转移相关基因干扰对肝癌转移潜能的影响

Effect ofsilence of associated metastasis gene on hepatocellular carcinoma metastasis

目的通过干扰高转移潜能肝癌细胞（MHCC97-H）中的骨桥蛋白（OPN）、肿瘤增殖性因子肿瘤生长因子β1（TGFβ1）基因,使之减少OPN、TGFβ1表达,观察MHCC97-H转移能力的变化情况。方法对MHCC97-H细胞行OPN、TGFβ1基因干扰,q PCR法检测干扰效果。MHCC97-H细胞迁移细胞学实验采用transwells法。肺转移动物模型制作方法：裸鼠尾静脉注射各组细胞,含细胞数5×10~6个/只,隔天连续注射3次,2周后观察肺脏组织。离体肺脏组织标本制作,DAPI染核处理10 min,甘油PBS封片,置于荧光显微镜下观察。免疫荧光法检测裸鼠动物模型整合素αvβ3表达。结果经基因干扰后MHCC97-H表达OPN和TGFβ1明显降低（P〈0.05）。细胞学实验显示与对照组比较,经OPN和TGFβ-1干扰组MHCC97-H细胞迁移数量明显减少（P〈0.01）。动物模型实验显示与对照组比较,OPN和TGFβ1干扰组肺组织中肝癌细胞较少,定量分析显示OPN和TGFβ1干扰组肺脏组织中肝癌细胞IOD值明显低于对照组（P〈0.05）。OPN和TGFβ1干扰组与对照组比较整合素αvβ3表达IOD值差异无统计学意义（P〉0.05）。结论经OPN和TGFβ1基因干扰的MHCC97-H细胞肺转移能力有明显的下降,尤其是TGFβ1基因干扰组下降更加明显,但经OPN和TGFβ1基因干扰的MHCC97-H细胞表达整合素αvβ3能力无变化,整合素αvβ3介导参与了OPN和TGFβ1共同作用肝癌转移行为的变化。

Objective To observe the change of metastasis ability of hepatocellular carcinoma by inhibiting OPN, TGFβ1 gene expression of the high metastatic potential of hepatocellular carcinoma cells （ MHCC97？H） . Methods OPN and TGFβ1 gene si？lencing were made in the MHCC97？H cells and qPCR was used to test the expression of MHCC97？H cells. MHCC97？H cells migration experiment used transwells assay. Lung metastasis animal model making method： MHCC97？H cells were injected into the tail vein of nude mice three times every two days, and 0.2 mL cells suspension contained 5 × 106 cells, and the lung tissue was observed after 2 weeks. Lung tissue samples：DAPI nuclear stainined for 10 min, glycerol PBS mounted, and observed under fluorescent microscope. Nude mice animal model of integrin αvβ3 expression was detected by immunofluorescence. Results OPN and TGFβ？1 expression of MHCC97？H by genes silencing significantly decreased （ P〈0.05） . Compared with the control group, the cells migration of MHCC97？H OPN and TGFβ？1 genes silencing groups significantly decreased （P〈0.01）. Animal model experiments showed that there were less tumor cells in the lung tissue of the OPN and the TGFβ1 gene silence group compared with the control group. The results of quantitative analysis showed that the IOD value of the blank control group and the negative control group was greater than the two experimental groups, and the difference was statistically significant （ P〈0.05） . Compared with the control group, the IOD of integrin alpha v beta 3 expression in two genes silencing groups wasn’ t significantly different. Conclusion From the level of cytology and animal model re？sults, there was significant decrease in the metastatic ability of MHCC97？H cell by genes silencing, especially in the TGFβ1 gene si？lencing group. The expression ability of related metastasis factorintegrinαvβ3 had no change, and it suggests that integrin αvβ3mediates the role of OPN and TGFβ1 in HCC metastasis.