CN1637137A - Method of inducing stem cell to differentiate to insulin-like cell and its application - Google Patents
Method of inducing stem cell to differentiate to insulin-like cell and its application Download PDFInfo
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- CN1637137A CN1637137A CNA2004100706478A CN200410070647A CN1637137A CN 1637137 A CN1637137 A CN 1637137A CN A2004100706478 A CNA2004100706478 A CN A2004100706478A CN 200410070647 A CN200410070647 A CN 200410070647A CN 1637137 A CN1637137 A CN 1637137A
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Abstract
The present invention aims at providing one method of obtaining great amount of insulin-like cell aggregates for application in cell treatment of diabetes. The technological scheme for reaching the said aim includes: 1) obtaining PDX-1 gene; 2) inserting the PDX-1 gene in prokaryotic expression vector for expressing and purifying PDX-1 protein or constituting virus vector containing PDX-1 gene; 3) separating, culturing and amplifying stem cell; 4) adding PDX-1 protein directly to the culture liquid, or mediating PDX-1 gene to express in stem cell with virus vector to induce stem cell differentiating into insulin-like cell; and 5) transplanting the obtained insulin-like cell via hepatic portal vein or abdominal cavity into diabetic body to exert. The present invention has simplified process to provide diabetics with new insulin cell source and lays foundation for the self stem cell treatment of diabetes.
Description
Technical field
The present invention relates to biomedical sector, specifically utilize the vital role of PDX-1 gene in the pancreas growth and development process, make up the virus vector that contains this gene, be used for infecting stem cell; Or construction of expression vector, purifying PDX-1 albumen, making an addition in the inducing culture liquid, induced dry-cell breaks up to insulin-like cell, and the cell that will obtain is implanted in diabetic subject's body and will be used for treatment of diabetes.
Background technology
In recent years, islet cell transplantation treatment diabetes had obtained some curative effects, and deficiency, serious difficulties such as immunological rejection have limited the application of this therapy greatly but the donor's cells originates.Stem cell has the cell of self and multidirectional differentiation potential as a class, the new resources that people seek islet cells have been become gradually, be easy to the separation and Culture amplification as the mescenchymal stem cell in the marrow (MSCs), genetic background is stable, body is implanted into a little less than the reaction, can be divided into multiple histocyte under specific inductive condition, be the first-selected seed cell of repairing tissue such as bone, cartilage or cell injury, also is the desirable target cell of gene therapy.
Stem cell is carried out suitable genetic modification, make it to become can excreting insulin cell, be one of strategy of treatment type i diabetes, and introduce the PDX-1 gene, i.e. pancreas duodenum homology frame albumen 1, might obtain can excreting insulin cell.The PDX-1 gene has been played the part of very important role in the pancreas growth and development process, it not only can regulate and control the expression of Regular Insulin, can also regulate and control glucose transporter (Glut-2), the isogenic expression of glucokinase (GK).
Because stem cell is relative immobilized a group cell, utilizes liposome transfection efficient very low, selects for use virus vector then can obtain higher transfection efficiency.The adenovirus carrier unconformability in karyomit(e), the exogenous gene expression level height that carries, expression time is short, is the only carrier of transfection PDX-1.Because PDX-1 is a regulatory gene, it can start the expression of autogene and downstream gene, and transient expression just is enough to bring into play function.
Utilize PDX-1 to have the characteristics of nexin transduction domain, construction of expression vector, purifying PDX-1 albumen make an addition in the inducing culture liquid, can overcome the unsafe factor of gene therapy.
Summary of the invention
The purpose of this invention is to provide a kind of a large amount of insulin-like cell group that obtains to be used for the method for diabetes cell therapy.By the following technical solutions:
1) structure contains PDX-1 Prokaryotic Expression carrier: expression, purifying PDX-1 albumen.
