CN102618500A

CN102618500A - Method for inducing human mesenchymal stem cells to differentiate into insulin-secreting cells in vitro

Info

Publication number: CN102618500A
Application number: CN2012100762519A
Authority: CN
Inventors: 王楠; 张同存; 俞如发
Original assignee: Tianjin University of Science and Technology
Current assignee: Tianjin University of Science and Technology
Priority date: 2012-03-21
Filing date: 2012-03-21
Publication date: 2012-08-01

Abstract

The invention relates to the field of biomedicine, in particular to a method for inducing human mesenchymal stem cells to differentiate into insulin-secreting cells in vitro, which includes, according to the technical scheme, constructing recombinant eukaryotic expression plasmids of SRF (serum response factor) gene; preliminarily inducing human mesenchymal stem cells into nestin cells first; further inducing the nestin cells into pancreatic progenitor cells; transfecting an eukaryotic expression vector containing the SRF gene to the cells above; and finally inducing the cells into insulin-secreting cells, wherein the eukaryotic expression vector comprises pEGFP (plasmid enhanced green florescence protein), pcDNA or pCMV. By the method, new seed cell sources for cell therapy of diabetes mellitus are provided, new theoretical and experimental basis is provided for directed induction of mesenchymal stem cells differentiating into insulin-secreting cells and clinical application of the insulin-secreting cells, and new models for drug development and screening related to the insulin-secreting cells are provided.

Description

A kind of external evoked human mesenchymal stem cell is divided into the method for insulin secretory cell

Technical field

The invention belongs to stem cell and induce the Study on Differentiation field, relate to a kind of method of induced dry-cell differentiation, especially a kind of external evoked human mesenchymal stem cell is divided into the method for insulin secretory cell.

Background technology

Mellitus are one group, and to increase with the blood glucose level be the metabolic disease of characteristic, mainly is because sugar, fat and the protein metabolism disorder property disease that relative or absolute deficiency of insulin secretion or insulin receptor effect defective cause.Distribution of diabetes is only second to cardiovascular and cerebrovascular and tumour, serious threat human life and health.Along with the rising year by year of its sickness rate, mellitus also will further increase the weight of the harm of human health.The World Health Organization estimates that the whole world has 1.8 hundred million people to suffer from mellitus, and this numeral will be doubled more than one times to the year two thousand thirty probably.Aspect treating diabetes, traditional treatment promote nothing more than β cell Regular Insulin secretion, increase peripheral tissues to the susceptibility of Regular Insulin and use the several aspects of exogenous insulin, yet these therapeutic strategies obviously fail to make the diabetic subject to obtain radical cure; Transplantation of pancreas and pancreatic islets transplantation are difficult to overcome not enough and this two large problems of immunological rejection of donor again, make its clinical application and effect also receive very big influence.Therefore the novel method of mellitus is cured in active demand.Stem cell is with its extremely strong self and multidirectional differentiation potential; Become the best seed cell source that people seek the islet cells that substitutes patient's autoimmunization system destruction, the regenerative therapy that with the stem cell transplantation is the master is that the radical cure of mellitus has brought new hope.

(mesenchymal stem cells MSCs), is the seed cell that application advantage is arranged at present most to mescenchymal stem cell.Mescenchymal stem cell has the very strong self duplication ability and the multipotency of differentiation; Domestic and international research shows and can be divided into insulin secretory cell according to particular environment; And mescenchymal stem cell have other stem cell incomparable advantage; As draw materials convenience, propagation and differentiation capability are strong, the transplanting non-immunogenicity, have also avoided the ethics problem that embryo stem cell transplantation faced simultaneously.

Induction scheme for the mesenchyma stem cell to pancreatic islet cytodifferentiation can be classified as following two types at present: most for concentrating the combined utilization of NGFF and activin, like bFGF, EGF etc.; Another kind method is the key gene in the growth of transfection islet cells, the insulin secretion process, like transcription factors such as PDX-1, Ngn3.But existing research induces the differentiation scheme that very big limitation is arranged, and differentiation efficiency is not high, and effect is undesirable during application.

