骨桥蛋白促进人肝癌细胞株SMMC-7721恶性表型的实验研究

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

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pcDNA3．1（-）／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、Westernblot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。

Objective To study the effects of osteopontin （OPN） on the phenotypes of human bepatocellular carcinoma cell line SMMC-7721. Methods Human bepatocellular 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 〈 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〈0.05）. Conclusion OPN might enhance the expression of MMP-2 and uPA to promote malignant phenotypes of SMMC-7721 cells.