骨桥蛋白促进人肝癌细胞株SMMC-7721恶性表型的实验研究

目的研究骨桥蛋白(OPN)对低侵袭性人肝癌细胞株SMMC-7721恶性表型的影响。方法pcDNA 3,1(-)/OPN重组质粒转染SMMC-7721细胞,以空质粒转染作对照,用RT- PCR反应、Western blot检测OPN表达水平,用ELISA检测细胞培养上清液OPN、MMP-2、-9、尿激酶纤溶酶原活化因子(uPA)水平,并用体外功能试验观察转染前后恶性表型的变化。结果重组质粒转染SMMC-7721后OPN表达明显升高,细胞培养上清液OPN为(3.02±0.12)ng/ml,对照组为(1.43±0.07)ng/ml,MMP-2重组质粒转染组为(43.04±3.06)ng/ml,对照组为(22.15±4.34)ng/ml、uPA重组质粒转染组水平明显高于空质粒转染组,分别为(4.78±0.70)ng/ml和(1.61±0.34)ng/ml,两组差异均有统计学意义,t值分别为19.89、6.81和7.03,P值均<0.01。MMP-9水平分别为(7.82±2.25)ng/ml和(7.70±1.92)ng/ml,两组差异无统计学意义。体外功能试验提示SMMC-7721转染OPN重组质粒后细胞黏附、运动和侵袭能力明显增强,细胞黏附率为75.33%±10.59%,对照组为57.34%±2.52%,t=2.86,P<0.05。运动试验透膜细胞数分别为(14.3±2.5)个和(6.3±1.5)个,t=4.70,P<0.05。侵袭试验透膜细胞数分别为(8.2±1.5)个和(4.1±1.3)个,t=4.11,P<0.05。而细胞增殖能力无明显改变。结论OPN可能是通过增加MMP-2、uPA分泌促进人肝癌细胞株SMMC-7721的恶性表型。

Osteopontin promotes the malignant phenotypes of human hepatocellular carcinoma cell line SMMC-7721

OBJECTIVE: To study the effects of osteopontin (OPN) on the phenotypes of human hepatocellular carcinoma cell line SMMC-7721. METHODS: Human hepatocellular carcinoma SMMC-7721 cells were transfected with plasmid pcDNA 3.1(-)/OPN and cells transfected with a mock plasmid served as controls. OPN expression was verified by RT-PCR and Western blot, and concentrations of OPN, MMP-2, MMP-9 and uPA were measured by ELISA. A series of functional assays in vitro were used to monitor the changes of SMMC-7721 malignant phenotypes. RESULTS: OPN expression of SMMC-7721 cells was elevated after transfection. Concentrations of OPN, MMP-2 and uPA in the medium of SMMC-7721 cells after transfection were higher than those of the controls [(3.02+/-0.12) ng/ml vs (1.43+/-0.07) ng/ml, (43.04+/-3.06) ng/ml vs (22.15+/-4.34) ng/ml, and (4.78+/-0.70) ng/ml vs (1.61+/-0.34) ng/ml respectively, P less than 0.01], but MMP-9 concentration did not increase [(7.82+/-2.25) ng/ml vs (7.70+/-1.92) ng/ml]. Functional assays in vitro indicated that SMMC-7721 cells after transfection showed higher adhesive, migrant and invasive capabilities than those of the controls (cell adhesion rates were 75.33%+/-10.59% vs 57.34%+/-2.52%; number of outer surface cells in migrant assay was 14.33+/-2.51 vs 6.34+/-1.53; cell number in the invasive assay was 8.23+/-1.53 vs 4.12+/-1.29 respectively, P less than 0.05). CONCLUSION: OPN might enhance the expression of MMP-2 and uPA to promote malignant phenotypes of SMMC-7721 cells.