Serum response factor (SRF) is a kind of high conservative and extensively is present in the intravital transcriptional regulator of multiple biology that it can combine with CArG box locus specificity on a plurality of gene promoters, and then specificity raises the transcriptional expression level of target gene.Find that at present its major function is the genetic transcription process of participating in regulating cell inner frame Actin muscle and some other scaffolding protein, and then participate in multiple Mammals physiological process that for example sarcostyle is grown and is divided into processes such as Skelettmuskel, unstriated muscle.The up-to-date binding site CArG box that contains SRF on the insulin gene promotor that discovers; And SRF and PDX-1 can work in coordination with and promote transcribing of insulin gene, but utilize SRF and the common inducing mesenchymal stem cell of combinations of. growth factors not to appear in the newspapers as yet to the smooth muscle cell differentiation.

Summary of the invention

The object of the present invention is to provide a kind of external evoked human mesenchymal stem cell to be divided into the method for insulin secretory cell, present method is utilized SRF genetic modification and the plain secretory cell differentiation of multiple growth factor co-induction mesenchyma stem cell to pancreatic islet.

For solving the problems of the technologies described above, the technical scheme that the present invention adopts is:

A kind of external evoked human mesenchymal stem cell is divided into the method for insulin secretory cell, may further comprise the steps:

(1) makes up the eukaryotic expression recombinant plasmid that contains the SRF gene;

(2) induce human mesenchymal stem cell to Nestin ⁺Cytodifferentiation: cell is removed original substratum when 70-80% merges in the step (1), adds the inducing culture I and cultivates 5～6 hours;

The inducing culture I is the DMEM-high glucose medium that contains 5mmol/L beta-mercaptoethanol and 2% foetal calf serum described in the said step (2), contains 25mM glucose;

(3) induce Nestin ⁺Cell breaks up to the pancreas progenitor cell: obtain to remove inducing culture I in the step (2) in the cell in step (2), add the inducing culture II and cultivated 7～8 days;

Inducing culture II described in the step (3) is the DMEM-high glucose medium that contains 5～20nmol/L bFGF, 5～20nmol/L EGF, 2%B27,0.1% beta-mercaptoethanol and 2% foetal calf serum, contains 25mM glucose;

(4) induce the pancreas progenitor cell to break up: to obtain in the cell in step (3) to insulin secretory cell; Remove inducing culture II in the step (2); Add the inducing culture III, the eukaryotic expression recombinant plasmid of the SRF gene in the transfection step (1) was cultivated 7～8 days simultaneously;

Inducing culture III described in the step (4) is the DMEM-high glucose medium that contains 5～20nmol/L Exendin-4,5～20mmol/L nicotinamide, 2%B27 and 2% foetal calf serum, contains 25mM glucose.

And, making up the eukaryotic expression recombinant plasmid that contains the SRF gene in the step (1), carrier for expression of eukaryon is pEGFP, pcDNA3.1 or pCMV.

And the human mesenchymal stem cell described in the step (2) comprises the mescenchymal stem cell in amniotic fluid, marrow, placenta, Cord blood or fatty tissue source.

And the SRF eukaryon expression plasmid described in the step (4) is for removing endotoxic plasmid, and plasmid superhelix banding pattern is clear, A260/A280=1.8～2.0.

And the inducing culture II described in the said step (3) is the DMEM-high glucose medium that contains 10nmol/L bFGF, 10nmol/L EGF, 2%B27,0.1% beta-mercaptoethanol and 2% foetal calf serum, contains 25mM glucose.

And the induced liquid substratum III described in the said step (4) is the DMEM-high glucose medium that contains 10nmol/L Exendin-4,10mmol/L nicotinamide, 2%B27 and 2% foetal calf serum, contains 25mM glucose.

Advantage of the present invention and positively effect are following:

1, the present invention is used as the modifying factor of inducing mesenchymal stem cell in the insulin secretory cell atomization with SRF first, and first transfection SRF and the common inducing mesenchymal stem cell of multiple combinations of. growth factors is broken up to insulin secretory cell.

2, the present invention utilizes the combinations of. growth factors inducing mesenchymal stem cell to be divided into pancreas progenitor cell (cell high expression level PDX-1 this moment); Pair cell carries out genetic modification simultaneously; The SRF that raises in the cell expresses; And being aided with the cell that growth factor and active substance possibly obtain excreting insulin, the foundation of this method is main " regenerative medicine " significant and practical value for research and development based on cellular replacement therapy.

3, Effectene Transfection Reagent transfection system transfection efficiency of the present invention is high, and security is good.

What 4, the present invention can be mescenchymal stem cell provides new theory and experimental basis to insulin secretory cell directional induction differentiation and its clinical application, and new model is provided for relevant with it drug development and screening.

