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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细胞侵袭和转移能力的影响。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(P<0.05);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(P<0.05);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(P<0.05);动物实验结果显示与对照组相比,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。
Objective To construct an eukaryotic expression vector of human ribonuclease inhibitor (RI) and investigate its effect on invasion and metastasis of B16 cells. Methods The total RNA of human 7703 cells was extracted. The RI coding region was amplified by RT-PCR. The recombinant plasmid was cloned into vector pIRES2-EGFP. Stably transfected into B16 cells, positive clones were screened by G418. The transfected plasmids were named as B16 cell group (untransfected group), empty plasmid control group (pIRES2-EGFP group) and experimental group (pIRES2-EGFP-RI group). The expression of RI mRNA in B16 cells was detected by RT-PCR. The RI protein level was detected by Western blot and immunocytochemistry. The morphological changes of HEK-293 cells were observed by HE staining. Adhesion, adhesion and migration were evaluated by scratch test and Transwell assay. And invasion ability. The expression of MMP-2, MMP-9, nm23-H1 and E-cadherin were detected by Western blot. The lung metastasis model of C57BL / 6 mice was established. Results The results of enzyme digestion and sequencing showed that the recombinant plasmid was successfully constructed. The mRNA and protein expression of RI in experimental group were significantly higher than those in B16 group and empty plasmid control group (P <0.05). HE staining showed that the cells spread well, (P <0.05). Compared with the two control groups, the expression of MMP-2 and MMP-9 in the experimental group was significantly lower than that in the control group The expression levels of nm23-H1 and E-cadherin were significantly increased (P <0.05). The results of animal experiments showed that compared with the control group, the lung metastases of the experimental group were significantly reduced. Conclusion The constructed RI eukaryotic expression plasmid can enhance the expression of RI gene and protein, and significantly inhibit the metastasis and invasion ability of B16 cells.