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目的开发高通量的细胞电融合平台,并通过此平台将小鼠胚胎干细胞(mouse embryonic stem cells,mESCs)与体细胞融合,探讨融合细胞的多能性。方法本实验室自主研发微流控芯片,将转染了绿色荧光的mESCs与转染了红色荧光的NIH3T3细胞进行电融合,并计算其排队率与融合率。通过流式细胞仪筛选出表达两种荧光的融合细胞,观察融合细胞的表型。RT-PCR检测NIH3T3、mESC以及融合细胞的多能性基因(Nanog、OCT4、SOX2和LIN28)mRNA的表达水平。结果自主研发了微流控芯片,构建了高通量的细胞电融合平台,通过此平台将mESCs与NIH3T3进行电融合,其排队率为(44.35±10.99)%,融合率为(59.88±20.03)%,融合细胞可形成类似于ESC样的克隆。RT-PCR法结果显示mESC和融合细胞均表达多能性基因Nanog、OCT4、SOX2和LIN28 mRNA,而NIH3T3不表达。结论通过自主研发的微流控芯片,可实现mESCs与NIH3T3的高效融合,使体细胞重编程为多能性干细胞。
Objective To develop a high-throughput cellular electrofusion platform and to fuse mouse embryonic stem cells (mESCs) with somatic cells to explore the pluripotency of fusion cells. Methods The microfluidic chip was independently developed in our laboratory. The mESCs transfected with green fluorescence and the NIH3T3 cells transfected with red fluorescence were electrofused, and the queuing rate and fusion rate were calculated. The fusion cells expressing the two kinds of fluorescence were screened by flow cytometry to observe the phenotype of the fusion cells. The mRNA expression levels of pluripotency genes (Nanog, OCT4, SOX2 and LIN28) of NIH3T3, mESC and fusion cells were detected by RT-PCR. Results The microfluidic chip was independently developed and a high-throughput cell electrofusion platform was constructed. The mESCs and NIH3T3 were electrofused by this platform, with the queuing rate of (44.35 ± 10.99)% and the fusion rate of (59.88 ± 20.03) %, Fused cells can form a clone similar to ESC-like. RT-PCR results showed that both mESC and fusion cells expressed pluripotency genes Nanog, OCT4, SOX2 and LIN28 mRNA, while NIH3T3 did not express. Conclusion The self-developed microfluidic chip can efficiently fuse mESCs with NIH3T3 and reprogram somatic cells into pluripotent stem cells.