Description of drawings:

Fig. 1 carries out pcr amplification for the present invention utilizes the upstream and downstream primer of SRF to the SRF gene, and the PCR product that amplification is obtained carries out agarose gel electrophoresis, and M is molecular weight marker, 1 representative amplification PCR product fragment;

Fig. 2 carries out the double digestion checking for the constructed SRF plasmid of the present invention, and M is molecular weight marker, and 1 is the electrophorogram after constructed plasmid carries out double digestion.

Fig. 3 induces the cellular form figure of human mesenchymal stem cell to the insulin secretory cell differentiation for the present invention utilizes inducing culture to make up three one-step inducing methods, and wherein, the form of the mescenchymal stem cell of group is not induced in Fig. 3-1 representative; Fig. 3-2 representative utilizes inducing culture I inducing mesenchymal stem cell to be divided into nestin ⁺The metamorphosis situation of cell; Fig. 3-3 representative utilizes the inducing culture II to induce nestin ⁺Cytodifferentiation is the metamorphosis situation of pancreas progenitor cell; Fig. 3-4 representative utilizes the inducing culture III to induce the pancreas progenitor cell to be divided into the metamorphosis situation of insulin secretory cell.

Fig. 4 induces human mesenchymal stem cell after the insulin secretory cell differentiation for the present invention utilizes inducing culture to make up three one-step inducing methods; Utilize dithizone dyeing to detect insulin secretory cell group; Wherein, Fig. 4-1 representative does not induce the mescenchymal stem cell of group to carry out dithizone dyeing; Dithizone dyeed after Fig. 4-2 representative utilized growth factor-induced, and the cell mass that occurs expression of insulin after utilizing the inducing culture combination to induce is described.

Fig. 5 utilizes inducing culture to make up three one-step inducing methods for the present invention and transcription factor SRF induces human mesenchymal stem cell jointly after the insulin secretory cell differentiation; Utilize RT-PCR to detect differentiation back cell expressing insulin gene mRNA level; After wherein on behalf of inducing culture, A make up the differentiation of three one-step inducing method inducing mesenchymal stem cells, insulin gene mRNA level; After on behalf of inducing culture, B make up three one-step inducing methods and transcription factor SRF to induce the human mesenchymal stem cell differentiation jointly, insulin gene mRNA level.The mRNA level of explanation Regular Insulin after inducing culture combination and transcription factor SRF coinduction is higher.

Fig. 6 utilizes inducing culture to make up three one-step inducing methods for the present invention and transcription factor SRF induces human mesenchymal stem cell jointly after the insulin secretory cell differentiation; Utilize the ELISA method to detect secretion of insulin situation in the substratum; After wherein on behalf of inducing culture, A make up the differentiation of three one-step inducing method inducing mesenchymal stem cells, secretion of insulin situation in the substratum; After on behalf of inducing culture, B make up three one-step inducing methods and transcription factor SRF to induce the human mesenchymal stem cell differentiation jointly, secretion of insulin situation in the substratum.Explanation insulin secretion after inducing culture combination and transcription factor SRF coinduction significantly raises.

Embodiment

Through specific embodiment the present invention is made further detailed description below, following examples are descriptive, are not determinate, can not limit protection scope of the present invention with this.

Among the present invention not specifically the per-cent of the unit of indicating all be weight percentage.The unspecified method of the present invention is techniques well known or known experimental technique, does not have singularity.

The present invention provides a kind of novel method of branch 3 one-step inducings that combined by multiple inductor; Wherein the SRF genetic modification is the method that adopts first in the plain secretory cell differentiation of mesenchyma stem cell to pancreatic islet; This method can be induced to differentiate into insulin secretory cell with human mesenchymal stem cell; And have higher amount of insulin secretion, specifically may further comprise the steps:

(1) separation of mescenchymal stem cell, cultivation

Mescenchymal stem cell comprises tissue-derived mescenchymal stem cells such as amniotic fluid, marrow, placenta, Cord blood, fat.

(2) make up the eukaryotic expression recombinant plasmid that contains the SRF gene, the preparation method who contains the eukaryon expression plasmid of SRF gene is the method for this area structure carrier for expression of eukaryon commonly used.

