CN102618491A - Culture solution for inducing mesenchymal stem cells to differentiate into islet-like cells, and inducing method and application of culture solution - Google Patents
Culture solution for inducing mesenchymal stem cells to differentiate into islet-like cells, and inducing method and application of culture solution Download PDFInfo
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Abstract
The invention discloses a culture solution for inducing mesenchymal stem cells to differentiate into islet-like cells, and an inducing method and application of the culture solution. The culture solution comprises the following materials: niacinamide, Conophylline, cell growth factor, betacellulin and a base medium. The base medium contains 97% of high glucose DMEM (Dulbecco Modified Eagle Medium), 2% of B-27 and 1% of N-2. The inducing method comprises the following steps of: preparing an inducing culture solution; preparing the human mesenchymal stem cells; taking the human mesenchymal stem cells, inoculating the human mesenchymal stem cells into a six-hole ultralow absorption culture plate by 1.5-2*105cells/hole, adding 3ml inducing culture medium into each hole and carrying out suspended induction; and changing liquid at every 3 days, collecting cell supernatant at the ninth day, and storing the cell supernatant at the temperature of -20 DEG C. The culture solution disclosed by the invention has the advantages that the human mesenchymal stem cells are induced to differentiate into the islet-like cells by utilizing the combination of the niacinamide and the Conophylline, so that the inducing cycle is shortened, the suspension cells are beneficial to being clustered to form cell clusters similar to natural islets, further the induced differentiation efficiency is obviously increased and the clinical application risk is reduced; and the function of inducing the secretion of the cell insulin is obviously improved.
Description
Technical field
The present invention relates to a kind of novel plant inductor Conophylline and nicotinamide combined highly effective of utilizing induces human adipose-derived stem cell to be divided into nutrient solution and the induction method and the purposes of functional islets like cell group.
Background technology
One type mellitus are called insulin-dependent diabetes mellitus again, are the cell-mediated autoimmune diseases of primary T, finally cause β cell considerable damage, hypoinsulinism, and blood sugar concentration is regulated unbalance, the serious harm human health.Pancreatic islets transplantation is the effective means of treatment mellitus, but the donorcells wretched insufficiency needs to seek new pancreatic islets transplantation surrogate.Mescenchymal stem cell is a kind of cell with multidirectional differentiation potential, and it can directional induction be various kinds of cell such as insulin-like cell, chondrocyte.And mesenchymal cell is present in the human multiple tissue; Especially the mescenchymal stem cell in umbilical cord, placenta, fatty tissue source; Have wide material sources, be easy to advantages such as collection, no ethics problem; But mass-producing is induced to differentiate into insulin-like cell, for the clinical treatment mellitus provide new technical scheme.
Nicotinamide is an ADP ribose synthetase inhibitors, can promote the differentiation of human fetal pancreas, and the normal reaction that in long-term high sugared environment, keeps islet cells that glucose is stimulated, thus be widely used in insulin secretory cell induce differentiation environment structure composition.Conophylline has and induces pancreas precursor cell model-AR42J cells in vitro to be divided into the ability of insulin secretory cell; And compare with Activin-A, the Conophylline molecular weight is little, is easy to infiltration; Apoptosis that can inducing cell is considered to a kind of plant inductor of mystery.The efficiency ratio Activin-A that existing bibliographical information Conophylline inducing bone mesenchymal differentiation of stem cells is an insulin secretory cell is higher.
At present mescenchymal stem cell is induced to differentiate into the adherent inductive mode of the many employings of insulin-like cell group.Because of mescenchymal stem cell has stronger adherent property, it is agglomerating in inducing process, to have hindered cell aggregation, so inductivity is generally lower.Two methods or three-step approach, inducible factor uses more, and induction duration was generally 14-21 days, and induction duration is long, the residual factor is of a great variety, has increased the risk of clinical application.
Summary of the invention
Technical problem to be solved by this invention is that nutrient solution and induction method and the purposes of a kind of inducing mesenchymal stem cell to the insulin-like cell differentiation are provided.
In order to solve the problems of the technologies described above, the technical scheme that the present invention adopts is: a kind of inducing mesenchymal stem cell comprises nicotinamide, Conophylline, ESC, cell regulin and basic medium to the nutrient solution of insulin-like cell differentiation.
Contain in every liter of basic medium:
Said ESC is HGF, bFGF or EGF.
