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Studies on Mesenchymal Stem Cell Isolation, Culture and Induced Differentiation to Insulin-Secreting Cells in Vitro

Author: YangFen
Tutor: YangNaiLong
School: Qingdao University
Course: Internal Medicine
Keywords: Bone marrow mesenchymal stem cells Cell culture Induced differentiation Insulin-secreting cells Appraisal
CLC: R329
Type: Master's thesis
Year: 2008
Downloads: 66
Quote: 0
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Abstract


Objective To study the adult bone marrow mesenchymal stem cells (MSCs) isolated and cultured in vitro and the insulin-secreting cells induced to differentiate and identification methods , provide a source of seed cells for the treatment of diabetes . Methods were used whole bone marrow culture method and Percoll density gradient centrifugation isolated from adult bone marrow MSCs, cultured in vitro under the same conditions , to compare two methods for adherent the cloned cell number , cell morphology , cell surface markers and the fat cell differentiation . The whole bone marrow culture of MSCs vitro cultured and passaged take three to five generations of cells has to be 2 - mercaptoethanol , glutamine , epidermal growth factor ( EGF ) , basic fibroblast growth factor (bFGF) and Nick amide through three stages to the islet - like cells induced to differentiate . 3 phase contrast microscope MSCs in induced morphology before and after the changes identified by immunofluorescence pancreatic duodenal homeobox gene (PDX-1) expression , dithizone staining islet β -like cell clusters ECLIA Determination of the amount of insulin secretion . Results 1 whole bone marrow culture adherent cells obtained significantly more than the number of clones Percoll density gradient centrifugation ( P <0.05 ) . MSCs morphology obtained by the two methods was no significant difference of CD44 expression rate and expression of CD34 negative rate was no significant difference ( P > 0.05 ) , dexamethasone , insulin-induced differentiation into adipocytes can . The 2.MSCs by 2 - mercaptoethanol , nicotinamide induced cells gradually rounded cells expressing PDX- 1 - induced two-stage , three - stage formation induced islet-like cell clusters , dithizone staining positive for glucose-stimulated insulin release ; uninduced MSCs fusiform adherent growth , PDX-1 expression and and dithizone staining negative insulin release . Conclusion 1 . Percoll density gradient centrifugation , whole bone marrow culture method is more simple and practical separation of MSCs in vitro method one . 2.2 -mercaptoethanol , glutamine , EGF, bFGF and nicotinamide can be induced in vitro the adult MSCs differentiate into insulin-secreting cells .

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CLC: > Medicine, health > Basic Medical > Human morphology > Human histology
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