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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。
Objective To investigate the effect of endostatin (ES) and doxycycline (DOX) on melanoma microcirculation and its molecular mechanism. Methods Mouse B16 melanoma xenograft model was established by injection of B16 melanoma cell suspension and treated with ES, DOX and ES + DOX, respectively. Meanwhile, a blank control group was established. The growth of tumor in each group was observed. The expression of matrix metalloproteinase (MMP) -9, MMP-2 and TIMP-2 in tumor tissues was detected by immunohistochemistry. The endothelium-dependent Blood vessel, mosaic vessel (MV) and vasculogenic mimicry (VM). Total protein was extracted from frozen fresh tumor tissue. The expression of MMP-9 and MMP-2 in tumor tissue was detected by gelatin zymography Pro-MMP-2, active MMP-2 activity changes. Results The tumors in ES group, DOX group and ES + DOX group grew more slowly than those in blank control group. The MV counts of ES group, DOX group and ES + DOX group were 3.20 ± 1.66, 4.17 ± 2.29 and 3.69 ± 1.70, respectively, and the VM counts were 2.00 ± 2.24, 5.08 ± 3.40 and 2.62 ± 1.80, respectively, which were significantly lower than those of the blank control group (12.21 ± 10.28 And 13.21 ± 8.19), the differences were statistically significant (P <0.05). The positive rates of MMP-2, MMP-9 and TIMP-2 in ES group, DOX group and ES + DOX group were lower than those in blank control group (P <0.05). The levels of MMP-9 and MMP-2 bands in ES group were slightly lower than those in control group, while the levels of MMP-9 and MMP-2 in DOX and ES + DOX groups were significantly lower In the control group. Conclusion ES and DOX can inhibit the expression of MMP in melanoma and affect the microcirculation pattern of melanoma blood supply.