CN102391982A - Method for in vitro induction of human amniotic mesenchymal stem cells (hAMSCs) differentiated into insulin-secreting cells (ISCs) - Google Patents
Method for in vitro induction of human amniotic mesenchymal stem cells (hAMSCs) differentiated into insulin-secreting cells (ISCs) Download PDFInfo
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- CN102391982A CN102391982A CN2011104045811A CN201110404581A CN102391982A CN 102391982 A CN102391982 A CN 102391982A CN 2011104045811 A CN2011104045811 A CN 2011104045811A CN 201110404581 A CN201110404581 A CN 201110404581A CN 102391982 A CN102391982 A CN 102391982A
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Abstract
The invention discloses a method for the in vitro induction of hAMSCs differentiated into ISCs. According to the method, the extracted hAMSCs are cultured for 7 days by a serum-free high sugar-DMEM induction medium containing niacinamide of 10mmol/L and an N2 supplement. The hAMSCs induced by the method of the invention express insulin gene mRNA and PDX-1 gene mRNA, and the content of insulin in the supernatant of the culture reaches above 330muIU/ml; and the method of the invention allows the in vitro induction of the hAMSCs differentiated into the ISCs to be realized.
Description
Technical field
The present invention relates to the method that a kind of external evoked human amnion mesenchymal stem cell is divided into insulin secretory cell.
Background technology
Human amnion mesenchymal stem cell (human amniotic mesenchymal stem cells; HAMSCs) have that polyphyly differentiation characteristic, reduced immunogenicity, source enrich, are easy to enrichment, the amplification ability by force, does not involve advantages such as medical ethics problems, in regenerative medicine, be with a wide range of applications.Proved already that hAMSCs can be divided into endothelium and liver epithelial like cell, myocardial cell's like cell, neuron cell, scleroblast etc. under suitable inductive condition, but can its B cells of pancreas that be divided into excreting insulin do not see relevant introduction so far.
The generation of IDDM is relevant with inherited genetic factors, environmental factors and immunologic mechanism, and its basic etiology and pathogenesis is that B cells of pancreas destruction causes hypoinsulinism.The IDDM patient must rely on insulinize throughout one's life and just can earn a bare living.Although the Perfected process that is expected to become the treatment IDDM is transplanted in pancreas or pancreatic islets transplantation, organize problems such as confession source, immunity rejection, technical difficulty to receive a lot of restriction because of it.HAMSCs has the polyphyly differentiation capability and exempts characteristic with immunity, and (insulin secreting cells ISCs) is worth for the treatment IDDM provides new donorcells to have important use undoubtedly to induce it to be divided into insulin secretory cell.
Summary of the invention
The technical problem that the present invention solves isOvercome the problem that prior art exists, the present invention provides a kind of hAMSCs of making to be divided into the induction method of ISCs.
The technical scheme that the present invention adopts: a kind of external evoked human amnion mesenchymal stem cell is divided into the method for insulin secretory cell, may further comprise the steps:
