CN102618491B - 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 nutrient solution and induction method and purposes of utilizing novel plant inductor Conophylline and nicotinamide combined highly effective induction human adipose-derived stem cell to be divided into functional islets like cell group.
Background technology
One type diabetes are called again insulin-dependent diabetes mellitus, are the cell-mediated autoimmune diseases of primary T, finally cause β cell considerable damage, hypoinsulinism, blood sugar concentration Imbalance, serious harm human health.Pancreatic islets transplantation is the effective means for the treatment of diabetes, but donorcells wretched insufficiency needs to find new pancreatic islets transplantation surrogate.Mescenchymal stem cell is a kind of cell with multi-lineage potential, and it can directional induction be the various kinds of cell such as insulin-like cell, chondrocyte.And mesenchymal cell is present in human multiple tissue; especially the mescenchymal stem cell of umbilical cord, placenta, adipose tissue-derived; there are wide material sources, be easy to gather, without advantages such as ethics problems; can mass-producing be induced to differentiate into insulin-like cell, for clinical treatment diabetes provide new technical scheme.
Nicotinamide is ADP ribose synthetase inhibitors, can promote the differentiation of human fetal pancreas, and the normal reaction that keeps islet cells to stimulate glucose in long-term high sugared environment, thereby is widely used in the induction differentiation environment structure composition of insulin secretory cell.Conophylline has the ability that induction diabetes model-AR42J cells in vitro is divided into insulin secretory cell, and compare with Activin-A, Conophylline molecular weight is little, be 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 insulin secretory cell is higher.
By mesenchyma stem cell differentiation induction, be the insulin-like cell group modes that adopt adherent induction at present more.Because mescenchymal stem cell has stronger adherent property, in Induction Process, hindered cell aggregation agglomerating, so inductivity is generally lower.Two methods or three-step approach, inducible factor is used more, and induction duration is 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 insulin-like cell differentiation are provided.
In order to solve the problems of the technologies described above, the technical solution used in the present invention is: a kind of inducing mesenchymal stem cell, to the nutrient solution of insulin-like cell differentiation, comprises nicotinamide, Conophylline, cell growth factor, cell regulin and basic medium.
In every liter of basic medium, contain:
Described cell growth factor is HGF, bFGF or EGF.
Described cell regulin is Betacellulin.
Described basic medium contains 97% DMEM in high glucose, 2% B-27,1% N-2, and described concentration is volume by volume concentration.
Inducing mesenchymal stem cell, to a method for insulin-like cell differentiation, comprises the following steps:
(1) preparation inducing culture liquid: take DMEM in high glucose as basic medium, the B-27 of interpolation 2%, 1% N-2 and inductor nicotinamide, Conophylline, HGF, Betacellulin, mix, and 4 degree are preserved, and described concentration is volume by volume concentration;
(2) prepare human mesenchymal stem cell;
(3) get human mesenchymal stem cell, with 1.5-2 * 10
5cells/ hole is inoculated in the 6 ultralow absorption culture plates in hole, and 3ml inducing culture is added in every hole, and induction suspends;
(4) within every 3 days, change liquid, collecting cell supernatant liquor in the time of 9 days ,-20 degree are preserved.
The application of the nutrient solution that contains inductor nicotinamide and Conophylline in inducing mesenchymal stem cell breaks up to insulin-like cell.
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, suspension cell is beneficial to agglomerating, forms the cell mass of similar natural pancreas islet, significantly improve induction differentiation efficiency; Significantly reduce cytokine and use kind, reduce clinical application risk; Significantly improve inducing cell insulin secretion function.Overcome cell in existing inductor and induction method and be difficult for the shortcomings such as gathering is agglomerating, induction duration is long, efficiency is low, gained cell quantity is few, function is poor, the easy apoptosis of cell.
Accompanying drawing explanation
Figure 1A is metamorphosis (40 *) in the primary culturing process of hADSCs.
Figure 1B is the hADSCs cultivation (P6) (4 *) of going down to posterity.
Fig. 1 C is hADSCs growth curve.
Fig. 2 hADSCs surface specific Protein Detection.
Adipocyte after Fig. 3 induction, 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 *).
The expression of genes involved in Fig. 6 islet cells atomization.
Fig. 7 glucose stimulates the secretion level (P<0.001) of lower Regular Insulin.
