。
尽管上面已经示出和描述了本发明的实施例，可以理解的是，上述实施例是示例性的， 不能理解为对本发明的限制，本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。

Claims (21)

  1. 一种重组干细胞，其特征在于，所述重组干细胞能够过表达Exendin-4蛋白。
  2. 根据权利要求1所述的重组干细胞，其特征在于，所述重组干细胞含有Exendin-4基因，所述重组干细胞相较于天然干细胞，所述Exendin-4基因的表达量至少提高了1000倍。
  3. 根据权利要求1所述的重组干细胞，其特征在于，所述重组干细胞含有Exendin-4基因，所述重组干细胞相较于天然干细胞，所述Exendin-4基因的表达量至少提高了2000倍。
  4. 根据权利要求1所述的重组干细胞，其特征在于，所述重组干细胞含有Exendin-4基因，所述重组干细胞相较于天然干细胞，所述Exendin-4基因的表达量至少提高了3000倍。
  5. 根据权利要求1所述的重组干细胞，其特征在于，将5×10 5个重组干细胞培养24小时后，所述重组干细胞至少能够表达300ng Exendin-4蛋白。
  6. 根据权利要求1所述的重组干细胞，其特征在于，将5×10 5个重组干细胞培养24小时后，所述重组干细胞至少能够表达400ng Exendin-4蛋白。
  7. 根据权利要求1所述的重组干细胞，其特征在于，将5×10 5个重组干细胞培养24小时后，所述重组干细胞至少能够表达500ng Exendin-4蛋白。
  8. 根据权利要求1所述的重组干细胞，其特征在于，所述重组干细胞为经过改造的干细胞，所述干细胞为间充质干细胞或者其他具有分化能力的干细胞。
  9. 根据权利要求8所述的重组干细胞，其特征在于，所述间充质干细胞为选自骨髓间充质干细胞，胎盘间充质干细胞，脐带间充质干细胞，脐带血间充质干细胞，羊水间充质干细胞，宫血间充质干细胞中的至少一种。
  10. 根据权利要求8所述的重组干细胞，其特征在于，所述其他具有分化能力的干细胞选自胚胎干细胞、诱导多能干细胞、造血干细胞、神经干细胞、肝脏干细胞中的至少一种。
  11. 根据权利要求1所述的重组干细胞，其特征在于，所述Exendin-4蛋白的序列为SEQ ID NO:1所示。
  12. 权利要求1～11中任一项所述的重组干细胞的制备方法，其特征在于，包括：
    利用Exendin-4编码基因与载体连接，以便获得含有Exendin-4编码基因的表达载体；
    将所述含有Exendin-4编码基因的表达载体导入到干细胞中，以便获得所述重组干细胞。
  13. 根据权利要求12所述的制备方法，其特征在于，所述载体为慢病毒载体，利用所述慢病毒载体制备所述重组干细胞包括：
    利用Exendin-4编码基因与所述慢病毒载体连接，以便获得含有Exendin-4编码基因的重组慢病毒表达载体；
    将所述含有Exendin-4编码基因的重组慢病毒表达载体在第一细胞中组装，以便获得慢病毒；
    利用所述慢病毒感染干细胞，以便获得所述重组干细胞。
  14. 根据权利要求13所述的制备方法，其特征在于，所述第一细胞为293FT细胞，所述慢病毒表达载体为pLV-IRES-Puro载体。
  15. 根据权利要求13所述的制备方法，其特征在于，利用所述慢病毒在聚凝胺的作用下感染所述干细胞。
  16. 一种治疗肥胖、脂肪肝和/或2型糖尿病及其并发症的药物，其特征在于，所述药物包括权利要求1～11中任一项所述的重组干细胞，还可以选择性地含有药学上可用的载体。
  17. 根据权利要求16所述的药物，其特征在于，所述药物为注射剂。
  18. 根据权利要求16所述的药物，其特征在于，所述药物中所述重组干细胞的含量为1×10 5～1×10 7个/kg人的体重。
  19. 权利要求1～11中任一项所述的重组干细胞在制备治疗肥胖、脂肪肝和/或2型糖尿病及其并发症的药物中的应用。
  20. 一种治疗肥胖、脂肪肝和/或2型糖尿病及其并发症的方法，其特征在于，所述方法包括给予患者有效量的重组干细胞，所述重组干细胞为权利要求1～11中任一项所述的重组干细胞。
  21. 根据权利要求20所述的方法，其特征在于，所述有效量的重组干细胞为1×10 5～1×10 7个/kg人的体重。
PCT/CN2019/104502 2019-09-05 2019-09-05 基因修饰MSCs治疗2型糖尿病 WO2021042321A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/CN2019/104502 WO2021042321A1 (zh) 2019-09-05 2019-09-05 基因修饰MSCs治疗2型糖尿病

