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| Dynamic Change of Lepus brachyurus Bone Marrow Mesenchymal Stem Cells (BMSCs) Induced and Differentiated into Islet Cells In vitro |
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Abstract The aim of this study was to explore the feasibility of rabbit bone marrow mesenchymal stem cells (BMSCs) into islet cells induced by growth factors in three phase induction method in vitro, and observe their dynamic change. BMSCs were obtained by red blood cell lysis, the CD90, CD44 and CD34 antigen expression were detected by immunocytochemistry technique. Growth factors three-step strategy was adopted to induce BMSCs to differentiate toward islet cells. The growth of BMSCs were observed under inverted microscope. The differentiated cells were stained with dithizone (DTZ). The express of inslet cells related gene(Pdx-1, Insulin, Nkx6.1) was detected by RT-PCR, and the expression of specific protein like Insulin in derived cells was investigated by immunocytochemistry technique. Insulin secretion was assayed by enzyme linked immunosorbent assay (ELISA). The result showed that the isolated BMSCs were shuttle or gathered into a vortex-like uniform growth, and positive for CD90、CD44 antigen, negative for CD34. At the end of the first-step strategy (3 d), the cell morphology had no obvious change, and part of cells become round, DTZ staining negative, any specific gene mRNA expression was detected. After inducted 7 d, BMSCs gradually merged to form cluster, DTZ staining positive, only Pdx-1 mRNA expression was observed, which was verified as insulin progenitor cells. At the end of second-step strategy (11 d), the number of differentiated cells cluster was incteased, RT-PCR showed that Pdx-1 and Insulin were expressed. After inducted 19 d, cell clusters looked like islet-like structure in terminal, DTZ staining positive and detected Pdx-1, Insulin and Nkx6.1 expression. Immunocytocheemistry analysis showed expression of Pdx-1 in the islet-like clusters. A lot of dark brown particles appeared in the cytoplasm of cells in the experimental group, the results showed positive reaction. The control group did not appear brown particles in the cytoplasm of cells, the results showed negative reaction. Insulin content in the culture supernatant detected by ELISA showed that there was no significant difference between the induced group and the control group (P＞0.05) on the 3rd day because there was no cell clusters like changes and insulin secretion, in addition, there was a significant difference on the 7th day, 11th day and 19th day(P＜0.05). Inducing BMSCs secrete insulin at the 3rd、7th、11th、15th、19th days, and the difference was significant (P＜0.05) between BMSCs and induced groups except the 3rd days. It illustrated that isolated and purified BMSCs in vitro could be inducted to differentiate into fuctional mature islet cells. The study provides basic data for transplanting autologous BMSCs to cure diabetes for diabetics.
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Received: 28 January 2015
Published: 01 June 2015
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