(1) separate hAMSCs: people's amnion is cut into fragment, adds the 0.05% tryptic digestion solution that contains 0.02%EDTA, in 37 ° of C, 200 rev/mins of rotations digested 10 minutes, abandoned supernatant; Again add Digestive system, in 37 ° of C, 200 rev/mins of rotations digested 30 minutes, abandoned supernatant; Leave and take indigested amnion fragment, use D-Hank ' s liquid flushing secondary, add the collagenase of 0.75 mg/ml that contains 0.075 mg/ml DNase I; In 37 ° of C, 200 rev/mins of about 2 h of rotation digestion are to organizing complete digestion, and 300 order steel meshes filter; Collecting cell filtrating, 1500 rev/mins centrifugal 10 minutes, the collecting cell deposition is original hAMSCs;
(2) purifying hAMSCs: isolating original hAMSCs is suspended in the LG-DMEM substratum again (contains 10% FBS; 2 mmol/L L-glutaminate; 1% non-essential amino acid, 55 μ mol/L 2 mercapto ethanols, 1 mmol/L Sodium.alpha.-ketopropionate; 100 U/ml penicillium mould and 100 mg/ml Streptomycin sulphates), with 1.25 * 10
5The cell density of/ml is inoculated in 6 well culture plates, and placing 37 ° of C, saturated humidity, volume(tric)fraction is 5% CO
2, CO
2Incubator was cultivated 36～48 hours, under inverted microscope, removed fully the not amniotic epithelial cells of adherent growth.The substratum that more renewed in the 3rd day; After treating that cell degree of converging reaches 80 ~ 90%, in 37 ° of C digestion 2 ~ 3 minutes, add substratum and stop trypsin acting with 0.25% trypsinase-0.02% EDTA solution; 1000 rev/mins, centrifugal 5 minutes, abandon supernatant; Cell precipitation suspends with substratum again, with 1 * 10
7The cell density of/L goes down to posterity;
(3) identify hAMSCs: get capable vimentin of the 2nd generation hAMSCs and CK19 immunohistochemical staining, hAMSCs expresses vimentin and does not express CK19; Detect CD29, CD44, CD166, CD34 and CD45 with flow cytometer, the phenotypic characteristic of hAMSCs is for being CD29
+CD44
+CD166
+CD34
-CD45
-
(4) induce hAMSCs to break up to ISCs: inductive differentiation medium is for containing 10mmol/L nicotinamide and N
2The serum-free HG-DMEM substratum of fill-in; Get the 2nd generation hAMSCs and suspend, with 2.5 * 10 with inductive differentiation medium
6Individual/ml cell density is inoculated in 6 well culture plates, and placing 37 ℃, saturated humidity, volume(tric)fraction is 5%CO
2Cultivated in the incubator 7 days, changed liquid 1 time with inductive differentiation medium on the 3rd day;
(5) evaluation of ISCs: 1. the hAMSCs inducing culture got culture supernatants after 7 days, adopted radioimmunology to detect insulin content; 2. collect the part cell; Adopt reverse-transcription polymerase chain reaction (PT-PCR) to detect insulin gene mRNA and the special-shaped box factor-1 of key transcription factor pancreas duodenum homology (pancreatic and duodenal homeobox factor-1, the PDX-1) expression of gene mRNA of regulating pancreas growth and B cell differentiation; HAMSCs promptly was divided into ISCs in 7 days through its inducing culture of above-mentioned inducing culture.
The beneficial effect that the present invention reaches: hAMSCs promptly was divided into ISCs in 7 days through above-mentioned inducing culture, and cell has insulin gene and PDX-1 genetic expression, and the insulin content in the culture supernatant reaches more than the 330 μ IU/ml; Both do not had the expression of pancreas islet plain gene mRNA without inductive hAMSCs, detected yet, explained that the inventive method can be divided into ISCs at external evoked hAMSCs less than Regular Insulin.The ISCs that produces through the inventive method and contain the ISCs compsn and can be used for the IDDM IDDM.Gordian technique of the present invention is to remove in the substratum to add nicotinamide and N
2Outside the fill-in, the high sugared environment of keeping cell growth is important to inducing hAMSCs to be divided into ISCs ten minutes.