Said cell regulin is Betacellulin.
Said basic medium contains 97% high sugared DMEM, 2% B-27,1% N-2, and said concentration is volume by volume concentration.
A kind of inducing mesenchymal stem cell may further comprise the steps to the method for insulin-like cell differentiation:
(1) preparation inducing culture liquid: with the sugared DMEM of height is basic medium, the B-27 of interpolation 2%, 1% N-2 and inductor nicotinamide, Conophylline, HGF, Betacellulin, and mixing, 4 degree are preserved, and said concentration is volume by volume concentration;
(2) preparation human mesenchymal stem cell;
(3) get human mesenchymal stem cell, with 1.5-2 * 10
5The cells/ hole is inoculated in the ultralow absorption culture plate in 6 holes, and the 3ml inducing culture is added in every hole, and suspension is induced;
(4) changed liquid in per 3 days, collecting cell supernatant in the time of 9 days ,-20 degree are preserved.
The nutrient solution that contains inductor nicotinamide and Conophylline is in the application of inducing mesenchymal stem cell in the insulin-like cell differentiation.
The invention has the beneficial effects as follows: utilize nicotinamide and Conophylline combined induction human mesenchymal stem cell to the differentiation of insulin-like cell group; Shorten induction duration; It is agglomerating that suspension cell is beneficial to, and forms the cell mass of similar natural pancreas islet, significantly improves and induce differentiation efficiency; Significantly reduce cytokine and use kind, reduce the clinical application risk; Significantly improve the inducing cell insulin secretion function.Shortcomings such as the difficult gathering of cell is agglomerating in existing inductor and the induction method, induction duration is long, efficient is low, the gained cell quantity is few, function is poor, the easy apoptosis of cell have been overcome.
Description of drawings
Figure 1A is a metamorphosis (40 *) in the former process of supporting of being commissioned to train of hADSCs.
Figure 1B is the hADSCs cultivation (P6) (4 *) of going down to posterity.
Fig. 1 C is the hADSCs growth curve.
Fig. 2 hADSCs surface specific Protein Detection.
Adipocyte after Fig. 3 induces, scleroblast identify (A, B:40 *; C:4 *; D:10 *).
The formation (10 *) of Fig. 4 hADSCs differentiation and insulin-like cell group.
Fig. 5 Insulin, c-peptide immunocytochemical assay (40 *).
Expression of Related Genes in Fig. 6 islet cells atomization.
Fig. 7 glucose stimulates secretion of insulin level (P＜0.001) down.
Fig. 8 induces not isogeneous induction group amount of insulin secretion after 9 days (P＜0.05vs (NIC+CNP)).
Embodiment
Below in conjunction with accompanying drawing and embodiment the present invention is done further explain:
Inducing mesenchymal stem cell of the present invention comprises nicotinamide, Conophylline, ESC, cell regulin and basic medium to the nutrient solution of insulin-like cell differentiation.
Contain in every liter of basic medium:
Said ESC is HGF, bFGF or EGF.
Said cell regulin is Betacellulin.
Said basic medium contains 97% high sugared DMEM, 2% B-27,1% N-2, and said concentration is volume by volume concentration.
A kind of inducing mesenchymal stem cell may further comprise the steps to the method for insulin-like cell differentiation:
(1) preparation inducing culture liquid: with the sugared DMEM of height is basic medium, the B-27 of interpolation 2%, 1% N-2 and inductor nicotinamide, Conophylline, HGF, Betacellulin, and mixing, 4 degree are preserved, and said concentration is volume by volume concentration;
(2) preparation human mesenchymal stem cell;
(3) get human mesenchymal stem cell, with 1.5-2 * 10
5The cells/ hole is inoculated in the ultralow absorption culture plate in 6 holes, and the 3ml inducing culture is added in every hole, and suspension is induced;
(4) changed liquid in per 3 days, collecting cell supernatant in the time of 9 days ,-20 degree are preserved.
The nutrient solution that contains inductor nicotinamide and Conophylline is in the application of inducing mesenchymal stem cell in the insulin-like cell differentiation.
The present invention carries out the single stage method suspension and induces, and adopts the differentiation of novel plant inductor Conophylline and nicotinamide combined induction mesenchyma stem cell to pancreatic islet like cell.
