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

Expression of human interleukin 10 in rat cardiomyocytes by retroviral vector

目的：建立一个在大鼠心肌细胞表达人白细胞介素１０（ＨｕｍａｎＩｎｔｅｒｌｅｕｋｉｎ１０，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１７５５ｎｇ／（１０６ｃｅｌｓ·２４ｈ）。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。

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 (6×10 5CFU/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/(10 6cells·24 h). Conclusion: Exogenous hIL 10 gene can be transferred into Rat CMC and be expressed stably. The retroviral vector PLX(hIL 10)SN shall lay foundation for researching hIL 10 mechanism and its application for curing heart diseases.