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目的初探水蛭提取液对新生血管生长的作用机制。方法采用酶联免疫法、放射免疫法检测不同时间点水蛭提取液对细胞培养上清液中MMP-2、IV型胶原表达的影响。结果 12 h各组间比较,空白组、水蛭组MMP-2的OD值分别为0.137±0.006、0.136±0.007,表达低于凝血酶组0.165±0.018,组间差异均有统计学意义(均为P<0.05)。空白组12 h的MMP-2含量要低于2 h、6 h,差异均有统计学意义(均为P<0.05);水蛭组2 h时MMP-2的分泌高于6 h、12 h,差异均有统计学意义(均为P<0.05)。2 h时空白组IV型胶原的含量(20.67±1.02)ng·mL-1高于凝血酶组(15.58±3.40)ng·mL-1、合药组(15.75±6.23)ng·mL-1,差异均有统计学意义(均为P<0.05);6 h时水蛭组IV型胶原的含量明显高于其他3组,差异均有统计学意义(均为P<0.05);12 h时观察,空白组IV型胶原的含量(21.33±2.75)ng·mL-1,明显高于合药组(16.49±1.59)ng·mL-1,差异有统计学意义(P<0.05),水蛭组IV型胶原的含量明显高于凝血酶组及合药组,差异有统计学意义(P<0.05)。空白组2 h、12 h IV型胶原的含量均较6 h高,差异均有统计学意义(均为P<0.05);凝血酶组IV型胶原的合成呈增加趋势,12 h与2 h相比,差异有统计学意义(P<0.05);水蛭组IV型胶原的合成明显增加,6 h、12 h IV型胶原的含量显著高于2 h,差异有统计学意义(P<0.05)。结论64 g·L-1水蛭提取液可以促进IV型胶原合成,抑制MMP-2的分泌,对新生血管的形成有抑制作用。
Objective To investigate the mechanism of leech extract on neovascularization. Methods The effect of leech extract on the expression of MMP-2 and type IV collagen in cell culture supernatants was detected by enzyme-linked immunosorbent assay and radioimmunoassay. Results At 12 h, the OD values of MMP-2 in the blank group and leeches group were 0.137 ± 0.006 and 0.136 ± 0.007, respectively, which were lower than those in the thrombin group (0.165 ± 0.018, both were statistically significant P <0.05). The level of MMP-2 in the blank group at 12 h was lower than that at 2 h and 6 h (both P <0.05). The secretion of MMP-2 at 2 h in the leeches group was higher than that at 6 h and 12 h, The differences were statistically significant (both P <0.05). The content of type IV collagen in the blank group (20.67 ± 1.02) ng · mL-1 was significantly higher than that in the thrombin group (15.58 ± 3.40 ng · mL-1) at 2 h and 15.75 ± 6.23 ng · mL- (All P <0.05). At 6 h, the content of type IV collagen in leeches was significantly higher than the other three groups (all P <0.05). At 12 h, The level of type IV collagen in the blank group (21.33 ± 2.75 ng · mL-1) was significantly higher than that in the combination group (16.49 ± 1.59 ng · mL-1), the difference was statistically significant (P <0.05) Collagen content was significantly higher than the thrombin group and the combination group, the difference was statistically significant (P <0.05). The contents of type IV collagen in the blank group at 2 h and 12 h were all significantly higher than those at 6 h (all P <0.05). The synthesis of type IV collagen in the thrombin group showed an increasing trend, (P <0.05). The synthesis of type IV collagen in leeches obviously increased, and the content of type IV collagen at 6 and 12 h was significantly higher than that in 2 h (P <0.05). Conclusion 64 g · L-1 leech extract can promote the synthesis of type IV collagen, inhibit the secretion of MMP-2 and inhibit the formation of new blood vessels.