Concrete is: select suitable carriers, carrier can be carrier for expression of eukaryon such as pcDNA3.1, pCMV.According to SRF full length gene sequence and selected carrier design restriction enzyme site; Utilize the method for PCR or synthetic to obtain the SRF full length gene; Utilize corresponding restriction enzyme to carry out endonuclease reaction; SRF gene fragment after with ligase enzyme enzyme being cut then is connected with carrier, further through the method for agarose gel electrophoresis, digestion with restriction enzyme evaluation, PCR evaluation and order-checking the plasmid that obtains is identified, obtains the SRF eukaryon expression plasmid.

(3) induce human mesenchymal stem cell to Nestin ⁺Cytodifferentiation

Cell kind in the step (1) is gone in 6 orifice plates; When waiting to grow to the 70-80% fusion; Remove original DMEM and 10% foetal calf serum substratum; Add inducing culture I 2mL and cultivated 5～6 hours, the inducing culture I is the high sugar of DMEM-(25mM glucose) substratum that contains 5mmol/L beta-mercaptoethanol and 2% foetal calf serum.The substratum add-on is relevant with use culture plate size, as uses 6 orifice plates, then adds 2ml, as uses 24 orifice plates, then adds 1mL.

(4) induce Nestin ⁺Cell breaks up to the pancreas progenitor cell

Obtain in the cell in step (3); Remove inducing culture I in the step (3); Add inducing culture II 2mL and cultivated 7～8 days, the inducing culture II is the high sugar of DMEM-(25mM glucose) substratum that contains 5～20nmol/L bFGF, 5～20nmol/L EGF, 2%B27,0.1% beta-mercaptoethanol and 2% foetal calf serum.

(5) induce the pancreas progenitor cell to break up to insulin secretory cell

Obtain in the cell in step (4); Remove inducing culture II in the step (4); Add inducing culture III 2mL; The eukaryotic expression recombinant plasmid of the SRF gene in the transfection step (2) was cultivated 7～8 days simultaneously, and the inducing culture III is the high sugar of DMEM-(25mM glucose) substratum that contains 5～20nmol/LExendin-4,5～20mmol/L nicotinamide, 2%B27 and 2% foetal calf serum.

(6) evaluation of differentiation back cell: the cell after utilizing methods such as cellular form observation, RT-PCR, dithizone dyeing to differentiation is identified.

Embodiment 1

A kind of external evoked human mesenchymal stem cell is divided into the method for insulin secretory cell, and what present embodiment adopted is human marrow mesenchymal stem cell, and step is following:

1. the separation of human marrow mesenchymal stem cell, cultivation

Obtain human marrow mesenchymal stem cell from the separation of healthy volunteer's hipbone; Collecting cell is cultivated when cell reaches the 70-80% fusion then, after cleaning with PBS, uses trysinization; Add the low sugar culture-medium (5.6mM glucose) of the DMEM that contains 10% foetal calf serum then and stop digestion; Blow and beat into single cell suspension, abandon supernatant after centrifugal, again with the low sugar culture-medium (5.6mM glucose) of the DMEM that contains 10% foetal calf serum suspension cell again; The cultivation of going down to posterity then;

2. the eukaryotic expression recombinant plasmid that contains the SRF gene makes up

(1) the genomic extraction of mouse: extract the heart tissue of mouse, tissue is placed on the ice cube, note aseptic technique simultaneously.Use pH value is 7.4 PBS, and heart tissue is moistening.Use eye scissors that heart tissue is cut the fritter as 0.5cm * 0.5cm; The heart tissue that shreds is put into homogenizer, add 1 milliliter of Trizol in the 10-20mg heart tissue, on ice as for grinding 5 minutes; The centrifuging and taking supernatant utilizes traditional method (chloroform/Virahol) to extract RNA.

(2) according to full length sequence information and the used pcDNA3.1 carrier information design primer of SRF in the Genebank DB among the NCBI, primer sequence is following:

Upstream primer 1:5 '-CATGGATCCCCATGTTACCGACCCAAGCT-3 '

Downstream primer 1:5 '-GGTCTCGAGGTTCACCACCTGTAGCTCGG-3 '

The PCR reaction system:

Utilize the EasyTaq polysaccharase at 94 ℃ of 5min; (94 ℃ of 1min; 53 ℃ of 30s, 72 ℃ of 3min) pcr amplification is carried out in 35 circulations under the reaction conditions of 72 ℃ of 10min; With PCR product electrophoresis (like Fig. 1) in 1% TBE sepharose that amplification obtains, utilize sepharose DNA fast purifying to reclaim the test kit purifying and recovering and obtain the SRF gene fragment.