Embodiment
Embodiment
A kind of external evoked human amnion mesenchymal stem cell of the present invention is divided into the method for insulin secretory cell, may further comprise the steps:
(1) separate hAMSCs: people's amnion is cut into fragment, adds the 0.05% tryptic digestion solution that contains 0.02%EDTA, in 37 ° of C, 200 rev/mins of rotations digested 10 minutes, abandoned supernatant; Again add Digestive system, in 37 ° of C, 200 rev/mins of rotations digested 30 minutes, abandoned supernatant; Leave and take indigested amnion fragment, use D-Hank ' s liquid flushing secondary, add the collagenase of 0.75 mg/ml that contains 0.075 mg/ml DNase I; In 37 ° of C, 200 rev/mins of about 2 h of rotation digestion are to organizing complete digestion, and 300 order steel meshes filter; Collecting cell filtrating, 1500 rev/mins centrifugal 10 minutes, the collecting cell deposition is original hAMSCs;
(2) purifying hAMSCs: with isolating original hAMSCs be suspended in again low sugar-DMEM substratum (available from Gibco company, article No.: 31600-026; Contain 1000mg/L glucose, 10% foetal calf serum, 100 U/ml penicillium mould and 100 mg/ml Streptomycin sulphates), with 1.25 * 10
5The cell density of/ml is inoculated in 6 well culture plates, and placing 37 ° of C, saturated humidity, volume(tric)fraction is 5% CO
2, CO
2Incubator was cultivated 36～48 hours, under inverted microscope, removed fully the not amniotic epithelial cells of adherent growth.The substratum that more renewed in the 3rd day; After treating that cell degree of converging reaches 80 ~ 90%, in 37 ° of C digestion 2 ~ 3 minutes, add substratum and stop trypsin acting with 0.25% trypsinase-0.02% EDTA solution; 1000 rev/mins, centrifugal 5 minutes, abandon supernatant; Cell precipitation suspends with substratum again, with 1 * 10
7The cell density of/L goes down to posterity;
(3) identify hAMSCs: get capable vimentin of the 2nd generation hAMSCs and CK19 immunohistochemical staining, hAMSCs expresses vimentin and does not express CK19; Detect CD29, CD44, CD166, CD34 and CD45 with flow cytometer, the phenotypic characteristic of hAMSCs is for being CD29
+CD44
+CD166
+CD34
-CD45
-
(4) induce hAMSCs to break up to ISCs: inductive differentiation medium is for containing 10mmol/L nicotinamide and N
2Fill-in (available from Gibco company, article No.: serum-free HG-DMEM substratum 17502048) (available from Gibco company, article No.: 12800-017; Glucose content is 4500mg/L).Get the 2nd generation hAMSCs and suspend, with 2.5 * 10 with inductive differentiation medium
6Individual/ml cell density is inoculated in 6 well culture plates, and placing 37 ℃, saturated humidity, volume(tric)fraction is 5%CO
2Cultivated in the incubator 7 days, changed liquid 1 time with inductive differentiation medium on the 3rd day; HAMSCs promptly was divided into ISCs in 7 days through its inducing culture of above-mentioned inducing culture.
(5) evaluation of ISCs: 1. the hAMSCs inducing culture got culture supernatants after 7 days, and adopting radioimmunology to detect insulin content is 331.6 μ IU/ml; 2. collect the part cell; Adopt reverse-transcription polymerase chain reaction (PT-PCR) to detect insulin gene mRNA and the special-shaped box factor-1 of key transcription factor pancreas duodenum homology (pancreatic and duodenal homeobox factor-1, the PDX-1) expression of gene mRNA of regulating pancreas growth and B cell differentiation.
Claims (1)
1. an external evoked human amnion mesenchymal stem cell is divided into the method for insulin secretory cell, it is characterized in that: may further comprise the steps:
(1) separate hAMSCs: people's amnion is cut into fragment, adds the 0.05% tryptic digestion solution that contains 0.02%EDTA, supernatant is abandoned in rotation digestion; Again add Digestive system, supernatant is abandoned in rotation digestion, leaves and takes indigested amnion fragment; Use the flushing of D-Hank ' s liquid, add the collagenase of 0.75 mg/ml that contains 0.075 mg/ml DNase I, rotation digestion is to organizing complete digestion; 300 order steel meshes filter; Collecting cell filtrating, centrifugal back collecting cell deposition promptly gets original hAMSCs;
(2) purifying hAMSCs: isolating original hAMSCs is suspended in the LG-DMEM substratum, with 1.25 * 10 again
