多能性转录因子mRNA转染山羊胎儿成纤维细胞条件优化

摘要

Objective]To optimize the conditions for transfecting pluripotent transcription factors oct4、sox2、klf4 and c-myc mRNA to GEF cells, this study focused on transfection reagents and transfection methods.[Method]GEF cells were separated from goat fetus leg muscle tissue via trypsin digestion method, and transcription factors then purified Using plasmid mini kits obtained from bacteriums. Then each downstream of the target genes in transcription carrier was extracted with a mono-restriction endonuclease enzyme. DNA purification kits were used to purify all the linear plasmids. Based on the linear plasmid, 5' end cap structure of mRNA was synthesised. TURBO DNase was used to eliminate the DNA template and the mMESSAGE mMACHINE® T7 Ultra Kit was use to add "tail" to the mRNA. Finally, MEGAclear™ Kit was used to purify the full mRNA. Five sheep derived recombinant plasmids: pCDNA3-oct4、pCDNA3-sox2、pCDNA3-klf4、pCDNA3-c-myc and pCDNA3-EGFP were linearized and then transcribed into mRNA in vitro respectively. GEF cells were transfected with different proportions of total mRNA (each of the five transcription factors mRNA was 0.2g). We used Lipofectamine 2000 by ratios of 1׃0.5、1׃1.0、1׃1.5 and 1׃2.0 to get the best transfection proportion. Using the best transfection proportions, transfection efficiencies were compared within the 2, 4, 6 generations of GEF cells. Immunofluorescence assay was performed to verify the transfection effect.[Result] The best transfection proportion was 1:1.0 (total mRNA/ Lipofectamine 2000), and the transfection efficiency of 2 generation was significantly higher than other generations of GEF cells under the best transfection proportion(P<0.05).This group of cells was good in both morphology and cell growth. Immunofluorescence assays showed that four pluripotent transcription factors mRNA were located in the nucleus of GEF cells, and obtained stable expression, while there was no expression of these four pluripotent transcription factors in the cytoplasm of GEF cells. After 24h of transfection by 4 pluripotent transcription factors, GEF cells turned slowly from fusiform to circular, and the cell activity was lower than the control group (normal GEF cells).[Conclusion] The best transfection condition for transfecting pluripotent transcription factors mRNA to GEF cells was at the 2 generation of GEF cells and under the 1:1.0 transfection proportion of total mRNA/ Lipofectamine 2000. This study provides proper parameters for transfecting cells or ES by mRNA , and also for obtaining iPS cells. We find indirect evidence for exogenous pluripotent mRNA in GEF cells and lay the foundation for further study of pluripotent transcription factors oct4、sox2、klf4 and c-myc.%【目的】为了优化外源多能性转录因子oct4、sox2、klf4、c-myc的mRNA转入山羊胎儿成纤维细胞（goat embryonic fibroblast，GEF）的条件，本试验针对转染试剂脂质体2000和转染方法进行研究。【方法】采用胰酶消化法，从山羊胎儿腿部肌肉组织中分离并获得了纯化的GEF细胞。根据mMESSAGE mMACHINE® T7 Ultra Kit试剂盒说明书，体外转录步骤主要有：①前期的质粒准备：首先用质粒小提试剂盒对摇菌获得的各体外转录载体质粒进行提取，随后对各体外转录载体目的基因下游进行单酶切，获得线性化质粒，最后用DNA纯化试剂盒对线性化的质粒进行纯化；②mRNA的“加帽”：以线性化DNA为模板，合成带有5′端帽子结构的mRNA，添加TURBO DNase以除去模板DNA；③mRNA的“加尾”：以ATP为原料，利用试剂盒自带的Poly(A)聚合酶，对已经“加帽”的mRNA进行“加尾”，以合成结构完整的mRNA；④完整mRNA的纯化：按照MEGAclearTM Kit试剂盒对mRNA进行纯化。随后，将体外转录获得各转录因子的mRNA进行浓度和OD值测定。分别比较总mRNA（EGFP和4种多能性转录因子的mRNA的质量分别为0.2µg）和脂质体2000的比例为1׃0.5、1׃1.0、1׃1.5、1׃2.0以及在最佳总mRNA与脂质体2000比例体系下第2、4、6代GEF细胞的mRNA转染效率。对最佳转染体系下转染多能性转录因子mRNA的GEF细胞进行免疫荧光检测，并对转染多能性转录因子mRNA后GEF细胞的形态和数目进行观察。【结果】mRNA和脂质体2000的比例为1׃1.0以及在最佳总mRNA与脂质体2000比例体系下第2代GEF细胞的mRNA转染效率均显著高于其他组（P＜0.05），且本组细胞在形态和生长方面相对较好；免疫荧光检测表明，4种多能性转录因子的mRNA在GEF细胞中定位于细胞核，并获得稳定表达，在细胞质中不见多能性转录因子oct4、sox2、klf4、c-myc的表达；GEF细胞在转染4种mRNA 24h后，细胞形态由梭状慢慢向圆形靠拢；细胞活力低于对照组（山羊成纤维细胞正常生长组）。【结论】GEF细胞在总mRNA和脂质体2000的比例为1׃1.0以及在最佳总mRNA与脂质体2000比例体系下第2代GEF的条件下更适合mRNA的转染。研究工作为mRNA转染体细胞或干细胞的研究及诱导性多能干细胞（induced pluripotent stem cells，iPS）的获得提供参考资料，并对mRNA在成纤维细胞中的去向提供间接证据，有利于更加深入地了解多能性转录因子oct4、sox2、klf4、c-myc的mRNA相关功能。