(3) with SRF gene and pcDNA3.1 plasmid with BamHI and XhoI double digestion after, connect through T4DNA ligase enzyme (Promega), get and connect product 10 μ l and join competent cell (conventional CaCl ₂Legal system is equipped with E.coliDH5 α competent cell) in; Rap tube wall and make the content mixing, ice bath 30min, 42 ℃ of water-bath thermal shock 90s; Rapid transposition ice bath 2min; Add 500 μ lLB substratum shaking culture, 300 μ l converted products are coated on the LB culture medium flat plate that contains penbritin, place 37 ℃ of incubator overnight cultures.Picking mono-clonal behind the cultivation 12h is inoculated in the liquid nutrient medium that 3ml contains penbritin respectively, and 37 ℃ of shaking culture are spent the night.Utilize the little extraction reagent kit of plasmid (Solarbio) to extract plasmid; With BamHI and XhoI double digestion checking (like Fig. 2); Agarose gel electrophoresis detects has the SRF purpose fragment about 1.6kb, and the DH5 α bacterium liquid that further will contain the pcDNA3.1-SRF plasmid is delivered to Shanghai and given birth to the evaluation of checking order of worker Bioisystech Co., Ltd, and sequencing result is compared in the Genebank DB; It is entirely true that proof is read frame, contains the SRF full-length gene in the pcDNA3.1-SRF plasmid.

(4) a large amount of extractions of DNA; With pcDNA3.1-SRF plasmid transformation escherichia coli DH5 α; Get bacterium liquid 30 μ l; Be inoculated into 3ml and contain in the LB substratum of penbritin, on 37 ℃ of shaking baths, shake overnight cultures, utilize and go the big extraction reagent kit of intracellular toxin plasmid (Solarbio) operation steps purifying extraction pcDNA3.1-SRF plasmid subsequent use.Plasmid after the extraction carries out agarose gel electrophoresis to be observed, and plasmid superhelix banding pattern is clear.Utilize nucleic acid-protein determinator (U.S. Bio-Rad company) that nucleic acid is carried out quantitative analysis simultaneously, A260/A280=1.8～2.0 are best.

3. inducing human mesenchymal stem cells breaks up to the pancreas progenitor cell

Wait to separate growth of mesenchymal stem cells to 70～80% that obtains long-term cultivation, add inducing culture I (the high sugar of the DMEM-of 5mmol/L beta-mercaptoethanol and 2% foetal calf serum (25mM glucose) substratum), put into 37 ℃, 5%CO ₂Constant incubator was cultivated 5～6 hours, mescenchymal stem cell was induced be Nestin ⁺Cell; Old substratum is removed in suction, adds inducing culture II (the high sugar of the DMEM-of 10nmol/L bFGF, 10nmol/L EGF, 2%B27,0.1% beta-mercaptoethanol and 2% foetal calf serum (25mM glucose) substratum), puts into 37 ℃, 5%CO ₂Constant incubator is cultivated, and 48h half amount first changes liquid, and full dose was changed liquid once in per afterwards 3 days, cultivated 7 days.

4. transfection SRF eukaryon expression plasmid in the pancreas progenitor cell, induce it to break up to insulin secretory cell:

According to Effectene Transfection Reagent transfection system the pancreas progenitor cell is gone in the SRF gene transfection; The concrete operations step is following: in 24 orifice plates, add 0.5mL in advance and contain serum but do not contain the low sugar culture-medium of antibiotic DMEM, place 37 ℃, 5%CO ₂Hatch in the incubator.Cell degree of converging before transfection reaches 60-70%, substratum is inhaled gone, and after the adding PBS rinsing once, adds 0.25% trypsin digestion cell, centrifugal cell harvesting, and with not containing Ca ²⁺And Mg ²⁺PBS rinsing cell, the centrifugal supernatant that goes adds resuspended damping fluid R re-suspended cell, making its final concentration is 1.0 * 10 ⁷Individual cell/mL.To the aseptic Eppendorf tube of 1.5mL, the pcDNA3.1 plasmid that will not contain the SRF gene by the every hole of 1 μ g/ respectively joins in the cell suspension with the pcDNA3.1-SRF expression plasmid that contains SRF, gently mixing with resuspended cell transfer.