Fig. 8 induces not isogeneous induction group amount of insulin secretion after 9 days (P<0.05vs (NIC+CNP)).
Embodiment
Below in conjunction with the drawings and specific embodiments, the present invention is described in further detail:
Inducing mesenchymal stem cell of the present invention, to the nutrient solution of insulin-like cell differentiation, comprises nicotinamide, Conophylline, cell growth factor, cell regulin and basic medium.
In every liter of basic medium, contain:
Described cell growth factor is HGF, bFGF or EGF.
Described cell regulin is Betacellulin.
Described basic medium contains 97% DMEM in high glucose, 2% B-27,1% N-2, and described concentration is volume by volume concentration.
Inducing mesenchymal stem cell, to a method for insulin-like cell differentiation, comprises the following steps:
(1) preparation inducing culture liquid: take DMEM in high glucose as basic medium, the B-27 of interpolation 2%, 1% N-2 and inductor nicotinamide, Conophylline, HGF, Betacellulin, mix, and 4 degree are preserved, and described concentration is volume by volume concentration;
(2) prepare human mesenchymal stem cell;
(3) get human mesenchymal stem cell, with 1.5-2 * 10
5cells/ hole is inoculated in the 6 ultralow absorption culture plates in hole, and 3ml inducing culture is added in every hole, and induction suspends;
(4) within every 3 days, change liquid, collecting cell supernatant liquor in the time of 9 days ,-20 degree are preserved.
The application of the nutrient solution that contains inductor nicotinamide and Conophylline in inducing mesenchymal stem cell breaks up to insulin-like cell.
The present invention carries out single stage method suspension induction, adopts novel plant inductor Conophylline and the differentiation of nicotinamide combined induction mesenchyma stem cell to pancreatic islet like cell.
Embodiment: the preparation of fat mesenchymal stem cell
The preparation of 1.1 fat mesenchymal stem cells
Under aseptic condition, get the byproduct of clinical operation or beauty and shaping---fatty tissue, in Biohazard Safety Equipment, fatty tissue is cleaned 2-3 time in PBS, thoroughly remove red corpuscle and fragment of tissue.Then the 0.1% NTx enzyme solution that adds preheating, puts 37 ℃ of water-bath concussion digestion 1h.The centrifugal 5min of 300g, removes upper strata adipocyte and collagenase solution.With PBS+1%BSA, clean bottom bulk precipitation, the centrifugal 5min of 300g, adds the appropriate DMEM re-suspended cell containing 10%FBS, through 100 μ m cell sieves, filters, and adjusts cell density and is seeded to Tissue Culture Flask, in 37 ℃, 5%CO
2in incubator, cultivate.After 48h, change liquid, remove not attached cell and residual red corpuscle.After this within every 3 days, change liquid.When cell reaches 80% fusion, the Trypsin-EDTA digestion attached cell with 0.25%, goes down to posterity by 1:2, observation of cell growth and morphological change characteristics under inverted microscope.Result shows, visible circular attached cell after primary hADSCs cultivation 24h, and 48h cell presents spindle shape, polygon, 72h cell quantity obviously increases, dense arrangement (Figure 1A).Passage cell (P6) rate of propagation is very fast, in the 3rd day, starts quantity showed increased, at the bottom of an about 6-7 days cell covers with culturing bottle, is mediation shape and arranges (Figure 1B).
Adopt Alamar Blue method to measure hADSCs growth curve, get P3, P4, P5 is for logarithmic phase cell, and pancreas enzyme-EDTA digestion is counted, and adjusting 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 going back commercial weight formula: go back commercial weight %=117216 * (T570)-80586 * (T600)/155677 * C600)-14652 * (C570) draw growth curve, calculate population doubling time.Result shows, P3, P4, P5 are all S-type for cell growth curve, and 1-3 days cells, in growth resting stage, inoculates cell after 4-5 days and entered the logarithmic proliferation phase, and approximately 7 days cell arrival plateaus, population doubling time is 60h(Fig. 1 C).
The evaluation of 1.2 fat mesenchymal stem cells
1.2.1 flow cytometry
By P1 fat subsitutes stem cell, after Trypsin-EDTA digestion, adjusting cell concn is 10
6cells/ml, with the antibody of FITC and PE mark on ice lucifuge hatch 30min, with the PBS re-suspended cell containing 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 demonstration of flow cytometer detected result, human adipose-derived stem cell phenotype is CD44
+, CD105
+, CD29
+, CD49d
+, CD106
-, CD31
-, CD34
-, CD45
-, HLA-DR
-(Fig. 2).