2) making up the virus vector that contains the PDX-1 gene, is example with the adenovirus carrier: make shuttle plasmid pAdtrack-CMV-PDX-1 and viral skeleton plasmid pAdEasy-1 homologous recombination in intestinal bacteria BJ5183 with electroporation.Utilize liposome-mediated recombinant adenoviral vector rotaring redyeing 293 cell, packing and amplification adenovirus.
3) induced dry-cell differentiation: by directly adding PDX-1 albumen, or transfection contains the virus vector of PDX-1 gene, can be divided into insulin-like cell by induced dry-cell.
4) insulin-like cell that obtains is implanted in diabetic subject's body with some amount, hypoglycemic activity can be brought into play in abdominal cavity or transplant through the hepatic portal arteries and veins.
Content of the present invention is unexposed to be delivered, and those skilled in the art can not obtain method of inducing differentiation of the present invention according to existing technology deduction as not spending creative work at all.
Embodiment
The proteic expression of embodiment 1, PDX-1, purifying
Design contains the PDX-1 gene upstream and downstream primer in NdeI, XhoI site, the pcr amplification full length fragment, and electrophoresis reclaims, be connected into pGEM-T easy carrier (Promega company product), after order-checking was correct, NdeI, XhoI double digestion purpose fragment and pET-24a (+) reclaimed, connect.IPTG abduction delivering, and this albumen of purifying.
Embodiment 2, pAdv-PDX-1 construction of recombinant adenovirus containing
Carrying the plasmid of PDX-1 gene is presented by U.S. Hui doctor HongXiang.PDX-1 inserts in the PBluscriptII KS carrier by the smaI site.Select BamHI and XhoI enzyme to cut at two ends, smaI site.BamHI, BglII are isocaudarners, so select BglII and XhoI enzyme to cut adenovirus shuttle plasmid pAdtrack-cmv.Enzyme is cut the product electrophoresis reclaim, 16 ℃ of connections are spent the night.Connect product transformed into escherichia coli DH5 α, that mycin resistant panel screening of card-coating is got 1 μ l bacterium liquid and is carried out the PCR evaluation.Extract PCR and be accredited as male bacterium liquid plasmid, the row double digestion is identified.Select BglII and XhoI site to carry out enzyme and cut evaluation, cut out the big fragment of small segment and the 9.7kb of 400bp.
Select for use the PmeI enzyme to cut 1 μ g shuttle plasmid pAdtrack-cmv-PDX-1,70% ethanol sedimentation plasmid adds 5 μ lH2O.Linearizing shuttle plasmid and 100ng super spirial plasmid pAdEasy-1 electroporation cotransformation are to the BJ5183 competence bacteria, and that mycin resistant panel of bacterium liquid card-coating is screened.Select 20 little clones, be inoculated in the LB nutrient solution that contains kantlex respectively, 37 ℃ are shaken bacterium and spend the night.Extract plasmid, recombinant plasmid is all greater than 40kb, but through 0.7% agarose electrophoresis preliminary evaluation.According to electrophoresis result, choose plasmid and do the evaluation of PacI restriction enzyme digestion.Recombinant plasmid can be digested goes out the small segment of 4.5kb and the big fragment of 38.6kb.
Get and identify the good capable PacI restriction enzyme digestion of recombinant virus plasmid 5 μ g.With Lipofectine 2000 (Invitrogen company product) parcel linearization plasmid rotaring redyeing 293 cell, remove transfection liquid behind the transfection 12h, adding the DMEM nutrient solution that contains 5% foetal calf serum continues to cultivate 7～10 days, when CPE appears in cell, collecting cell,-70 ℃ and 37 ℃ of multigelations three times, the centrifuging and taking supernatant.Infect 293 cells once more with viral supernatant, collect supernatant.Measure virus titer (with reference to AdEasy Vector Systerm working method) with the TCID50 method, titre is 6.3 * 10
7PFU/mL.