Open Neon ^TMInstrument switch is at Neon ^TMAdd the 3mL electrolytic buffer in the pipe, then it is inserted Neon ^TMIn the pipettor suction nozzle frame, use Neon ^TMNeon of pipettor gripping ^TMSuction nozzle guarantees that pipettor and suction nozzle are seamless.With Neon ^TMSuction nozzle immerses in cell-plasmid mixture, draws the 10uL mixture.Then vertical insertion of pipettor is positioned at Neon ^TMNeon on the pipettor suction nozzle frame ^TMIn the pipe.It is voltage 990V that electricity commentaries on classics parameter is set on image display, burst length 40ms, and number of clicks 1 time is pressed the START key then.As screen displaying Complete, explain that electricity changes completion.Pipettor is taken off, and the sample transfer in the suction nozzle is to adding in advance in the orifice plate that inducing culture III (the high sugar of the DMEM-of 10nmol/L Exendin-4,10mmol/L nicotinamide, 2%B27 and 2% foetal calf serum (25mM glucose) substratum) hatches.Rock culture plate gently, guarantee that cell distribution is even.Culture plate is placed 37 ℃, 5%CO ₂Continue in the incubator to cultivate 48 hours, carry out half amount and change liquid, full dose was changed liquid once in per afterwards 3 days, induced it to break up to insulin secretory cell.

5. detect the morphological change of differentiation back cell

In inducing atomization, order utilizes inducing culture liquid I, inducing culture liquid II, inducing culture liquid III to carry out the differentiation of SRF genetic modification inducing cell simultaneously, experience Nestin ⁺Cell, pancreas progenitor cell, insulin secretory cell three phases utilize inverted phase contrast microscope to carry out morphological observation, and the result is as shown in Figure 3, and considerable change takes place on morphology cell, become circle and form cell cluster.

6. utilize the dithizone dyeing process to identify insulin secretory cell

At first dispose the dithizone staining agent, 50mg dithizone (DTZ) adds 5mL DMSO dissolving as storing solution, uses PBS dilution final concentration to be 0.1mg/mL, and the suction filtration degerming is formulated as staining fluid.For the cell of inducing differentiation with do not induce the cell of differentiation,, add staining fluid, microscopy behind 37 ℃ of incubation 15min with PBS rinsing 2 times.The result is as shown in Figure 4, induces differentiation back cell painted darker, is red-brown.

Embodiment 2

A kind of external evoked human mesenchymal stem cell is divided into the method for insulin secretory cell, present embodiment uses be pCMV-Tag2B as carrier, concrete grammar is following:

1. the separation of human marrow mesenchymal stem cell, cultivation is with embodiment 1.

2. make up the pCMV-SRF eukaryon expression plasmid

(1) be masterplate with the pcDNA3.1-SRF plasmid that builds among the embodiment 1; With pCMV-Tag2B as carrier; Adopt the method for the PCR SRF gene fragment that from the pcDNA3.1-SRF plasmid, increase, design primer according to full length sequence information and the used pCMV carrier information of SRF, primer sequence is following:

Upstream primer 2:5 '-ATCGGGATCCATGTTACCGACCCAAGCT-3 '

Downstream primer 2:5 '-GGTCTCGAGGTTCACCACCTGTAGCTCGG-3 '

Carry out pcr amplification, after the PCR product utilization agarose gel electrophoresis that amplification is obtained detects, utilize the gel extraction method purifying and recovering to obtain the SRF gene fragment.

(2) with SRF gene and pCMV-Tag2B plasmid with BamHI and XhoI double digestion after; Connect through T4DNA ligase enzyme (Promega); Get and connect product transformed into escherichia coli DH5 α (method for transformation is with embodiment 1); 300 μ l converted products are coated on the LB culture medium flat plate that contains penbritin, placed 37 ℃ of incubator overnight cultures.Picking mono-clonal behind the cultivation 12h is inoculated in the liquid nutrient medium that 3ml contains penbritin respectively, and 37 ℃ of shaking culture are spent the night.Utilize the little extraction reagent kit of plasmid (Solarbio) to extract plasmid; With BamHI and the checking of XhoI double digestion; Agarose gel electrophoresis detects has the SRF purpose fragment about 1.6kb, and the DH5 α bacterium liquid that further will contain the pCMV-SRF plasmid is delivered to Shanghai and given birth to the evaluation of checking order of worker Bioisystech Co., Ltd, and sequencing result is compared in the Genebank DB; It is entirely true that proof is read frame, contains the SRF full-length gene in the pCMV-SRF plasmid.