Embodiment: the preparation of fat mesenchymal stem cell
1.1 the preparation of fat mesenchymal stem cell
Get the sub product of clinical operation or beauty and shaping---fatty tissue under the aseptic condition, in the Biohazard Safety Equipment fatty tissue is cleaned 2-3 time in PBS, thoroughly remove red corpuscle and fragment of tissue.The 0.1%I Collagen Type VI enzyme solution that adds preheating is then put 37 ℃ of water-bath concussion digestion 1h.The centrifugal 5min of 300g removes upper strata adipocyte and collagenase solution.Clean bottom bulk deposition with PBS+1%BSA, the centrifugal 5min of 300g adds the DMEM re-suspended cell that contains 10%FBS in right amount, filters through 100 μ m cells sieve, and the adjustment cell density is seeded to Tissue Culture Flask, in 37 ℃, 5%CO
2Cultivate in the incubator.Change liquid behind the 48h, remove not attached cell and residual red corpuscle.After this changed liquid in per 3 days.When cell reached 80% fusion, the Trypsin-EDTA digestion attached cell with 0.25% was gone down to posterity by 1: 2, observation of cell growth and morphological change characteristics under the inverted microscope.The result shows, visible circular attached cell behind the former generation hADSCs cultivation 24h, and the 48h cell presents spindle shape, polygon, the 72h cell quantity obviously increases, dense arrangement (Figure 1A).Passage cell (P6) rate of propagation is very fast, and in the 3rd day beginning quantity showed increased, about 6-7 days cell covered with at the bottom of the culturing bottle, is the mediation shape and arranges (Figure 1B).
Adopt Alamar Blue method to measure the hADSCs growth curve, get P3, P4, P5 are for the logarithmic phase cell, and pancreas enzyme-EDTA digestion is counted, and adjustment single cell suspension concentration is 10
4Cells/ml is inoculated in 24 orifice plates, every hole 1ml; Be divided into 8 groups; Every group three multiple holes, according to commercial weight formula also: commercial weight %=117216 * (T570)-80586 * (T600)/155677 * C600)-14652 * (C570) draw growth curve also, calculate population doubling time.The result shows, P3, P4, P5 are all S-type for cell growth curve, and 1-3 days cells are in growth resting stage, inoculate that cell gets into the logarithmic proliferation phase after 4-5 days, and about 7 days cell arrival plateaus, population doubling time is 60h (Fig. 1 C).
1.2 the evaluation of fat mesenchymal stem cell
1.2.1 flow cytometry
After Trypsin-EDTA digestion, the adjustment cell concn is 10 with P1 fat subsitutes stem cell
6Cells/ml; With the antibody of FITC and PE mark on ice lucifuge hatch 30min; With the PBS re-suspended cell that contains 2.5%FBS, the expression of its surface specific antigens c of flow cytometry D29-PE, CD44-FITC, CD31-PE, CD34-PE, CD45-FITC, CD49d-PE, CD106-FITC, CD105-PE and HLA-DR-FITC.The flow cytometer detected result shows that the human adipose-derived stem cell phenotype is CD44
+, CD105
+, CD29
+, CD49d
+, CD106
-, CD31
-, CD34
-, CD45
-, HLA-DR
-(Fig. 2).1.2.2hADSCs multidirectional differentiation capability
Get P4 and be incubated at in fat inducing culture and the osteogenic induction substratum, observe its metamorphosis for hADSCs, and to inducing the cell after the differentiation to carry out the dyeing of oil red O stain and sodium alizarinsulfonate respectively.Become fat to induce cellular form generation considerable change in the process, cell becomes round gradually by spindle shape.Circular fat occurs in the down visible born of the same parents of 2-3 days inverted microscopes and drip, drip quantity and be on the increase with inducing fate to increase fat, induce 14 days after, being full of fat in 80% cell drips, nucleus is pressed to the edge, oil red O stain positive (Fig. 3 A).The immunocytochemistry experiment shows the specific proteins Leptin (Fig. 3 C) of the cell expressing adipocyte after inducing; Analyze through RT-PCR; Induce back cell expressing LPL, AP2 gene, and AP2 has faint expression among the hADSCs, (Fig. 3 E) raised in genetic expression after becoming fat to induce.