The separation and Culture of embodiment 3, mesenchymal stem cells MSCs and amplification
Under the aseptic condition, extrude the marrow in the rib, rib can be by obtaining behind the non-disease in the blood system thoracic surgery; Or by non-disease in the blood system or normal people's posterior superior iliac spine extraction marrow, about 5ml.In bone marrow fluid, add α-MEM (the Gibico company product) thorough mixing that contains 10% foetal calf serum, the centrifugal 5min of 1500r/min, abandon supernatant, add the density that is added to gently behind the 5ml complete culture solution mixing again and be on 1.073 the Percoll parting liquid, the centrifugal 20min of 1500r/min, get the above part of white corpuscle rete, add complete culture solution and wash twice.By 1 * 10
6/ ml density is inoculated in the perfect medium, puts 37 ℃, 5%CO
2Cultivate in the incubator of saturated humidity.Change nutrient solution after cultivating 72h, later every 4d changes liquid once.Cell reached 80% fusion back and uses 25% trysinization, by 1: 3 continuation amplification cultivation that goes down to posterity.
Embodiment 4, the differentiation of PDX-1 inducing bone mesenchymal stem cell to pancreatic islet like cell
Directly add PDX-1 albumen in nutrient solution; Or select the suitable MOI (MOI=150) of virus infection MSCs, according to cell count, calculate required virus quantity and infect MSCs, hatch 2h after, add the α-MEM nutrient solution that contains 10% foetal calf serum, continue to cultivate for 1 week, induce differentiation.
The evaluation of embodiment 5, insulin-like cell group
Utilize RT-PCR to identify nidogen (nestin), neural plain 3 (ngn3), PDX-1, GK, Glu2, Regular Insulin, the hyperglycemic-glycogenolytic factor expression of gene of generating.Utilize primer 3 software design gene primers, sequence is as follows:
| Gene | Sense primer | Antisense primer | Annealing temperature | Length |
| ??nestin | ?AGAGGGGAATTCCTG?GAG | ??CTGAGGACCAGGACTCTCTA | ????54℃ | ?495bp |
| ??ngn3 | ?CTTCGCCCACAACTACATC ?TG | ??ATCTGAGAAAGCCAGACTGC ??CTG | ????58℃ | ?259bp |
| ??PDX-1 | ?CCCATGGATGAAGTCTACC | ??GTCCTCCTCCTT?TTTCCAC | ????52℃ | ?262bp |
| ??GK | ?AGGTAGAGCAGATCCTGGC ?A | ??TCACCTTCTCCCACCTTCAC | ????60℃ | ?241bp |
| ??Glut-2 | ?CAATGACAGAAGATAAGGT ?CAC | ??TGCTACTAACATGGCTTTGA | ????47℃ | ?395bp |
| Regular Insulin | ?ACCCAGCCGCAGCCTTTGT ?G | ??TTCCACAATGCCACGCTTCTG ??C | ????56℃ | ?223bp |
| Hyperglycemic-glycogenolytic factor | ?AGGCAGACCCACTCAGTG ?A | ??AACAATGGCGACCTCTTCTG | ????56℃ | ?308bp |
The result shows:
The cell of transfection PDX-1 gene (1 week) weak expression nestin, ngn3, Glut2 gene, high expression level PDX-1, GK, Regular Insulin, hyperglycemic-glycogenolytic factor gene.
Utilize immunohistochemical methods to identify Regular Insulin, the antigenic expression of hyperglycemic-glycogenolytic factor.The result shows: inductive cell expressing Regular Insulin, hyperglycemic-glycogenolytic factor, Somatostatin albumen.