1.2.2hADSCs Multidirectional Differentiation ability
Get P4 and be incubated at in fat inducing culture and osteogenic induction substratum for hADSCs, observe its metamorphosis, and the cell after induction differentiation is carried out respectively to oil red O stain and Alizarin red staining.Become cellular form generation considerable change in fat Induction Process, cell becomes round gradually by spindle shape.Under 2-3 days inverted microscopes, in visible born of the same parents, occur that circular fat drips, drip quantity be on the increase with induction number of days increase fat, induce after 14 days, in 80% cell, be full of fat and drip, nucleus is pressed against edge, oil red O stain positive (Fig. 3 A).Immunocytochemistry experiment shows specific proteins Leptin(Fig. 3 C of the cell expressing adipocyte after induction), through RT-PCR, analyze, cell expressing LPL, AP2 gene after induction, and in hADSCs, AP2 has faint expression, after becoming fat induction, (Fig. 3 E) raised in genetic expression.
Change after skeletonization inducing culture, Growth of Cells is quick, presents flakey, and connects in flakes, forms colony, and cell is Multi layer Growth subsequently, and calcium deposition increases, and after approximately 21 days, under mirror, can be observed calcium tubercle, Alizarin red staining positive (Fig. 3 B).Immunocytochemistry result shows the cell expressing Osteopontin albumen (Fig. 3 D) after induction, PT-PCR is detected as osteocyte specific gene AKP, Osteopontin, Osteonectin, after induction, cell all has expression, and the hADSCs of induction 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 are divided into insulin-like cell group and adopt the single stage method induction that suspends, by P5 for hADSCs with 1.5-2 * 10
5cells/ hole is inoculated in the 6 ultralow absorption culture plates in hole, nutrient solution composition: 97% DMEM in high glucose 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, within every 3 days, change liquid, induce 9 days.Described concentration is volume by volume concentration.
The evaluation of insulin-like cell after 2.2 induction differentiation
2.2.1 dithizone staining reaction
Get the insulin-like cell group of induction after 9 days and carry out dithizone dyeing, the painted situation of observation of cell.Result demonstration induction group cellular form is compared generation considerable change with control group.After bed board, cell is individual cells suspension growth, and in whole Induction Process, cell is constantly assembled formation cell mass, induces after 9 days 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 Immuncytochemical detection
Insulin-like cell group is transferred to the cover glass overnight incubation that is coated with poly-lysine, makes cell mass adherent.Fixing 30min under 4% paraformaldehyde room temperature, PBS adds 0.5%Triton X-100 to penetrate 15min after cleaning.With 4%PBS-BSA, seal 30min (RT), prevent non-specific binding.Add primary antibodie, 4 ℃ are spent the night, and PBS adds two to resist after cleaning, hatch 30min for 37 ℃.Then hatch 15min with 5 μ g/mlDAPI37 ℃, primary antibodie: mouse-anti people Leptin (1:300) wherein, the anti-human Osteopontin of rabbit (1:200), mouse-anti people Insulin (1:300), the anti-human C-peptide of rabbit (1:500), two is anti-: sheep anti-mouse igg, IgM, Alexafluor488 (1:800), goat anti-rabbit igg, Cy3 (1:500).
Suspend after induction, the expression of the functional protein Insulin of Immuncytochemical detection insulin-like cell group, C-peptide, in visible cell group, there is Insulin, C-peptide positive cell, show to have produced the cell mass (Fig. 5) with insulin secretion function after induction differentiation.
2.2.3RT-PCR gene expression analysis
Application TRIzol reagent extracts hADSCs, and cell total rna after induction is got the synthetic cDNA of reverse transcription test kit for 1 μ gRNA.PCR reaction system is as follows: 94 ℃ of denaturation 5min, and 94 ℃ of 30s, 55-60 ℃ of 30s, 72 ℃ of 30s, 72 ℃ are extended 10min, and 30 circulations, get pcr amplification product, 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 transcription regulaton factor important in pancreas islet growth course, islet cell aysmature syndrome, propagation and differentiation are had to vital role, after induction, this gene expression amount raises, and insulin promoter factor Ipf-1 cells after induction only, marker gene Insulin, somatostatin, the glucagon of ripe islet cells all can detect in cell after induction, and not breaking up somatostatin in hADSCs has faint expression (Fig. 6).