After changing the skeletonization inducing culture, the cell growth presents flakey fast, and connects in flakes, forms colony, and cell is the multilayer growth subsequently, and calcium deposition increases, and can be observed the calcium tubercle under the mirror after about 21 days, sodium alizarinsulfonate stained positive (Fig. 3 B).Immunocytochemistry result shows the cell expressing Osteopontin albumen (Fig. 3 D) after inducing; PT-PCR is detected as osteocyte specific gene AKP, Osteopontin, Osteonectin; Induce the back cell that expression is all arranged, and inductive hADSCs does not express said gene (Fig. 3 F).
2, external evoked fat mesenchymal stem cell breaks up to insulin-like cell
2.1 external evoked hADSCs is divided into insulin-like cell group and adopts single stage method to suspend to induce, with P5 for hADSCs with 1.5-2 * 10
5The cells/ hole is inoculated in the ultralow absorption culture plate in 6 holes, the nutrient solution composition: 97% high sugared DMEM substratum, 2%B-27; 1%N-2,5-10mM nicotinamide, 80-100 μ g/LConophylline; 2nM Activin-A, 6-10 μ g/L Betacellulin, 80-100pM HGF; Press column split and induce (1) NIC+BTC+HGF (2) NIC+CNP+BTC+HGF (3) CNP+BTC+HGF (4) Activin-A+BTC+HGF (5) NIC+Activin-A+BTC+HGF, changed liquid in per 3 days, induced 9 days.Said concentration is volume by volume concentration.
2.2 induce the evaluation of differentiation back insulin-like cell
2.2.1 dithizone staining reaction
Get the insulin-like cell group that induces after 9 days and carry out dithizone dyeing, the painted situation of observation of cell.Result's demonstration induces the group cellular form to compare the generation considerable change with control group.Cell is the individual cells suspension growth behind the bed board, whole inducing in the process, and cell is constantly assembled the formation cell mass, induces after 9 days the cell mass edge gradually smooth, and diameter 50～150 μ m are similar to natural pancreas islet, and dithizone dyeing presents brick-red (Fig. 4).
2.2.2 immunocytochemistry detects
Insulin-like cell group is transferred to the deckglass overnight cultures that is coated with poly-lysine, makes cell mass adherent.Fixing 30min under the 4% Paraformaldehyde 96 room temperature, PBS clean the back and add 0.5%Triton X-100 and penetrate 15min.Seal 30min (RT) with 4%PBS-BSA, prevent non-specific binding.Add one and resist, 4 ℃ are spent the night, and it is anti-that PBS cleans back adding two, hatches 30min for 37 ℃.Hatch 15min for 37 ℃ with 5 μ g/mlDAPI then, wherein one is anti-: mouse-anti people Leptin (1: 300), the anti-people Osteopontin of rabbit (1: 200); Mouse-anti people Insulin (1: 300), the anti-people C-peptide of rabbit (1: 500), two is anti-: sheep anti-mouse igg; IgM; Alexafluor488 (1: 800), goat anti-rabbit igg, Cy3 (1: 500).
After suspension is induced; Immunocytochemistry detects the expression of the functional protein Insulin of insulin-like cell group, C-peptide; There are Insulin, C-peptide positive cell in the visible cell group, show to have produced cell mass (Fig. 5) after inducing differentiation with insulin secretion function.
2.2.3RT-PCR gene expression analysis
Use TRIzol reagent and extract hADSCs, induce the back cell total rna, get 1 μ gRNA with the synthetic eDNA of reverse transcription test kit.The PCR reaction system is following: 94 ℃ of preparatory sex change 5min, and 94 ℃ of 30s, 55-60 ℃ of 30s, 72 ℃ of 30s, 72 ℃ are extended 10min, and pcr amplification product is got in 30 circulations, and 2.5% agarose gel electrophoresis analysis is taken a picture and record.
RT-PCR detects the expression of islet cells development related gene; The undifferentiated hADSCs of result expresses Isl-1; Isl-1 is a transcription regulaton factor important in the pancreas islet growth course; Islet cell aysmature syndrome, propagation and differentiation are had vital role, and this gene expression amount raises after inducing, and insulin promoter factor Ipf-1 only expresses in inducing the back cell; The marker gene Insulin of ripe islet cells, somatostatin, glueagon all can detect in inducing the back cell, do not break up that somatostatin has faint expression (Fig. 6) among the hADSCs.
