[体外转染人脐带间充质干细胞后合成的增强绿色荧光蛋白信使核糖核酸的稳定性]
[The stability of synthesized EGFP mRNA in vitro transfected into human umbilical cord mesenchymal stem cells].
作者信息
Wang Xiaoli, Hu Pei, Liu Zhixiang, Yuan Yahong, Li Dongsheng
机构信息
Hubei Key Laboratory of Embryonic Stem Cell Research, Taihe Hospital, Hubei University of Medicine, Shiyan 442000, China.
出版信息
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2013 Oct;29(10):1094-7.
OBJECTIVE
To identify human umbilical cord mesenchymal stem cells (hUCMSCs) and increase the stability of the in vitro synthesized EGFP mRNA through modification.
METHODS
Immunophenotype of hUCMSCs was examined by flow cytometry. Adipogenic and osteogenic differentiations were determined by oil red O and alkaline phosphatase staining, respectively. In vitro synthesized EGFP mRNA was modified through polyA tailing, capping and adding base analogues, and then transfected into the hUCMSCs. After transfection, the fluorescence intensity was detected by flow cytometry and cell viability was determined by Trypan blue staining.
RESULTS
Flow cytometry revealed that the hUCMSCs were positive for CD29, CD44, CD105, and were negative for CD34, CD45 and HLA-DR. They had the capacity of differentiating into adipocytes and osteoblasts. Through the modification the in vitro synthesized mRNA was more stable than unmodified mRNA, and mRNA transfection had lower cytotoxicity than DNA.
CONCLUSION
Modification can highly improve the stability of in vitro synthesized mRNA, which can be translated into protein in hUCMSCs.
目的
鉴定人脐带间充质干细胞（hUCMSCs），并通过修饰提高体外合成的增强绿色荧光蛋白（EGFP）mRNA的稳定性。
方法
采用流式细胞术检测hUCMSCs的免疫表型。分别通过油红O染色和碱性磷酸酶染色检测成脂和成骨分化。对体外合成的EGFP mRNA进行聚腺苷酸化、加帽和添加碱基类似物修饰，然后转染到hUCMSCs中。转染后，通过流式细胞术检测荧光强度，通过台盼蓝染色测定细胞活力。
结果
流式细胞术显示，hUCMSCs对CD29、CD44、CD105呈阳性，对CD34、CD45和HLA-DR呈阴性。它们具有分化为脂肪细胞和成骨细胞的能力。通过修饰，体外合成的mRNA比未修饰的mRNA更稳定，且mRNA转染的细胞毒性低于DNA。
结论
修饰可显著提高体外合成mRNA的稳定性，其可在hUCMSCs中翻译为蛋白质。
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