逆转录病毒介导的shRNA对大鼠原代肝细胞PPARα基因表达的抑制.docVIP

逆转录病毒介导的shRNA对大鼠原代肝细胞PPARα基因表达的抑制* 刘辉1 王力2** 蚌埠医学院 生物科学系，安徽 蚌埠 233030；2.蚌埠医学院 预防医学系，安徽 蚌埠 233030) 摘要：目的：包装能够干扰大鼠氧化物酶增殖物激活受体αperoxisome proliferater activated receptor α, PPARα）基因表达的逆转录病毒，探索该病毒体外侵染SD大鼠原代肝细胞后，PPARα基因及其下游基因表达的变化情况。方法：构建针对大鼠PPARα基因的RNAi载体，通过PLAT-E细胞系包装逆转录病毒，侵染SD大鼠原代肝细胞后抽提总RNA，用荧光定量PCR检测PPARα基因及其下游基因cte1的表达。结果：PPARα基因及其下游基因cte1的表达均被抑制，其他基因的表达未受影响。结论：逆转录病毒载体介导的shRNA能够有效地干扰大鼠原代肝细胞的PPARα基因的转录，为后续的肝细胞的脂代谢研究提供了方便的转基因细胞模型。 关键词：逆转录病毒；病毒包装；原代肝细胞；PPARα；RNAi； Inhibition effect of retroviral-mediated shRNA on expression of PPARα gene in rat primary hepatocytes Liu Hui1, Wang Li2 (1 Department of Bioscience, Bengbu Medical College, Bengbu 233030, Anhui, China) (2 Department of Preventive Medicine, Bengbu Medical College, Bengbu 233030, Anhui, China) [Abstract] Objective: Retrovirus that can knock down the expression of PPARα (peroxisome proliferater activated receptor) gene of rat (Rattus norvegicus) was packaged; and the mRNA expression variations of the PPARα and its downstream gene were investigated. Methods: The retroviral RNAi vectors pGFP-V-RS that contain shRNA were constructed, and packaged with PLAT-E cell line. Rat primary hepatocytes were infected by the retrovirus, and total RNA was extracted by Trizol reagent, the expression of PPARα and the downstream gene cte1was assayed by real time PCR. Results: the expression of PPARα and the downstream gene cte1 were knockdown, while the nontargeted genes were not interfered. Conclusions: PPARα targeted retroviral-mediated shRNA can effectively knock down the expression of PPARα and the downstream gene cte1; the research provide a very convenient transgenic cell models for the subsequent study of hepatocyte lipid metabolism. [Key words] retrovirus；virus package；primary hepatocytes；PPARα；RNAi； 肝脏是人体物质代谢的重要器官，也是对许多内源性和外源性的有毒有害物质解毒的重要器官，所以肝脏就必然成为药理学研究和毒理学研究的重要靶器官。原代肝细胞培养较肝癌细胞系有更多优点，目前仍然是肝脏模型的“金标准”[1]。对于肝脏代谢研究的一个重要方向就是脂类代谢的研究，很多外源性的有毒有害物质如全氟有机物对肝脏的损害都是通过脂代谢起作用，PPAR是脂类代谢途径中一类重要的基因家族，目前有很多研究推测这类物质可能通过PPAR影响脂类代谢[2-3]。但是，