清开灵注射液对HepG2细胞u-PAR表达影响的实验研究

The Influence of Qing Kailing Injection on the Expression of u-PAR in HepG2 Cells

目的：尿激酶型的纤溶酶原活化剂受体（u-PAR）与恶性肿瘤细胞周围基质的纤溶状况密切相关，因此与肿瘤细胞的侵袭和转移有关。本实验观察了中药清开灵注射液对肝癌细胞HepG2培养上清液中u—PAR表达的影响，以了解清开灵注射液对肝癌细胞周围纤溶状态的影响。方法：取清开灵冻干粉200mg，用注射用水逐步稀释为从10到30000倍的9个稀释倍数，加入含10％胎牛血清的RPMI-1640培养液中分别培养等量细胞，以等量PBS液作为阴性对照。人肝癌细胞株HepG2，置37℃，5％CO2培养，3～5天传代1次，倒置显微镜观察，待细胞生长旺盛（达指数生长期）后用MTT法分别测定各浓度药液的细胞增殖抑制率，然后用含各浓度药液的10％胎牛血清RPMI-1640培养液刺激细胞株48h，分别取各组细胞上清进行u—PAR表达的ELISA检测，观察各浓度药物对细胞生长活力和u—PAR表达的影响。结果：清开灵注射液对HepG2细胞具有显著的增殖抑制作用，同时可使细胞培养上清中u—PAR水平下降。结论：清开灵注射液的运用可降低肝癌细胞的增殖活力，同时药物可通过降低癌细胞周围纤溶状态抑制肝癌细胞的周围组织浸润和转移，临床上可考虑作为治疗肝癌，防止癌细胞转移的药物选择之一。

Objective： urokinase- type plasminogen activator receptor （u -PAR） is related to the fibrenolysis situation around malignant tumor cells, and involved with the invasion and metastasis of tumor cells. In this research, we observed the effects of Qing Kailing injection on the level of u - PAR activation in the supernatant fluid of HepG2 cells to find the effect of Qing Kailing Injection on the fibrenolysis situation around liver cancer cells. Methods ： 2mL of Qing Kailing injection was diluted with injection water to 9 diluted liquids include 10 to 30000 times, added into RPMI - 1640 culture medium supplemented with 10% fetal bovine serum and cultured the same amount of cells. And the equal quantity of PBS fluid was used as the negative control. Fully RPMI - 1640 culture medium supplemented with 10% fetal bovine serum cultured HepG2 cells in a humidified incubator at 37℃ and 5% CO2. Generatied the cells every 3 to 5 days, observed with invert microscope till the cells grew vigorously （arrived to the logarithmic period）. A MTT assay was performed when the living cells were above 80%. Then treated the cells with PBS fluid or the varied concentration of Qing Kailing injection for 48 hours respectively. All kinds of the supernatant fluid were collected for ELISA essay to decide the activities of u - PAR. Results ： The laboratory research showed that Qing Kailing injection inhibited the proliferation of HepG2 cells, and have inhibited effects on the level of u - PAR activities. Conclusion ： It suggests that the clinic use of Qing Kailing injection can inhibit the proliferation of cells of liver cancer and inhibit the invasion and metastasis of liver cancer cells through decline fibrenolysis activities around the cells and can be used as one of the supplement theraputic methods of liver caner treatment.