The experiment 2.2.4 glucose stimulates insulin secretion
Approximately 200 inductions of picking cell mass (50-150 μ m) of 9 days, to 1.5ml centrifuge tube, cleans 2 times with PBS.Add 1ml sugar-free DMEM preculture 3-6h, then with 300ul, containing the DMEM of 5.6mmol/L glucose, 16.7mmol/L glucose, cultivate successively 2h, 37 degree.Collect supernatant liquor, by ELISA method, detect the secretory volume of the lower Regular Insulin of different glucose stimulation in supernatant, in cellular control unit supernatant, almost can't detect Regular Insulin, not reflection in the drawings, and cell mass after induction has a small amount of secretion under 5.56mmol/L glucose stimulates, amount of insulin secretion obviously raise (P < 0.001) after 25mmol/L glucose incubation 2h, be about lower 2.5 times of low sugar condition, from result, after induction, insulin-like cell group stimulates responsive to glucose, the secretion of its Regular Insulin is subject to the adjusting (Fig. 7) of external environment sugar.
Induce after 9 days, collect 5 groups of cell conditioned medium liquid, by ELISA method, detect the secretory volume of Regular Insulin in supernatant, result show NIC, CNP, ACT separately when use and NIC and ACT combined induction amount of insulin secretion there is no notable difference (P > 0.05), and nicotinamide and Conophylline combined induction have significantly improved to insulin-like cell and have rolled into a ball the ability (P < 0.05) of breaking up (Fig. 8)
In sum, and present method has been used Conophylline at human adipose-derived stem cell first in insulin-like cell induction, and Conophylline and the promoter action of nicotinamide combined induction to differentiation efficiency have been observed, result shows, cell reaches peak value in the secretory volume of induction Regular Insulin after 9 days, having shortened induction duration, improved induction differentiation efficiency, is that a kind of more effective induction human adipose-derived stem cell is to the method for insulin-like cell differentiation.
Content of the present invention is not limited in the above-described embodiment, and the knowledgeable people in same area can propose easily other embodiment within technical director's thought of the present invention, but this embodiment comprises within the scope of the present invention.
Claims (6)
1. a nutrient solution for induced lipolysis mesenchyma stem cell to pancreatic islet like cell differentiation, is characterized in that, comprises nicotinamide, Conophylline, cell growth factor, cell regulin and basic medium; In every liter of basic medium, contain:
2. the nutrient solution of induced lipolysis mesenchyma stem cell to pancreatic islet like cell differentiation according to claim 1, is characterized in that, described cell growth factor is HGF, bFGF or EGF.
3. the nutrient solution of induced lipolysis mesenchyma stem cell to pancreatic islet like cell differentiation according to claim 1, is characterized in that, described cell regulin is Betacellulin.
4. the nutrient solution of induced lipolysis mesenchyma stem cell to pancreatic islet like cell differentiation according to claim 1, is characterized in that, described basic medium contains 97% DMEM in high glucose, 2% B-27,1% N-2, and described concentration is volume by volume concentration.
5. a method for induced lipolysis mesenchyma stem cell to pancreatic islet like cell differentiation, is characterized in that, comprises the following steps:
(1) preparation inducing culture liquid: take DMEM in high glucose as basic medium, the B-27 of interpolation 2%, 1% N-2 and inductor nicotinamide, Conophylline, HGF, Betacellulin, mix, and 4 degree are preserved, and described concentration is volume by volume concentration;
(2) prepare fatty human mesenchymal stem cell;
(3) get fatty human mesenchymal stem cell, with 1.5-2 * 10
5cells/ hole is inoculated in the 6 ultralow absorption culture plates in hole, and 3ml inducing culture is added in every hole, and induction suspends;
(4) within every 3 days, change liquid, collecting cell supernatant liquor in the time of 9 days ,-20 degree are preserved.
6. a nutrient solution that contains inductor nicotinamide and the Conophylline application in the differentiation of induced lipolysis mesenchyma stem cell to pancreatic islet like cell.
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