目的:观察野生型鼠和膜1型基质金属蛋白酶(MT1-MMP)基因敲除鼠的成纤维细胞重新编程为诱导多能干细胞(iPSCs)后,其细胞特性是否具有胚胎干细胞(ESCs)相似的潜能.方法:利用逆转录病毒介导Oct3/4、Sox2、c-Myc和Klf4四种基因,转染到野生型鼠和MT1-MMP基因敲除鼠的成纤维细胞中.iPSCs克隆形成后,用免疫细胞化学检测ESCs特异性标志的表达情况.体外将iPSCs通过"悬滴法"向内皮细胞和心肌细胞分化,以检测其分化能力.结果:转染野生型鼠和MT1-MMP基因敲除鼠的成纤维细胞2周后,可见ESCs样克隆开始形成.iPSCs明显表达ESCs特异性标志物碱性磷酸酶(AP)、阶段特异性胚胎抗原-1(SSEA-1)和八聚体结合转录因子3/4(OCT3/4).诱导分化的内皮细胞表达早期内皮标志物Flk-1/KDR;诱导分化的心肌细胞出现跳动,表达心肌标志物肌钙蛋白I.结论:野生型鼠和MT1-MMP基因敲除鼠的成纤维细胞可被重新编程为iPSCs,iPSCs具有ESCs的特征及增殖分化能力,因此可能为再生医学的研究和临床上进行细胞移植治疗提供理想的种子细胞来源.%AIM: To determine the difference of biological potency between induced pluripotent stem cells ( iP-SCs ) and embryonic stem cells ( ESCs ) by investigating the induction of reprogramming of wild type ( WT ) and membrane - type 1 matrix metalloproteinase ( MT1 - MMP ) - deficient mouse fibroblasts into iPSCs in vitro. METHODS: WT and MT1 - MMP - deficient mouse fibroblasts were reprogrammed to iPSCs by retroviral introduction of the 4 transcription factors Oct3/4, Sox2, c - Myc and Klf4. The expression of ESC - specific markers alkaline phosphatase ( AP ), stage specific embryonic antigen -1 ( SSEA - 1 ) and Oct3/4 was detected in iPSCs by immunocytochemistry. The method of " hanging drop" was applied to differentiate iPSCs into endothelial cells ( Ecs ) and cardiomyocytes for determining the differentiation potency of the cells. RESULTS: The formation of iPSCs colonies became visible approximately 2 weeks after the retroviral infection. The expression of ESC - specific markers AP, SSEA - 1 and Oct3/4 in iPSCs was obviously observed. The Ecs, which differentiated from iPSCs, expressed early endothelial marker Flk - 1/KDR. The expression of cardiomyocyte - typical marker cardiac troponin I in the cardiomyocytes differentiated from iPSCs was also detected by immunocytochemical staining when the beating cells were growing. CONCLUSION: WT and MT1 - MMP - deficient mouse fibroblasts can be re-programmed to iPSCs. The morphology, proliferation and multilineage differentiation potency of iPSCs are indistinguishable from those of ESCs, indicating that iPSCs will become optimal source of seed cells for regenerative medicine and specific cell therapy.
展开▼