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逆转录病毒载体介导的人白细胞介素10在大鼠心肌细胞的表达 |
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李锦华 陈永雄 张仕光 廖晓星
摘 要 目的：建立一个在大鼠心肌细胞表达人白细胞介素10(Human Interleukin 10, hIL-10)的重组逆转录病毒载体基因转移系统，为下一步的研究工作做准备。方法：将构建的表达人白细胞介素10的逆转录病毒重组体pLX(hIL-10)SN经PA317细胞包装，G418筛选，NIH3T3细胞进行病毒滴度测定，选滴度最高的克隆(6×105CFU／ml)作为感染大鼠心肌细胞的感染细胞；用重组逆转录病毒感染大鼠心肌细胞，应用聚合酶链反应(PCR)及反转录聚合酶链反应(RT-PCR)检测转染大鼠心肌细胞DNA及mRNA，应用ELISA测定hIL-10在心肌细胞的表达。结果：外源性hIL-10基因已整合到心肌细胞染色体DNA并有效地转录和翻译，表达水平最高达1 755 ng／(106cells*24 h)。结论：外源性hIL-10基因可以转移到大鼠心肌细胞并稳定表达，为今后研究hIL-10的作用机制及在治疗疾病中的应用打下了基础。
关键词 基因转移 逆转录病毒载体 人白细胞介素10 大鼠心肌细胞
中国图书分类号 Q789
Expression of human interleukin 10 in rat cardiomyocytes by retroviral vector
LI Jin-Hua, CHEN Yong-Xiong, ZHANG Shi-Guang et al. Emergency Department, the First Affiliated Hospital, Sun-yat-sen University of Medical Sciences, Guangzhou 510080
Abstract Objective: To establish a model expressing human interleukin 10 in rat cardiomyocytes(Rat CMC) by retrovial vector for further study. Methods: PLX(hIL-10)SN was introduced into packaging cell PA317, then to select PA317 by G418,to determine retrovirus producer cells titre by NIH3T3 for identification of producer clones making high-titre virus. Exogenous gene hIL-10 was transferred into Rat CMC by the highest titre producer cells clone (6×105CFU／ml). After gene transfer 72 h, DNA and RNA were prepared from transfected Rat CMC for the polymerase chain reaction (PCR) and the reverse transcription polymerase chain reaction (RT-PCR). Expression of hIL-10 in transfected Rat CMC was checked by ELISA. Results: Exogenous hIL-10 gene has been subcloned into retroviral vector pLXSN successfully, exogenous hIL-10 gene has been integrated into the chromosal DNA of the transfected Rat CMC. RT-PCR and ELISA showed that exogenous hIL-10 gene was expressed in the transfected Rat CMC after transfection 48 h and the highest expression level was 1 755 ng／(106cells*24 h). Conclusion: Exogenous hIL-10 gene can be transferred into Rat CMC and be expressed stably. [1] [2] [3] 下一页
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