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2019/104502 WO2021042321A1 (zh) 2019-09-05 2019-09-05 基因修饰MSCs治疗2型糖尿病

Publications (1)

Publication Number Publication Date
WO2021042321A1 true WO2021042321A1 (zh) 2021-03-11

Family

ID=74852690

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2019/104502 WO2021042321A1 (zh) 2019-09-05 2019-09-05 基因修饰MSCs治疗2型糖尿病

Country Status (1)

Country Link
WO (1) WO2021042321A1 (zh)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102154369A (zh) * 2011-01-27 2011-08-17 中国人民解放军第三军医大学 一种重组慢病毒载体、重组慢病毒及含有该重组慢病毒的干细胞
CN103191445A (zh) * 2013-04-19 2013-07-10 吉林大学 间充质干细胞的用途及其制取方法
CN104313054A (zh) * 2014-09-24 2015-01-28 中国人民解放军第三军医大学 表达人外源胰岛素原的人造血干细胞及其应用
WO2016172479A1 (en) * 2015-04-22 2016-10-27 Cedars-Sinai Medical Center Enterically delivered bitter oligopeptides for the treatment for type 2 diabetes
CN107502594A (zh) * 2017-07-14 2017-12-22 中国药科大学 一种稳定表达外源性ex－4基因的骨髓源间充质干细胞株

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102154369A (zh) * 2011-01-27 2011-08-17 中国人民解放军第三军医大学 一种重组慢病毒载体、重组慢病毒及含有该重组慢病毒的干细胞
CN103191445A (zh) * 2013-04-19 2013-07-10 吉林大学 间充质干细胞的用途及其制取方法
CN104313054A (zh) * 2014-09-24 2015-01-28 中国人民解放军第三军医大学 表达人外源胰岛素原的人造血干细胞及其应用
WO2016172479A1 (en) * 2015-04-22 2016-10-27 Cedars-Sinai Medical Center Enterically delivered bitter oligopeptides for the treatment for type 2 diabetes
CN107502594A (zh) * 2017-07-14 2017-12-22 中国药科大学 一种稳定表达外源性ex－4基因的骨髓源间充质干细胞株

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
DATABASE Protein NCBI; 7 May 1993 (1993-05-07), "exendin-4 [Heloderma suspectum, venom, Peptide, 39 aa]", XP055788458, Database accession no. AAB22006 *

Similar Documents

Publication Publication Date Title
CN110628723B (zh) 基因修饰MSCs治疗2型糖尿病
JP5546453B2 (ja) 脂肪肝疾患の治療におけるインターロイキン−２２の使用
JP5221959B2 (ja) 代謝障害の状態を治療するためのｉｌ−２２の使用
US6608038B2 (en) Methods and compositions for treatment of diabetes and related conditions via gene therapy
JP6817943B2 (ja) 分化転換方法及びその使用方法
Burger et al. Molecular basis of cardioprotection by erythropoietin
CN109589337B (zh) 心肌细胞制剂及其制备方法和应用
US20090038022A1 (en) IGF-1 Novel peptides
CN114790160B (zh) 一种茴香霉素衍生物以及茴香霉素和其衍生物作为glp-1r激动剂的用途
JP2016521559A (ja) 細胞の集団、分化転換の方法及びその使用方法
CN109675016A (zh) 非酒精性脂肪性肝病的治疗药物
CN110302362B (zh) 一种蛋白在制备预防和治疗糖尿病并发症的药物中的应用
CN109562145B (zh) 含胰岛素样生长因子-2的药物组合物及其应用
WO2021042321A1 (zh) 基因修饰MSCs治疗2型糖尿病
CN112143705B (zh) 一种双基因修饰的干细胞及其用途
CN109705207A (zh) 一种长效glp-1及其制备方法和应用
CN111808186B (zh) 一种人源性分泌型fndc5蛋白及其制备方法和用途
US10448662B2 (en) Composition for preventing or treating fatty liver or insulin resistance syndrome including extracellular domain of delta-like 1 homolog
Chen et al. An overview of hypoglycemic biological drugs
Berg Leptin is a potent anti-diabetic in mice with lipodystrophy and insulin resistance
CN114652717B (zh) 盐酸萘甲唑啉的药物用途
CN114642668B (zh) 拉坦前列素的药物新用途
CN113616792B (zh) 提高smurf1蛋白表达量的试剂在制备防治钙化性主动脉瓣疾病的药物中的应用
Zhang et al. Oral administration of human FGF21 expressed by mycelium of Cordyceps militaris improves blood glucose and lipid in type II diabetes mellitus
CN111939260B (zh) 嗅觉受体or12d3作为糖尿病防治靶点的应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 19944586

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 19944586

Country of ref document: EP

Kind code of ref document: A1