RNAi沉默pyk2基因对鼠脑胶质瘤细胞增殖、侵袭能力的影响

目的 探讨pyk2小干扰RNA表达载体对小鼠脑胶质瘤G422细胞增殖、侵袭能力的影响.方法 用以pGenesil-1质粒为载体,以pyk2为靶基因,构建短发夹状小干扰RNA表达载体.用pyk2短发夹状小干扰RNA表达载体转染体外培养的人脑胶质瘤G422细胞,反转录聚合酶链反应和Western blot法检测pyk2基因和蛋白表达的改变,噻唑蓝(MTT)比色法检测转染后细胞的增殖情况,Transwell小室法测定转染细胞体外侵袭能力.结果 构建了pGenesil-1-pyk2重组质粒,并成功转染G422细胞.逆转录-聚合酶链反应(RT-PCR)显示pyk2 siRNA转染G422细胞后显著降低pyk2mRNA的表达,以48 h为著,下降近70%;Western blot证实转染后pyk2蛋白的表达下降近65%;G422细胞的活力降低为(67.1±5.2)%;Transwell小室测定转染细胞体外侵袭能力明显下降.结论 pGenesil-1-pyk2 siRNA重组质粒明显下调pyk2在胶质瘤细胞中的表达,并抑制肿瘤细胞增殖,抑制其侵袭能力.

The effects of RNA interference to pyk2 gene on proliferation and invasiveness of mouse glioma cell line G422

Objective To inverstigate the effects of RNA interference to pyk2 gene on proliferation and invasiveness of mouse glioma cell line G422.Methods Two pairs of DNA template coding siRNA was synthesized against G422 to reconstruct pGenesil-l-pyk2,which was transfected into G422 cells.G422 cells were transfected with pGenesil-l-pyk2 siRNA,and the expression of pyk2 was detected by RTPCR and Western blot.MTr was used to observe the proliferation of G422 ceils,and the invasiveness of G422 cells was tested by Transwell cabin.Results pGenesil-l-pyk2 siRNA was constructed and transfected into G422 cells.RT-PCR results revealed the transfection of pyk2 siRNA into G422 cells could significandy reduce the expression of pyk2 mRNA,most significant at 48 h.In G422 cells transfected with recombinant plasmid pGenesil-l-pyk2 siRNA,Western blot analysis showed that the expression of pyk2 protein was decreased by nearly 65%,MTT demonstrated that the viability of G422 cells was decreased to (67.11 ±5.2)%,and Transwell cabin suggested that the invasiveness of G422 cells was significantly decreased.Conclusion pGenesil-l-pyk2 siRNA could significantly inhibit pyk2 expression,suppress the growth of G422 cells and decrease their invasiveness.