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

WANG Yu-Mei; SHENG Guang-Yao

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Chinese Journal of Contemporary Pediatrics ›› 2010, Vol. 12 ›› Issue (12) : 979-983.

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

WANG Yu-Mei, SHENG Guang-Yao

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Abstract

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.［Chin J Contemp Pediatr, 2010, 12 (12):979-983］

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Lentiviral vector / PTEN gene / RNA interference escape strategy construct rescue / T-lymphocyte

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WANG Yu-Mei, SHENG Guang-Yao. Cell proliferation and signal pathway after knockdown and RESC concurrent rescue of RNAi lentiviral vector on human PTEN gene in T-lymphocytes[J]. Chinese Journal of Contemporary Pediatrics. 2010, 12(12): 979-983

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