PTEN基因敲减及RESC救援后人T淋巴细胞增殖和信号通路的变化

Cell proliferation and signal pathway after knockdown and RESC concurrent rescue of RNAi lentiviral vector on human PTEN gene in T-lymphocytes

目的构建人PTEN基因RNA干扰(RNAi)及其逃避RNAi策略结构(RESC)救援的慢病毒载体。观察PTEN基因敲减前后及RESC救援后人T淋巴细胞信号通路、细胞增殖和细胞周期的变化,为进一步研究淋巴细胞性白血病的发病机制提供依据。方法利用慢病毒载体系统,构建人PTEN基因RNAi及其RESC救援的慢病毒载体,转染T淋巴细胞,建立PTEN基因敲减及RESC救援的细胞模型T-LC-shPTEN和T-LC-rrshPTEN;分别应用Westernblot法、MTT法和流式细胞术检测PTEN基因敲减前后及RESC救援后PTEN蛋白的表达和AKT通路的活化情况、细胞增殖及细胞周期的变化。结果慢病毒介导的RNAi能有效下调PTEN基因的表达。PTEN基因表达下调后,T淋巴细胞生长受到促进,G0/G1期细胞减少,S期细胞和G2/M期细胞增多,PI3K/AKT通路激活。而RESC救援则能够完全恢复PTEN基因RNAi所造成的现象。结论成功构建了人PTEN基因RNAi及其RESC救援的慢病毒载体。PTEN基因敲减后,通过激活PI3K/AKT通路促进T淋巴细胞生长。

Objective To construct the lentiviral expression vectors of human PTEN gene for RNA interference（RNAi） and concurrent rescue of RNAi escape strategy construct（RESC） and to observe the changes of signal pathway,cell proliferation and cell cycle after PTEN gene knockdown and RESC concurrent rescue in human T-lymphocytes,in order to provide an experimental basis for a further research into the pathogenesis of acute lymphoblastic leukemia in children.Methods Using lentiviral vector systems to construct lentiviral vectors of human PTEN gene for RNAi and its RESC concurrent rescue,human T-lymphocytes were transfected with the lentiviruses.The cell models were established with PTEN gene knockdown（T-LC-shPTEN） and RESC concurrent rescue（T-LC-rrshPTEN）.After knockdown and RESC concurrent rescue of PTEN gene,the expression of PTEN protein and the activation of AKT signal pathway,cell proliferation and cell cycle were detected by Western blot,MTT assay and flow cytometry respectively.Results The RNAi-mediated lentiviruses can down-regulate the expression of the human PTEN gene effectively.After the down-regulation of PTEN gene,the T-lymphocytes grew faster.The phase G0/G1 cells decreased and the phases S and G2/M cells increased significantly.The PI3K/AKT signal pathway was activated.All RNAi phenomenon caused by PTEN gene knockdown were recovered fully by RESC concurrent rescue of RNAi.Conclusions The lentiviral expression vectors of human PTEN gene for RNAi and RESC concurrent rescue of RNAi are constructed successfully.The PI3K/AKT signal pathway can be activated and the proliferation of human T-lymphocytes can be promoted after PTEN gene knockdown.