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

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摘要目的：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Western blot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。［中国当代儿科杂志，2010，12(12)：979-983］

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	王玉梅
	盛光耀

关键词 ：慢病毒载体, PTEN基因, 逃避RNA干扰策略结构救援, T淋巴细胞

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］

Key words： Lentiviral vector PTEN gene RNA interference escape strategy construct rescue T-lymphocyte

PACS:

R-33

引用本文:

王玉梅,盛光耀. 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[J]. 中国当代儿科杂志, 2010, 12(12): 979-983.

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]. CJCP, 2010, 12(12): 979-983.

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http://www.zgddek.com/CN/ 或 http://www.zgddek.com/CN/Y2010/V12/I12/979

	［1］王颖超, 殷楚云, 徐学聚, 赵晓明, 方营旗, 盛光耀. 儿童急性淋巴细胞白血病128例免疫分型［J］. 郑州大学学报（医学版）, 2009, 44(6):1238-1240.
	［2］Li J, Yen C, Liaw D, Podsypanina K, Bose S, Wang SI, et al. PTEN, a putative protein tyrosine phosphatase gene mutated in human brain, breast, and prostate cancer［J］. Science,1997, 275(5308):1943-1947.
	［3］Dahia PL, Aguiar RC, Alberta J, Kum JB, Caron S, Sill H, et al. PTEN is inversely correlated with the cell survival factor Akt/PKB and is inactivated via multiple mechanismsin haematological malignancies［J］. Hum Mol Genet, 1999, 8(2):185-193.
	［4］Aggerholm A, Gr-nbaek K, Guldberg P, Hokland P. Mutational analysis of the tumour suppressor gene MMAC1/PTEN in malignant myeloid disorders［J］. Eur J Haematol, 2000, 65(2):109-113.
	［5］Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells［J］. Nature, 2001, 411(6836): 494-498.
	［6］王玉梅，盛光耀. 人PTEN基因RNA干扰及其RESC救援慢病毒载体的构建与鉴定［J］. 郑州大学学报（医学版）, 2010, 43(5):443-447.
	［7］Astier A, Trescol-Biémont MC, Azocar O, Lamouille B, Rabourdin-Combe C. Cutting edge: CD46, a new costimulatory molecule for T cells, that induces p120CBL and LAT phosphorylation［J］. Immunol, 2000, 164(12): 6091-6095.
	［8］李付广，李沛，靳静，董子明.外周血T淋巴细胞的培养、纯化及鉴定［J］.郑州大学学报（医学版）, 2006, 41(4):605-607.
	［9］Chu EC, Tarnawski AS. PTEN regulatory functions in tumor suppression and cell biology［J］. Med Sci Monit, 2004, 10(10):235-241.
	［10］李德渊, 屈艺. 肿瘤抑制基因PTEN在脑发育和分化中的作用［J］.中国当代儿科杂志, 2009, 11(2):159-161.
	［11］Bussaglia E, del Río E, Matías Guiu X, Prat J. PTEN mutations in endometrial carcinomas: a molecular and clinicopathologic analysis of 38 cases［J］. Hum Pathol, 2000, 31(3):312-317.
	［12］Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans［J］. Nature, 1998, 391(6669):806-811.
	［13］何柳芳, 陈克正. HIF-1α特异性RNA干扰表达载体的构建及体外效应研究［J］. 中国当代儿科杂志, 2008, 10(1):60-64.
	［14］Echeverri CJ, Perrimon N. High-through put RNAi screening in cultured cells: a user′s guide［J］. Nat Rev Genet, 2006, 7(5):373-384.
	［15］Romano G. Current development of lentiviral-mediated gene transfer［J］. Drug News Perspect, 2005, 18(2):128-134.
	［16］Stewart SA, Dykxhoorn DM, Palliser D, Mizuno H, Yu EY, An DS, et al. Lentivirus-delivered stable gene silencing by RNAi in primary cells［J］. RNA, 2003, 9(4):493-501.
	［17］Birmingham A, Anderson EM, Reynolds A, IlsleyTyree D, Leake D, Fedorov Y, et al. 3′UTR seed matches, but not overall identity, are associated with RNAi off-targets［J］. Nat Methods, 2006, 3(3):199-204.
	［18］Martin SE, Caplen NJ. Mismatched siRNAs downregulate mRNAs as a function of target site location［J］. FEBS Lett, 2006, 580(15):3694-3698.