Utilize radio immunoassay to detect insulin level.The cell mass in picking transfection PDX-1 1 week of gene is put in 24 orifice plates, divides 3 holes, about 90～100 cell masses in every hole, the about 150 μ m of diameter.Each hole adds the KRBB damping fluid that 1mL contains 5.6mmol/L glucose, puts in 37 ℃ of incubators and hatches 1h.Discard old damping fluid, hatch 1h successively, collect supernatant with the KRBB damping fluid that contains 5.6mmol/L glucose, 16.7mmol/L glucose.Collect the cell mass in each hole, add sour ethanolic soln, 4 ℃ are spent the night, and with cell Ultrasonic Cell Disruptor broken cell, supernatant is stored in-20 ℃.Detect insulin content in each supernatant with radio immunoassay.Detect total protein content in the cell with BCA (Bicinchoninic acid, bicinchoninic acid) assay method.The result: inductive insulin-like cell group insulin content in 5.6mmol/L glucose concn lower eyelid is (54.45 ± 9.14) ng/mg albumen; And insulin content is (130.14 ± 12.24) ng/mg albumen in 16.7mmol/L glucose concn lower eyelid.It is reactive to have certain sugar.
Transplantation experiments in embodiment 6, the body
At first make diabetes rat model.Get adult Wistar rats, male and female are not limit, the about 180-200g of body weight.Press 70mg/kg dosage and give every rats by intraperitoneal injection U-9889.The U-9889 pulvis is mixed with liquid with 0.1M citrate buffer solution (pH=4.5) and uses, and is now with the current.When rat blood sugar raise (〉=16.7mmol/L) and stablize a week, show that diabetes model builds up.Under the aseptic condition, following or 1000 insulin-like cell groups of hepatic portal arteries and veins subbranch injection to diabetes rat kidney packing.Postoperative, periodic observation blood sugar situation.Result: diabetes rat blood sugar 5.6mmol/L that on average descends during second week after implanting cell.Show that inductive insulin-like cell group has hypoglycemic activity.
Claims (10)
1. an induced dry-cell is characterized in that utilizing transcription factor PDX-1 induced dry-cell to break up to insulin-like cell to method and application thereof that insulin-like cell breaks up, and the insulin-like cell of acquisition can be used in treatment of diabetes.
2. according to the described induction scheme of claim 1, it is characterized in that in inducing culture liquid, directly adding the PDX-1 transcription factor, or in stem cell transfection PDX-1 gene, can break up to insulin-like cell by induced dry-cell.
3. according to the described scheme of claim 2, it is characterized in that selecting to make up containing PDX-1 Prokaryotic Expression carrier, express and this albumen of purifying, directly add in the nutrient solution by 100nmol/L～1 μ mol/L concentration, induced dry-cell breaks up to insulin-like cell.
4. according to the described scheme of claim 2, it is characterized in that selecting to make up the virus vector that contains the PDX-1 gene, comprise adenovirus carrier, gland relevant viral vector, lentiviral vectors, retroviral vectors etc. are expressed in stem cell by these carrier mediated PDX-1 genes, and induced dry-cell breaks up to insulin-like cell.
5. according to the described virus vector of claim 4, adenovirus carrier is the first-selected virus vector of transfection PDX-1 gene.
6. according to the described stem cell of claim 1, it is characterized in that being used to the inductive stem cell and can comprise embryonic stem cell, pancreatic stem cells, liver stem cells, hemopoietic stem cell, mescenchymal stem cell, neural stem cell etc.
7. according to the described scheme of claim 1, it is characterized in that selecting for use serum-free inducing culture liquid, and, can induce the proteic stem cell to pancreatic islet like cell differentiation of transfection PDX-1 gene or transduction PDX-1 to wherein adding nutrient substance or cytokines such as glucagon-like-peptide-1, nicotinamide.
8. according to the described scheme of claim 7, it is characterized in that glucagon-like-peptide-1≤20nmol/L of adding, nicotinamide≤20mmol/L.
9. according to the described scheme of claim 1, it is characterized in that the insulin-like cell group that obtains can express pancreas islet genes involved and albumen, as glucose transporter, glucokinase, Regular Insulin, hyperglycemic-glycogenolytic factor, Somatostatin etc.