(3) large scale extracting method of pCMV-SRF plasmid is with embodiment 1.

3. inducing human mesenchymal stem cells is to the differentiation of pancreas progenitor cell, with embodiment 1.

4. transfection SRF eukaryon expression plasmid in the pancreas progenitor cell, induce it to break up to insulin secretory cell: the transfection plasmid is the pCMV-SRF plasmid, and other are with embodiment 1.

5. utilize the dithizone dyeing process to identify insulin secretory cell, with embodiment 1.

6. utilize the mRNA expression of the method detection insulin secretory cell specific gene insulin of RT-PCR.After inducing differentiation, utilize the Trizol method to extract total RNA of culturing cell, total RNA of extraction detects with the nucleic acid quantification appearance and is quantitative.With mRNA is template, and adopting Oligo (dT) is primer, utilizes M-MLV reversed transcriptive enzyme rt to be cDNA.The primer of design insulin, synthetic by Invitrogen biotech company.Primer sequence is following:

Forward primer 3:5 '-CAGCCGCAGCCTTTGTGAA-3 '

Reverse primer 3:5 '-TCCACAATGCCACGCTTCTG-3 '

Utilize cDNA that reverse transcription obtains for template at 95 ℃ of 5min, 32 cyclic amplifications (95 ℃ of 30s, 55.1 ℃ of 45s, 72 ℃ of 60s) carry out pcr amplification under the condition that 72 ℃ are extended 10min.The result is as shown in Figure 5: after (A) representing inducing culture to make up the differentiation of three one-step inducing method inducing mesenchymal stem cells, and insulin gene mRNA level; (B) represent inducing culture to make up three one-step inducing methods and transcription factor SRF to induce the human mesenchymal stem cell differentiation jointly after, insulin gene mRNA level.

The mRNA level of presentation of results Regular Insulin after inducing culture combination and transcription factor SRF coinduction is higher.

Embodiment 3

A kind of external evoked human mesenchymal stem cell is divided into the method for insulin secretory cell, and what present embodiment adopted is people's umbilical cord blood mesenchymal stem cells, and concrete steps are following:

1. the separation of people's umbilical cord blood mesenchymal stem cells, cultivation is with embodiment 1.

Get anti-freezing bleeding of the umbilicus and isopyknic PBS mixing, utilize the Ficoll parting liquid to separate the mononuclearcell layer, PBS cleans, and cell counting is with 2 * 10 ⁷Cell density inoculation, placing 37 ℃, volume(tric)fraction is 5%CO ₂, cultivate in the saturated humidity incubator.After adherent 2 hours, change fresh medium, treat cell reach 80% when merging with 0.25% pancreatin and 0.02%EDTA1: 1 mixes joke, the cultivation of going down to posterity.

2. the eukaryotic expression recombinant plasmid that contains the SRF gene makes up, with embodiment 1.

3. induce people's umbilical cord blood mesenchymal stem cells to the differentiation of pancreas progenitor cell, with embodiment 1.

4. transfection SRF eukaryon expression plasmid in the pancreas progenitor cell, induce it to break up to insulin secretory cell: the transfection plasmid is the pcDNA-SRF plasmid, with embodiment 1.

5. utilize the mRNA expression of the method detection insulin secretory cell specific gene insulin of RT-PCR, with embodiment 1.

6. utilize Regular Insulin (INS) enzyme immunoassay (ELISA) experiment to detect amount of insulin secretion in the substratum

Induce after the differentiation completion, draw inducing culture, PBS fine laundering 3 times is used serum-free H-DMEM culture medium culturing 12h instead.Collect supernatant of culture medium, preserve to be equipped with for-80 ℃ and survey.

