| 摘要: |
| 【目的】研究牛诱导性多能干细胞（Bovine induced pluripotent stem cells,biPSCs）向心肌细胞的分化能力。【方法】将biPSCs悬浮培养制备类胚体，采用类胚体介导体外自由分化与定向诱导分化（添加RA、DMSO和RA+DMSO-3种诱导物）的方法，使biPSCs在体外分化，比较了biPSCs在3种不同心肌诱导条件下定向分化为心肌细胞的诱导效率；分别提取biPSCs、类胚体及不同诱导体系分化细胞的总RNA，用RT-PCR检测心肌细胞发育标志基因的表达。【结果】biPSCs在悬浮条件下培养7 d，能够形成典型球形和囊腔样类胚体。类胚体再贴壁培养7 d，经过免疫细胞化学检测，贴壁细胞大量分化为α-actin阳性的心肌样细胞。在体外诱导分化体系中，RA+DMSO共同诱导的效率较RA和DMSO单独诱导显著增高(P＜0.05)。RT-PCR结果显示，各组分化后的细胞中心肌细胞发育特异性基因ACTA2与GATA4均有高水平表达。【结论】biPSCs在体外悬浮培养能够形成类胚体，类胚体贴壁培养后可以分化为α-actin阳性的心肌样细胞；分化形成的心肌样细胞均表达心肌特异性基因。 |
| 关键词: 诱导性多能干细胞 细胞分化 心肌细胞 牛 |
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| 基金项目:国家自然科学基金项目(30671067)；陕西省重大科技项目(2006KZ05-G1) |
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| Induced differentiation of cardiomyocytes from bovine induced pluripotent stem cells |
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| Abstract: |
| 【Objective】The first evidence that bovine iPS cells could be induced to differentiate into cardiomyocytes by RA and DMSO in vitro was reported.【Method】The biPSCs were cultured using the hanging drop and suspension culture method for embryoid body (EBs) formation.Incubation of EBs with RA/DMSO and RA+DMSO was used to induce cardiac differentiation.The marker proteins of cardiomyocytes was examined by immunocytochemistry staining.The induction efficiency was compared in three different cardiac induced conditions.The marker genes of cardiomyocytes including GATA4 and ACTA2 were examined by reverse transcription polymerase chain reaction (RT-PCR).【Result】biPSCs could formate EBs after 7 days in suspension culture.A maximum level of spontaneously differentiation of cardiac cell clusters was observed within the outgrowths of embryoid bodies (EBs) after 7 days in adherent culture.Induction with both RA and DMSO resulted in a significantly higher elevation in differentiation rate than RA or DMSO alone (P＜0.05).RT-PCR confirmed that the differentiated cells derived from biPSCs could express cardiomyocytes marker genes GATA4 and ACTA2. 【Conclusion】The results revealed that the established biPSCs can effectively differentiate into cardiomyocyte like cells.Cardiomyocytes were evident as they expressed cardiac cell specific genes and protein markers including GATA4,ACTA2 and alpha actin when analyzed by PT-PCR and immunohistochemical staining. |
| Key words: induced pluripotent stem cells (iPSCs) differentiation potential cardiomyocytes bovine |
