核糖核酸酶抑制因子过表达抑制B16细胞的侵袭和转移.docVIP

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 重庆医科大学细胞生物学遗传学教研室，分子医学与肿瘤研究） 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）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；；；肿瘤侵润 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，，(Department of Cell Biology and Genetics, Chongqing Medical University；Molecular Medicine and Cancer Research Center, Chongqing400016, 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 assay,and for invasion ability by transwell invasion assay.The expression of MMP-2, MMP-9 and nm23-H1 and E-Cadherin were detected by western blot; The changes of lung metastasis nodule were observered by constructing the mode of pulmonary metastasis o