The experiment 2.2.4 glucose stimulates insulin secretion
About 200 cell masses (50-150 μ m) of inducing 9 days of picking clean 2 times with PBS to the 1.5ml centrifuge tube.Add 1ml sugar-free DMEM and cultivate 3-6h in advance, the DMEM that contains 5.6mmol/L glucose, 16.7mmol/L glucose with 300ul then cultivates 2h, 37 degree successively.Collect supernatant; Stimulate secretion of insulin amount down with different concns glucose in the ELISA method detection supernatant, almost detect in the cellular control unit supernatant less than Regular Insulin, reflection in the drawings; And the cell mass after inducing has a small amount of secretion under 5.56mmol/L glucose stimulates; Amount of insulin secretion obviously raises (P＜0.001) behind 25mmol/L glucose incubation 2h, is about following 2.5 times of low sugar condition, can be known by the result; It is responsive to induce back insulin-like cell group that glucose is stimulated, and its secretion of insulin receives the adjusting (Fig. 7) of external environment sugar.
After inducing 9 days; Collect 5 groups of cell conditioned medium liquid; Detect secretion of insulin amount in the supernatant with the ELISA method; The result show NIC, CNP, ACT separately when use and NIC and ACT combined induction amount of insulin secretion do not have notable difference (P＞0.05), and nicotinamide and Conophylline combined induction have significantly improved to the ability (P＜0.05) of insulin-like cell group differentiation (Fig. 8)
In sum; And present method has been used Conophylline at human adipose-derived stem cell first in insulin-like cell is induced, and has observed Conophylline and the nicotinamide combined induction promoter action to differentiation efficiency, and the result shows; Cell secretion of insulin amount after inducing 9 days reaches peak value; Having shortened induction duration, improved and induced differentiation efficiency, is a kind of more effective method of inducing human adipose-derived stem cell to the insulin-like cell differentiation.
Content of the present invention is not confined in the above embodiments, and the knowledgeable people in the same area can propose other embodiment easily within technical director's thought of the present invention, but this embodiment is included within the scope of the present invention.
Claims (7)
1. an inducing mesenchymal stem cell is characterized in that to the nutrient solution that insulin-like cell breaks up, and comprises nicotinamide, Conophylline, ESC, cell regulin and basic medium.
2. inducing mesenchymal stem cell according to claim 1 is characterized in that to the nutrient solution of insulin-like cell differentiation, contains in every liter of basic medium:
3. inducing mesenchymal stem cell according to claim 1 and 2 is characterized in that to the nutrient solution of insulin-like cell differentiation said ESC is HGF, bFGF or EGF.
4. inducing mesenchymal stem cell according to claim 1 and 2 is characterized in that to the nutrient solution of insulin-like cell differentiation said cell regulin is Betacellulin.
5. inducing mesenchymal stem cell according to claim 2 is characterized in that to the nutrient solution of insulin-like cell differentiation said basic medium contains 97% high sugared DMEM, 2% B-27,1% N-2, and said concentration is volume by volume concentration.
6. an inducing mesenchymal stem cell is characterized in that to the method that insulin-like cell breaks up, and may further comprise the steps:
(1) preparation inducing culture liquid: with the sugared DMEM of height is basic medium, the B-27 of interpolation 2%, 1% N-2 and inductor nicotinamide, Conophylline, HGF, Betacellulin, and mixing, 4 degree are preserved, and said concentration is volume by volume concentration;
(2) preparation human mesenchymal stem cell;
(3) get human mesenchymal stem cell, with 1.5-2 * 10
5The cells/ hole is inoculated in the ultralow absorption culture plate in 6 holes, and the 3ml inducing culture is added in every hole, and suspension is induced;
(4) changed liquid in per 3 days, collecting cell supernatant in the time of 9 days ,-20 degree are preserved.
7. a nutrient solution that contains inductor nicotinamide and Conophylline is in the application of inducing mesenchymal stem cell in the insulin-like cell differentiation.
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| CN116574670A (en) * | 2023-05-12 | 2023-08-11 | 广东华夏健康生命科学有限公司 | Method for inducing mesenchymal stem cells to differentiate into islet-like cells and application thereof |
| CN116574670B (en) * | 2023-05-12 | 2024-01-02 | 中科中銮生物科技(广东)有限公司 | Method for inducing mesenchymal stem cells to differentiate into islet-like cells and application thereof |
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