| Objective:With triptolide、cisplatin acting on nude mice xeograftes of cisplatin-resistant ovarian carcinoma,to analyze the expression of caspase-3、survivin protein and apoptosis rate in nude mice xeografts,to investigate the anti-apoptosis effect and the probable mechanism of triptolide on cisplatin-resistant human ovarian carcinoma cells in vivo;to analyze the expression of VEGF、MMP-2 protein in nude mice xeografts of cisplatin-resistant ovarian carcinoma and investigate the effect of invasion and metastasis by triptolide on cisplatin-resistant human ovarian carcinoma cells in vivo.Methods:1.cisplatin-resistant human ovarian carcinoma models transplanted subcutaneously in nude mice were established and divided into four groups randomly:TPL combined with DDP group,TPL group,DDP group,NS group,every group has five nude mice.2.Treatment methods of each group:TPL combined with DDP group:every nude mice received intraperitoneal injection of TPL（0.15 mg/kg/d）once a day for 2 weeks and DDP（0.4mg/kg/d）once a week;TPL group:TPL（0.15 mg/kg/d）was administered to nude mice by intraperitoneal injection once a day,for 2 weeks;DDP group:DDP（0.4mg/kg/d）was administered to nude mice by intraperitoneal injection once a week,;NS group:NS（50ml/kg/d）was administered to nude mice by intraperitoneal injection once a day,for 2 weeks.nude mice were killed by method of cervical dislocation after drug withdrawal for 24 hours,using paraffin embedding into wax block after stipped tumors.3.The cel apoptosis in tumor tissues was detected by TUNEL,Immunohistochemistry(Elivision TMplus)was employed for detecting the expression of caspase-3、survivin、VEGF、MMP-2 protein in tumor tissue.To calculate the mean density of different protein in differentimmunohistochemical image by software of Image Proplus6.0,Which could reflect the expression of protein,the greater the mean density is,the more expression of protein in tumor tissues.Results:1、Each group’s apoptosis rate detected by TUNEL was TPL combined with DDP group:42.03%±3.10,TPL group:24.48%±2.46,DDP group:14.14%±2.32,NS group:3.48%±1.15.The Comparison of the apoptosis rate of every group on cisplatin-resistant human ovarian carcinoma in nude mice xeografts was TPL combined with DDP group>TPL group>DDP group>NS group,the comparison of every two group has statistical significance（P﹤0.01）.2、The expression of caspsase-3 protein in each group by Immunohistochemistry,the mean density was:TPL combined with DDP group:0.241±0.020,TPLgroup:0.182±0.024,DDPgroup0.116±0.016,NS group:0.059±0.019.The mean denstiy of TPL combined with DDP group、TPL group and DDP group increased significantly compared with NS group,The mean density of TPL combined with DDP group increased significantly compared with TPL group、DDP group,the similar with the comparison between TPL group and DDP group,suggesting that the expression of caspase-3 in every group was TPL combined with DDP group>TPL group>DDP group>NS group,had significant difference between the every two group（P﹤0.01）.3、The expression of survivin protein in each group by Immunohistochemistry,the mean density was:TPL combined with DDP group:0.050±0.013,TPL group:0.104±0.016,DDP group:0.147±0.017,NS group:0.204±0.021.compared with NS group,the mean density of TPL combined with DDP group、TPL group and DDP group decreased significantly,the mean density of TPL combined with DDP group decreased significantly compared with TPL group、DDP group,the mean density of TPL group decreased significantly compared with DDP group,suggesting that the expression of survivin protein in every group was TPL combined with DDP group<TPL group<DDP group<NS group,had significant difference between every two group（P﹤0.01）.4、The expression of VEGF protein in each group by Immunohistochemistry,the mean density was:TPL combined with DDP group:0.087±0.020,TPL group:0.113±0.015,DDP group:0.151±0.017,NS group:0.220±0.021.The mean denstiy of TPL combined with DDP group、TPL group and DDP group decreased significantly compared with NS group,the mean density of TPL combined with DDP group decreased significantly compared with TPL group、DDP group,the mean density of TPL group decreased significantly compared with DDP group,suggesting that the expression of VEGF protein in every group was TPL combined with DDP group<TPL group<DDP group<NS group,had a significant difference between two each group（P﹤0.01,P﹤0.05）.5、The expression of MMP-2 protein in each group by Immunohistochemistry,the mean density was:TPL combined with DDP group:0.087±0.020,TPL group:0.113±0.015,DDP group:0.151±0.017,NS group:0.220±0.021.the mean denstiy of TPL combined with DDP group、TPL group and DDP group decreased significantly compared with NS group,the mean density of TPL combined with DDP group decreased significantly compared with TPL group、DDP group,the mean density of TPL group decreased significantly compared with DDP group,suggesting that the expression of MMP-2 protein in every group was TPL combined with DDP group<TPL group<DDP group<NS group,had a significant difference between two each group（P﹤0.01,P﹤0.05）.Conclusion:1、The apoptosis rate of TPL group was higher than NS control group,the apoptosis rate of TPL combined with DDP group was higher than their respective single-drug group,indicating that triptolide could induce the apoptosis of cisplatin-resistant human ovarian carcinoma in vivo,and could enhance the sensitivity of cisplatin-resistant human ovarian carcinoma xenograft to cisplatin chemotherapy.2、The expression of caspase-3 protein in the group of TPL was higher than the group of DDP and NS control,and the expression of survivin protein in the group of TPL group was lower than the group of DDP and NS control,the expression of caspase-3 protein in the group of TPL combined with DDP was higher than DDP group and NS group and the expression of survivin protein in the group of TPL combined with DDP was lower than the group of DDP and NS control group,suggesting that the mechanism of apoptosis by triptolide promoted cisplatin-resistant human ovarian carcinoma cells in vivo and enchance the sensitivity of cisplatin-resistant human ovarian carcinoma xenograft to cisplatin chemotherapy may be related to the up regulation of caspase-3 protein,inhibition of survivin protein.3、The expression of VEGF and MMP-2 protein in the TPL group was obviously lower than DDP group and NS group,the expression of VEGF and MMP-2protein in TPL combined with DDP group was lower than their respective single-drug group,suggesting that triptolide can inhibit tumor angiogenesis,prevent tumor cell adhesion and migration by down regulate the expression of VEGF、MMP-2 protein,which may be one of the mechanism of inhibit invasion and metastasis on human cisplatin-resistant ovarian cancer and enchance the sensitivity of cisplatin-resistant human ovarian carcinoma xenograft to cisplatin chemotherapy. |
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