10. according to the described scheme of claim 1, it is characterized in that the insulin-like cell group that obtains implants in diabetic subject's body by modes such as hepatic portal arteries and veins or abdominal cavities, can bring into play certain function of blood sugar reduction.
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102311940A (en) * | 2010-07-09 | 2012-01-11 | 中国人民解放军军事医学科学院野战输血研究所 | Method for inducing differentiation of stem cells into islet-like cells |
| CN101348512B (en) * | 2007-07-20 | 2012-08-15 | 郑州威瑞生物技术有限公司 | Antineoplastic ad virus preparation |
| CN103194424A (en) * | 2013-03-28 | 2013-07-10 | 于涛 | Method for inducing embryonic stem cell into pancreatic tissue-like cells |
| CN104928233A (en) * | 2015-07-02 | 2015-09-23 | 广州赛莱拉干细胞科技股份有限公司 | Culture solution and method of islet-like cells |
| CN108342353A (en) * | 2008-11-04 | 2018-07-31 | 韦尔赛特公司 | Stem cell aggregate suspension composition and its differentiation method |
| CN111875675A (en) * | 2018-09-03 | 2020-11-03 | 洛阳轩智生物科技有限公司 | Improved method for differentiation of epidermal stem cells into pancreatic cells |
| CN113174408A (en) * | 2021-04-28 | 2021-07-27 | 吉林大学 | Islet cells differentiated from stem cells, method, compound and application |
| CN116751735A (en) * | 2023-07-04 | 2023-09-15 | 重庆市铂而斐细胞生物技术有限公司 | Serum-free culture method of umbilical cord mesenchymal stem cells |
| WO2023227068A1 (en) * | 2022-05-25 | 2023-11-30 | Hangzhou Reprogenix Bioscience, Inc. | A new site for transplantation |
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2004
- 2004-07-28 CN CNA2004100706478A patent/CN1637137A/en active Pending
Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101348512B (en) * | 2007-07-20 | 2012-08-15 | 郑州威瑞生物技术有限公司 | Antineoplastic ad virus preparation |
| CN108342353A (en) * | 2008-11-04 | 2018-07-31 | 韦尔赛特公司 | Stem cell aggregate suspension composition and its differentiation method |
| CN108342353B (en) * | 2008-11-04 | 2022-01-11 | 韦尔赛特公司 | Stem cell aggregate suspension compositions and methods of differentiation thereof |
| CN102311940A (en) * | 2010-07-09 | 2012-01-11 | 中国人民解放军军事医学科学院野战输血研究所 | Method for inducing differentiation of stem cells into islet-like cells |
| CN103194424A (en) * | 2013-03-28 | 2013-07-10 | 于涛 | Method for inducing embryonic stem cell into pancreatic tissue-like cells |
| CN104928233A (en) * | 2015-07-02 | 2015-09-23 | 广州赛莱拉干细胞科技股份有限公司 | Culture solution and method of islet-like cells |
| CN104928233B (en) * | 2015-07-02 | 2018-07-13 | 广州赛莱拉干细胞科技股份有限公司 | The culture solution and cultural method of islet-like cells |
| CN111875675A (en) * | 2018-09-03 | 2020-11-03 | 洛阳轩智生物科技有限公司 | Improved method for differentiation of epidermal stem cells into pancreatic cells |
| CN113174408A (en) * | 2021-04-28 | 2021-07-27 | 吉林大学 | Islet cells differentiated from stem cells, method, compound and application |
| WO2023227068A1 (en) * | 2022-05-25 | 2023-11-30 | Hangzhou Reprogenix Bioscience, Inc. | A new site for transplantation |
| CN116751735A (en) * | 2023-07-04 | 2023-09-15 | 重庆市铂而斐细胞生物技术有限公司 | Serum-free culture method of umbilical cord mesenchymal stem cells |
| CN116751735B (en) * | 2023-07-04 | 2024-03-15 | 重庆市铂而斐细胞生物技术有限公司 | Serum-free culture method of umbilical cord mesenchymal stem cells |
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