内皮抑素和多西环素对黑色素瘤微循环模式的影响及其机制

目的探讨内皮抑素(ES)和多西环素(DOX)对黑色素瘤微循环模式的影响及其分子机制。方法通过注射B16黑色素瘤细胞悬液建立小鼠B16黑色素移植瘤模型,分别给予ES、DOX、ES DOX处理,同时设空白对照组。观察各组小鼠肿瘤的生长情况;免疫组化检测肿瘤组织基质金属蛋白酶(MMP)-9、MMP-2和组织金属蛋白酶抑制剂-2(TIMP-2)的表达;计数肿瘤中内皮依赖性血管、马赛克血管(MV)和血管生成拟态(VM)3种微循环血管;从冻存的新鲜肿瘤组织提取总蛋白,用明胶酶谱法检测各组肿瘤组织中MMP-9、MMP-2酶原(pro-MMP-2)、活性MMP-2的活性变化。结果ES组、DOX组和ES DOX组均较空白对照组肿瘤生长缓慢。ES组、DOX组和ES DOX组MV计数分别为3.20±1.66、4.17±2.29和3.69±1.70,VM计数分别为2.00±2.24、5.08±3.40和2.62±1.80,与空白对照组(12.21±10.28和13.21±8.19)比较,差异均有统计学意义(P<0.05)。ES组、DOX组和ES DOX组MMP-2、MMP-9和TIMP-2表达的阳性率均低于空白对照组(P<0.05)。ES组MMP-9和活性MMP-2条带酶解量灰度值略低于对照组,而DOX组和ES DOX组MMP-9和活性MMP-2条带酶解量灰度值明显低于对照组。结论ES和DOX可以抑制黑色素瘤MMP的表达,影响黑色素瘤血液供应的微循环模式形成。

Effects of endostatin and doxycycline on microcirculation patterns in melanoma and their relevant molecular mechanisms

Objective To investigate the effects of endostatin and doxycycline on microcirculation patterns in melanoma and their molecular mechanisms. Methods To establish mouse B16 melanoma model by subcutaneous injection of B16 melanoma cell suspension. The mice were divided into 3 experimental groups and 1 control group. To treat the mice in the 3 experimental groups with endostatin, doxycycline, endostatin and doxycycline, respectively, and the control group without any treatment. The tumor volume was measured and recorded to make comparison of their growth rate. To assess the expression of MMP-2, MMP-9 and TIMP-2 by immunohistochemical staining. The three microcirculation patterns of endothelium-dependent vessels, mosaic vessels and vasculogenic mimicry were counted. The activity of MMP-2, MMP-9 between different groups was examined by gelatin zymography. Results Tumor growth in the three experimental groups was statistically significantly slower than that in the control group. The expression of MMP-2, MMP-9 and TIMP-2 in each treated group was significantly different with that in the control group. The amount of three microcirculation patterns in three experimental groups was less than that of the control group, and the amount of MV and VM in each experimental group was significantly less than that in the control group. By gelatin zymography, the enzyme activity of MMP-9, actived-MMP-2 and MMP-2/pro-MMP-2 in ES, DOX and ES DOX group was lower than that in the control group, but the enzyme activity of pro-MMP-2 among the four groups was not significantly different. Conclusion The combined use of doxycycline and endostatin in melanoma can inhibit the expression of MMPs, influencing the formation of different microcirculation patterns in melanoma.