Wang Yu-Mei, Sheng Guang-Yao
Department of Pediatrics, First Affiliated Hospital of Zhengzhou University, Zhengzhou 450007, China. Email:
Zhongguo Dang Dai Er Ke Za Zhi. 2010 Dec;12(12):979-83.
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.
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.
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.
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.
构建用于RNA干扰（RNAi）的人PTEN基因慢病毒表达载体及RNAi逃逸策略构建体（RESC）的共挽救载体，并观察人T淋巴细胞中PTEN基因敲低及RESC共挽救后信号通路、细胞增殖和细胞周期的变化，为进一步研究儿童急性淋巴细胞白血病的发病机制提供实验依据。
利用慢病毒载体系统构建用于RNAi的人PTEN基因慢病毒载体及其RESC共挽救载体，将慢病毒转染人T淋巴细胞。建立PTEN基因敲低（T-LC-shPTEN）及RESC共挽救（T-LC-rrshPTEN）的细胞模型。PTEN基因敲低及RESC共挽救后，分别采用蛋白质免疫印迹法、MTT法及流式细胞术检测PTEN蛋白表达、AKT信号通路激活情况、细胞增殖及细胞周期。
RNAi介导的慢病毒能有效下调人PTEN基因表达。PTEN基因下调后，T淋巴细胞生长加快，G0/G1期细胞减少，S期和G2/M期细胞显著增多，PI3K/AKT信号通路激活。PTEN基因敲低引起的所有RNAi现象均被RESC共挽救完全恢复。
成功构建了用于RNAi及RESC共挽救的人PTEN基因慢病毒表达载体。PTEN基因敲低后可激活PI3K/AKT信号通路并促进人T淋巴细胞增殖。
