【作者】 何康霞；

【导师】 颜江华；

【作者基本信息】 厦门大学 ， 生物化学与分子生物学， 2009， 硕士

【摘要】 背景:hSHIP(Human SH2 domain-containing inositol 5′-phosphatase,人含SH2结构域肌醇磷酸酯酶)属于肌醇5’磷酸酯酶家族,主要在造血细胞中表达。研究显示,SHIP能通过PI3K/Akt信号通路对造血细胞增殖、存活及终端细胞活化起到负调节作用。本文主要探究hSHIP是否诱导PI3K/Akt信号通路异常活化的非造血肿瘤细胞(HeLa)的细胞周期发生阻滞作用及凋亡。方法:以人外周血单个核细胞总RNA为模板,RT-PCR扩增hSHIP基因的全长cDNA编码区序列,将其插入pcDNA3.1-GFP中,构建pcDNA3.1-GFP-hSHIP真核表达载体;用脂质体法转染HeLa细胞,经G418筛选,单克隆化细胞培养及采用荧光显微镜观察和Western blotting进一步鉴定以获得稳定表达目的基因的HeLa细胞株;流式细胞术检测细胞周期;caspase-3活性检测实验及Hoechst33258/PI双染色法分析细胞凋亡;MTT法及裸鼠体内成瘤实验检测细胞增殖与存活;半定量RT-PCR及Western blotting检测Akt的表达情况及ELISA法检测Akt磷酸化水平。结果:成功构建了pcDNA3.1-GFP-hSHIP真核表达载体,并成功建立了稳定转染HeLa细胞株(HeLa/hSHIP-GFP,HeLa/GFP)。与对照组(HeLa/GFP)相比,细胞处于S期的比例从27.3±4.50%上升到47.2±7.78%(P＜0.05),G2/M期从14.5±1.91%减少到2.6±0.71%(P＜0.05),显示hSHIP将HeLa细胞的细胞周期阻滞于S期;Hoechst33258/PI和caspase-3活性检测显示了hSHIP对HeLa细胞起促凋亡作用;MTT法显示96小时后hSHIP对HeLa细胞的生长抑制率高达80.91%(P＜0.05),裸鼠体内成瘤实验显示HeLa/hSHIP-GFP细胞生长显著减缓,瘤体重量比为255.0±50mg vs 117.5±35mg(P＜0.05),实验组的抑瘤率为53.92%(P＜0.05);进一步检测表明,总Akt蛋白及Akt1/2 mRNA的表达及Akt磷酸化水平明显下调。结论:本文研究提示,hSHIP能显著抑制HeLa细胞的增殖及存活,诱导HeLa细胞阻滞于S期及凋亡,其机制可能与Akt表达及磷酸化水平下调有关。这些研究工作为进一步探讨hSHIP的抗肿瘤机制及为其他PI3K/Akt信号通路异常活化的非造血肿瘤的抑癌作用的研究奠定基础。更多还原

【Abstract】 Background:Human SH2-containing inositol-5-phosphatase（hSHIP）belongs to inositol phosphatase family and restricts to hematopoietic cells.The studies showed that hSHIP acts as a negative regulator of proliferation,survival and end cell activation in hematopoietic cells.To explore whether hSHIP induces apoptosis and cell cycle arrest in nonhaemopoietic tumors with abnormal activation of PI3K/Akt.Methods:The full-length SHIP cDNA fragment was amplified by RT-PCR from the human peripheral blood mononuclear cells and subcloned into pcDNA3.1-GFP vector to construct pcDNA3.1-hSHIP-GFP full-length vector.pcDNA3.1-hSHIP-GFP and pcDNA3.1-GFP（control）were transfected into HeLa cells by Lipofectamine^TM2000,The stable transfected HeLa cell lines were then established by screening culture with G418,and the expression of hSHIP-GFP and GFP were identified by Flurescent microscope and Western blotting.Cell cycle was examined by Flow cytometry.Apoptosis was detected by Hoechst33258/ PI and caspase3 activity assay.Cell growth was determined by MTT assay and tumorigenicity in nude mice.The levels of Akt expression were measured by semi-quantitative RT-PCR and Western blotting,and phosphorylation of Akt（Ser473）was abserved by ELISA.Results:The eukaryotic expression vector pcDNA3.1-hSHIP-GFP were contracted successfully,and stable transfected HeLa cell lines were established.Flow cytometry analysis showed that expression of hSHIP remarkably increased the proportion of HeLa cells in S-phase（from 27.3±4.50%to 47.2±7.78%,P＜0.05）and decreased the proportion of cells in G₂/M phase（from 14.5±1.91%to 2.6±0.71%, P＜0.05）,these data showed that hSHIP significantly induced S-phase arrest of HeLa cells.hSHIP-induced apoptosis reflected by Hoechst33258/PI and caspase-3 activity assay.The cell growth was reduced by 80.19%（P＜0.05）at 96h,and the tumorigenicity in nude mice was reduced evidently（255.0±50mg vs 117.5±35mg, P＜0.05）,the ratio of inhibition of tumor was 53.92%（P＜0.05）,Further tests showed that hSHIP leads to down-regulate expression of Akt and phosphorylation of Akt.Conclusion:In this study,hSHIP can significantly inhibit proliferation and survival of HeLa cells,and induce S arrest and apoptosis.The possible mechanism is related to downregulation of the expression and phosphorylation of Akt.These results would contribute to the further study of its anti-tumor biological functions and the suppression of other nonhaemopoietic tumors with abnormal activation of PI3K/Akt by hSHIP.更多还原