核糖核酸酶抑制因子过表达抑制B16细胞的侵袭和转移.doc

核糖核酸酶抑制因子过表达抑制B16细胞的侵袭和转移 潘湘阳,李红彦，姚 雪，熊冬梅，陈俊霞（400016 重庆，重庆医科大学细胞生物学与遗传学教研室，分子医学与肿瘤研究中心） ［摘要］目的 构建人核糖核酸酶抑制因子（ribonuclease inhibitor，RI）真核表达载体，检测其对B16细胞迁移和侵袭能力的影响。方法 提取人7703细胞中的总RNA，RT-PCR特异性扩增RI编码区序列，利用基因重组技术将其克隆到载体pIRES2-EGFP中，稳定转染B16细胞以后，经G418筛选出阳性克隆。根据转染质粒不同分别命名为B16细胞组(未转染组)、空质粒对照组（pIRES2-EGFP组）和实验组（pIRES2-EGFP-RI组）。RT-PCR检测B16细胞中RI mRNA水平的表达,并通过Western blot和细胞免疫化学检测RI蛋白水平的表达；HE染色观察细胞形态的改变，黏附实验、划痕实验和Transwell法检测细胞黏附、迁移和侵袭能力的变化；Western blot检测MMP-2、MMP-9和nm23-H1以及E-Cadherin的表达；建立C57BL/6小鼠肺转移模型，观察肺上肿瘤转移灶的变化。结果 酶切和测序结果证明重组质粒构建成功，实验组细胞RI的mRNA和蛋白的表达较B16细胞组和空质粒对照组显著升高（P0.05）；HE染色结果显示，细胞铺展良好，核质比减小；转染pIRES2-EGFP-RI质粒的实验组细胞的黏附，迁移及侵袭能力明显降低（P0.05）；与两对照组相比，实验组细胞MMP-2和MMP-9的表达水平明显降低，nm23-H1和E-Cadherin的表达水平明显升高（P0.05）；动物实验显示与对照组相比，实验组的肺转移灶明显减少。结论 成功构建的RI真核表达质粒能升高RI基因及蛋白水平的表达，显著抑制了B16细胞的转移和侵袭能力。 [关键词] 核糖核酸酶抑制因子；B16黑色素瘤细胞；细胞黏附；迁移细胞运动；肿瘤转移；肿瘤侵润 侵袭 [中图法分类号] R394.2；R739.5 ［文献标志码］ A The Overexpression of Human ribonuclease inhibitor Inhibiting Invasion and Metastasis of B16 Melanoma Cells Pan Xiangyang ,Li Hongyan，Yao Xue，Xiong Dongmei, Chen Junxia(Department of Cell Biology and Genetics, Chongqing Medical University；Molecular Medicine and Cancer Research Center, Chongqing 400016, China) [Abstract] Objective To construct the human ribonuclease inhibitor(RI) eukaryotic expression vector and investigate the effect of RI overexpression on the invasion and metastasis of B16 cells. Methods Total RNA was extracted from human 7703 cells by Trizol reagents,the RI cDNA fragment was amplified by RT-PCR and the DNA fragment was inserted into the eukaryotic expression vector pIRES2-EGFP with DNA recombinant techniques.Then the vectors were transfected into B16 cells and positive clones were screened with G418.The efficiency of RI genes overexpression was determined by RT-PCR,Western blot and immunocytochemical method.B16 cells were observed for morphological changes by HE staining,and determined for adhesion ability by adhesion test ,for metastasis ability by wound healing