It is following to utilize insulinase linked immune analysis test kit (R&D) to detect step:

(1) dilution of standard substance and application of sample: encapsulate at enzyme mark and to establish 10 holes, standard substance hole on the plate, in first, second hole, add standard substance 100 μ L respectively, in first, second hole, add standard substance diluent 50 μ L then, mixing; From first hole, second hole, respectively get 100 μ L then and be added to the 3rd hole, the 4th hole respectively, add standard substance diluent 50 μ L, mixing again in the 3rd, the 4th hole respectively; In the 3rd, the 4th hole, respectively get 50 μ L earlier then and discard, respectively get 50 μ L again and be added to respectively in the 5th, the 6th hole, add 50 μ L standard substance diluents more respectively, mixing; Get respectively from the 5th, the 6th hole behind the mixing that 50 μ L join the 7th respectively, in the octal, again the 7th, add standard substance diluent 50 μ L respectively, mixing in the octal; Respectively get 50 μ L in the air from the seven or eight behind the mixing and be added to the 9th, the tenth in the air respectively, add standard substance diluent 50 μ L mixings; Respectively getting 50 μ L behind the mixing discards.(not having hole application of sample amount after the dilution all is 50 μ l, and concentration is respectively 12mU/L, 8mU/L, 4mU/L, 2, mU/L, 1mU/L), all the other steps with below identical, the drawing standard curve.

(2) application of sample: establish blank well (blank well does not add sample and enzyme marking reagent, and all the other steps are identical), testing sample hole respectively.Encapsulate at enzyme mark that the testing sample hole adds sample diluent 40 μ L earlier on the plate, and then add testing sample 10 μ L.Application of sample is added on bottom, enzyme plate hole, mixing with sample.

(3) incubation: be placed on 37 ℃ with shrouding film shrouding, 30min.

(4) dosing: 30 times of concentrated cleaning solutions are subsequent use after with 30 times of dilutions of zero(ppm) water.

(5) washing: carefully take the shrouding film off, discard liquid, dry, fill it up with washings, discard after leaving standstill 30s, repeat 5 times.

(6) enzyme-added: every hole adds enzyme marking reagent 50 μ L, except the blank well.

(7) incubation: with 3.

(8) washing: with 5.

(9) colour developing: every hole adds developer A50 μ L earlier, adds developer B 50 μ L again, mixing gently, 37 ℃ of lucifuges colour developing 15min.

(10) stop: every hole adds stop buffer 50 μ L, termination reaction (this moment, blue upright commentaries on classics was yellow).

(11) measure: with the blank well zeroing, the 450nm wavelength is measured each hole absorbancy in regular turn.

(12) calculate: the insulin content that calculates every hole sample according to typical curve.

The result is as shown in Figure 6; Utilize the cell of differentiation that the inventive method is induced to produce Regular Insulin; And; Compare with only utilizing three kinds of inducing culture inducing cell differentiation groups, the noble cells generation amount of insulin that the while pair cell carries out behind the SRF genetic modification significantly raises, and rises to 12.98 ± 1.86mU/L from 5.64 ± 1.04mU/L.

Claims

1. an external evoked human mesenchymal stem cell is divided into the method for insulin secretory cell, it is characterized in that: may further comprise the steps:

2. external evoked human mesenchymal stem cell according to claim 1 is divided into the method for insulin secretory cell; It is characterized in that: make up the eukaryotic expression recombinant plasmid that contains the SRF gene in the said step (1), carrier for expression of eukaryon is pEGFP, pcDNA3.1 or pCMV.

3. external evoked human mesenchymal stem cell according to claim 1 is divided into the method for insulin secretory cell, it is characterized in that: the human mesenchymal stem cell described in the step (2) comprises the mescenchymal stem cell in amniotic fluid, marrow, placenta, Cord blood or fatty tissue source.

4. external evoked human mesenchymal stem cell according to claim 1 is divided into the method for insulin secretory cell; It is characterized in that: the SRF eukaryon expression plasmid described in the step (4) is for removing endotoxic plasmid; Plasmid superhelix banding pattern is clear, A260/A280=1.8～2.0.

5. external evoked human mesenchymal stem cell according to claim 1 is divided into the method for insulin secretory cell; It is characterized in that: the inducing culture II described in the said step (3) is the DMEM-high glucose medium that contains 10nmol/L bFGF, 10nmol/L EGF, 2%B27,0.1% beta-mercaptoethanol and 2% foetal calf serum, contains 25mM glucose.

6. external evoked human mesenchymal stem cell according to claim 1 is divided into the method for insulin secretory cell; It is characterized in that: the induced liquid substratum III described in the said step (4) is the DMEM-high glucose medium that contains 10nmol/L Exendin-4,10mmol/L nicotinamide, 2%B27 and 2% foetal calf serum, contains 25mM glucose.