5The cell density of/ml is inoculated in 6 well culture plates, and placing 37 ° of C, saturated humidity, volume(tric)fraction is 5% CO
2, CO
2Incubator is cultivated, and under inverted microscope, removes fully the not amniotic epithelial cells of adherent growth, the substratum that more renewed in the 3rd day; After treating that cell degree of converging reaches 80 ~ 90%; With 0.25% trypsinase-0.02% EDTA solution digestion 2 ~ 3 minutes, add substratum and stop trypsin acting, the 10 centrifugal supernatants of abandoning; Cell precipitation suspends with substratum again, with 1 * 10
7The cell density of/L goes down to posterity;
(3) identify hAMSCs: get capable vimentin of the 2nd generation hAMSCs and CK19 immunohistochemical staining; Detect CD29, CD44, CD166, CD34 and CD45 with flow cytometer;
(4) induce hAMSCs to break up to ISCs: inductive differentiation medium is for containing 10mmol/L nicotinamide and N
2The serum-free HG-DMEM substratum of fill-in; Get the 2nd generation hAMSCs and suspend, with 2.5 * 10 with inductive differentiation medium
6Individual/ml cell density is inoculated in 6 well culture plates, and placing 37 ℃, saturated humidity, volume(tric)fraction is 5%CO
2Cultivated in the incubator 7 days, changed liquid 1 time with inductive differentiation medium on the 3rd day; HAMSCs promptly was divided into ISCs in 7 days through above-mentioned its inducing culture of differentiation culture of inducing;
(5) evaluation of ISCs: 1. the hAMSCs inducing culture got culture supernatants after 7 days, adopted radioimmunology to detect insulin content; 2. collect the part cell, adopt reverse-transcription polymerase chain reaction (PT-PCR) to detect the expression of insulin gene mRNA and the special-shaped box factor-1 of the key transcription factor pancreas duodenum homology gene mRNA of regulating pancreas growth and B cell differentiation.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105062952A (en) * | 2015-08-06 | 2015-11-18 | 深圳爱生再生医学科技有限公司 | Method for transformation of mesenchymal stem cells into islet cells |
| CN106635976A (en) * | 2016-11-08 | 2017-05-10 | 华南生物医药研究院 | Method and kit for acquiring amniotic mesenchymal stem cells |
| CN106834218A (en) * | 2017-01-06 | 2017-06-13 | 庞希宁 | People's amnioic epithelium stem cell serum-free culture medium and its cultural method |
| CN110423720A (en) * | 2019-07-23 | 2019-11-08 | 南昌大学 | A kind of amnioic epithelium stem cell is induced to differentiate into the method and its application of functional islets β cell |
| CN112063577B (en) * | 2020-08-14 | 2023-05-16 | 中国医科大学附属第一医院 | Combined culture medium for islet culture and preparation method thereof |
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| CN1536075A (en) * | 2003-04-09 | 2004-10-13 | 中国人民解放军军事医学科学院野战输 | Method for inducing bone marrow mesenchymal stem cells to differentiate into insulin-like cells |
| CN101186901A (en) * | 2007-10-17 | 2008-05-28 | 暨南大学 | Method for inducing differentiation of human embryo mesenchymal stem cells into pancreatic islet beta-like cell |
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| CN1536075A (en) * | 2003-04-09 | 2004-10-13 | 中国人民解放军军事医学科学院野战输 | Method for inducing bone marrow mesenchymal stem cells to differentiate into insulin-like cells |
| CN101186901A (en) * | 2007-10-17 | 2008-05-28 | 暨南大学 | Method for inducing differentiation of human embryo mesenchymal stem cells into pancreatic islet beta-like cell |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105062952A (en) * | 2015-08-06 | 2015-11-18 | 深圳爱生再生医学科技有限公司 | Method for transformation of mesenchymal stem cells into islet cells |
| CN106635976A (en) * | 2016-11-08 | 2017-05-10 | 华南生物医药研究院 | Method and kit for acquiring amniotic mesenchymal stem cells |
| CN106635976B (en) * | 2016-11-08 | 2020-04-28 | 华南生物医药研究院 | Method and kit for obtaining amniotic mesenchymal stem cells |
| CN106834218A (en) * | 2017-01-06 | 2017-06-13 | 庞希宁 | People's amnioic epithelium stem cell serum-free culture medium and its cultural method |
| CN110423720A (en) * | 2019-07-23 | 2019-11-08 | 南昌大学 | A kind of amnioic epithelium stem cell is induced to differentiate into the method and its application of functional islets β cell |
| CN112063577B (en) * | 2020-08-14 | 2023-05-16 | 中国医科大学附属第一医院 | Combined culture medium for islet culture and preparation method thereof |
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Application publication date: 20120328 |