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目的:通过体内实验探究蜂毒素通过调控组织蛋白酶S的表达抑制人肝癌Mock/MHCC97-H细胞肿瘤血管生成的作用.方法:分别建立人肝癌Mock/MHCC97-H细胞和沉默组织蛋白酶的shRNA-CatS/MHCC97-H细胞裸鼠原位移植瘤模型,模型小鼠随机分组共4组,A1组:小鼠接种shRNA-CatS/MHCC97-H细胞并给予蜂毒素治疗;A2组:小鼠接种shRNA-CatS/MHCC97-H细胞给予生理盐水;B1组:小鼠接种Mock/MHCC97-H细胞给予蜂毒素治疗;B2组:小鼠接种Mock/MHCC97-H细胞给予生理盐水.A1和B1治疗组为腹腔内注射蜂毒素80 mg/kg·d;A2和B2组为腹腔内注射生理盐水0.2 mL/d.给药25天,用药结束后隔天处死动物.观察并计录各组裸鼠肿瘤的大小和瘤重,并将取出的肝移植瘤组织进行CD34蛋白的免疫组化染色,用Western blot检测蜂毒素对裸鼠肝移植瘤中组织蛋白酶S、VEGF-A、p-VEGFR2、Ras、Raf、p-Raf、MEK1、p-MEK1、ERK1/2和p-ERK1/2蛋白的作用.结果:与B2组相比,B1组的肿瘤体积明显缩小,重量明显减轻(均P<0.001);而A1组和A2组在肿瘤体积和重量相比,均无明显差异.B2组肿瘤的中CD34阳性微血管数目明显多于A2组(P<0.001),A1组肿瘤的中CD34阳性微血管数目明显多于B1组(P<0.001).蜂毒素能明显的抑制接种Mock/MHCC97-H细胞模型组组中CatS、VEGF-A、p-VEGFR2、Ras、Raf、p-Raf、MEK1、p-MEK1、ERK1/2和p-ERK1/2蛋白的表达,而对接种shRNA-CatS/MHCC97-H细胞模型组的上述蛋白的表达无明显作用.结论:蜂毒素可以通过抑制组织蛋白酶S引导的VEGF-A信号通路,进而抑制组织蛋白酶S诱发的肿瘤生长和新生血管生成.“,”Objective:To study the anti-angiogenesis effect of melittin on human hepatoma Mock/MHCC97-H cells by regulating the expression of cathepsin S (CatS) in vivo. Methods: Models of in situ transplantation tumor of Mock/MHCC97-H cells and silencing cathepsin shRNA-CatS/ MHCC97-H cells in nude mice were established. The model mice were randomly divided into four groups. In the A1 group, the mice were inoculated with shRNA-CatS/MHCC97-H cells and treated with melittin. In the A2 group, the mice were inoculated with shRNA-CatS/MHCC97-H cells and treated with saline. In the B1 group, the mice were inoculated with Mock/MHCC97-H cells and treated with melittin. In the B2 group, the mice were inoculated with Mock/MHCC97-H cells and treated with saline. The A1 and B1 group were injected with melittin (80 mg/kg) intraperitoneally every day. The A2 and B2 group were injected with 0.2 mL normal saline intraperitoneally every day. After administration for 25 days, the animals were sacrificed. The tumor size and weight in nude mice in each group were recorded. The expression of CD34 protein in the xenograft tumor tissues was detected by immunohistochemistry. The expression of Cat S, VEGF-A, p-VEGFR2, Ras, Raf, p-Raf, MEK1, p-MEK1, ERK1/2 and p-ERK1/2 proteins were detected by western blot. Results:The B1 group had significantly smaller tumor volumes and lower tumor weights than the B2 group (both P<0.001). There was no significant difference between the A1 group and A2 group in tumor volumes and weights. The number of CD34-positive microvessels in the B2 group was significantly higher than that in the A2 group (P < 0.001). The number of CD34-positive microvessels in the B1 group was significantly lesser than that in the A1 group (P < 0.001). Most strikingly, in the model featuring inoculation of Mock/MHCC97-H cells, CatS, VEGF-A, p-VEGFR2, Ras, Raf, p-Raf, MEK1, p-MEK1, ERK1/2 and p-ERK1/2 expression were inhibited when treated with melittin. However, in the model featuring the inoculation of shRNA-CatS/MHCC97-H cells, no such effects were observed with similar treatments. Conclusion:Melittin can inhibit the growth of tumors and angiogenesis by blocking the CatS-VEGf-A signaling pathway.