| Objective:Through the establishment of xenograft model of human epithelial ovarian cancer(HEOC)and cisplatin in nude mice.To investigate the effect of triptolide(TPL)on the immune and angiogenesis of tumor cells.To provide experimental evidence for TPL as an effective or DDP sensitization durg for the treatment of cisplatin resistant ovarian cancer.Methods:1.Culture SKOV3/DDP cell:cultivate SKOV3/DDP cell with 10%FBS and90%RPMI-1640 complete nutritive medium,putting it in culture flask.Cultivate it in constant temperature incubator with the condition of 37℃、5%CO2.2.Establishment of the Netherlands cisplatin resistant ovarian cancer xenografts in nude mice and divided into groups:with the cultured SKOV3/DDP cell(2×10~7/ml×0.2ml)were inoculated into female nude mice to establishment of the Netherlands cisplatin resistant ovarian cancer xenografts in nude mice.When tumors size achieve 100 mm~3,divided into four groups:combination group(TPL+DDP),TPL group,DDP group,NS group,every group has six nude mice.3.Treatment methods of each group:combination group:TPL(0.15 mg/kg/d)to every nude mice by intraperitoneal injection once a day for 14 times and DDP(4mg/kg/d)to every nude mice by intraperitoneal injection once a week for 2times;TPL group:TPL(0.15 mg/kg/d)to every nude mice by intraperitoneal injection once a day for 14 times;DDP group:DDP(4mg/kg/d)to every nude mice by intraperitoneal injection once a week for 2 times;NS group:NS(50ml/kg/d)to every nude mice by intraperitoneal injection once a day for 14 times.They were killed by exsanguination after drug withdrawal for 24 hours,and persist the peripheral blood and the tumors.4、Detect the levels of IL-2 and TNF-a in nude mice peripheral blood:ELISA method was used to detect the expression of IL-2 and TNF-a in the peripheral blood serum of the experimental group with cisplatin resistant human epithelial ovarian cancer xenograft in nude mice:After the nude mice peripheral blood centrifugation(4000r/minutes×15 minutes),take the supernatant,according to ELISA kit instructions operation,testing the expression of IL-2 and TNF-a in each group.5、The effect of TPL on cellular immunity:Flow cytometry was used to detect the changes of CD4~+and CD8~+in peripheral blood after the intervention of tumor cells in the peripheral blood of each group:First,take part in nude mouse transplanted tumor tissue,using mechanical scatter method into single cell suspension,then stay healthy adult women outside week blood 20ml,with lymphocyte separation liquid extract the lymphocyte,then the Transwell chamber in human lymphocytes and tumor cells are moved to the Chinese culture,the upper chamber on tumor cells(1×10~5),room temperature on lymphocyte(2×10~6).Finally,at 12h,24h,48h with flow cytometry to detect the changes of CD4~+,CD8~+in room.6.The changes of NK cells and angiogenesis in different experimental groups were detected by immunohistochemistry technique:5mm×5mm×5mm tumor tissue was placed in 4%formalin fixed,using immunohistochemistry to detect the expression of NK,CD56 and vascular endothelial growth factor related CD31,CD105 expression of CD16 cells in the tumor.The average gray value of each test index in each experimental group was analyzed by Proplus6.0 Image image analysis software,the greater the average gray value,the less expression of the protein in the tissue.Results:1、The concentration of each experimental group mice peripheral blood supernatant IL-2 were as follows:the combination group(133.40±8.60pg/ml),TPL group(73.40±5.52 pg/ml),DDP group(74.28±8.99 pg/ml),NS group(31.65±2.73pg/ml).Tip in each experimental group IL-2 concentration:combination group>DDP group>TPL group>NS group,in addition to TPL group and DDP group,the difference between groups have significant statistical significance(P<0.01).2、Each experimental group mice peripheral blood supernatant TNF-a concentrations were as follows:the combination group(70.46±6.00pg/ml),TPL group(155.36±13.57pg/ml),DDP group(164.45±19.84 pg/ml),NS group(293.72±29.06pg/ml).Tip in each experimental group TNF-a concentration:NS group>DDP group>TPL Group>combination group,in addition to TPL group and DDP group,the differences between groups have significant statistical significance(P<0.01).3、Cisplatin resistant ovarian carcinoma xenografts intervention CD4~+/CD8~+ratio of the normal adult female peripheral blood 12h、24h、48h experimental groups as follows:12h is the combination group(29.48±2.34),TPL group(21.00±2.24),DDP group(13.93±1.11),NS group(9.23±1.40);24h is the combination group(38.62±2.17),TPL group(26.25±1.07),DDP group(20.74±1.90),NS group(5.31±1.92);48h is the combination group(47.87±2.01),TPL group(30.78±2.68),DDP group(26.66±2.39),NS group(1.77±0.59).prompted CD4~+/CD8~+ratio in each experimental group:the combination group>TPL group>DDP group>NS group,the difference between groups have significant statistical significance(P<0.05).4、Cisplatin resistant ovarian carcinoma xenografts interfere with the normal adult female peripheral blood 12h、24h、48h,CD4~+/CD8~+ratio NS groups are as follows:12h(9.23±1.40),24h(5.31±1.92),48h(1.77±0.59);DDP groups are as follows:12h(13.93±1.11),24h(20.74±1.90),48h(26.66±2.39);TPLgroups are as follows:12h(21.00±2.24),24h(26.25±1.07),48h(30.78±2.68);combined groups are as follows:12h(29.48±2.34),24h(38.62±2.17),48h(47.87±2.01).Prompted each time point CD4~+/CD8~+ratio:NS groups is12h>24h>48h,differences between groups were significant statistical significance(P<0.05);DDP groups、TPLgroups and NS groups are 48h>24h>12h,differences between groups were significant statistical significance(P<0.05).5、The grey value of CD16 and CD56 Cin the transplanted tumor of nude mice in each group was:CD16 is the combination group(115.03±10.21),TPL group(128.31±7.25),DDP group(126.73±6.23),NS group(174.72±8.61);CD56 is the combination group(88.37±7.76),TPL group(111.84±2.89),DDP group(137.70±5.86),NS group(153.55±8.43).The expression of CD16 in each experimental group was suggested:Combination group>DDP group>TPL group>NS group,in addition to the TPL group and the DDP group,the expression of CD16 in each group were significantly different(P<0.05);The expression of CD56 in each experimental group was suggested:Combination group>TPL group>DDP group>NS group,the expression of CD56 in each group were significantly different(P<0.01).6、The grey value of CD 31 and CD105 in the transplanted tumor of nude mice in each group was:CD31 is the combination group(175.68±4.41),TPL group(164.87±7.06),DDP group(155.76±8.89),NS group(128.47±13.30);CD105is the combination group(159.13±7.40),TPL group(160.44±8.17),DDP group(159.53±15.18),NS group(117.79±13.43).The expression of CD31 in each experimental group was suggested:NS group>DDP group>TPL group>Combination group,in addition to the TPL group and the DDP group、the TPL group and the combination group,the expression of CD31 in each group were significantly different(P<0.01);The expression of CD105 in each experimental group was suggested:NS group>Combination group>DDP group>TPL group,compared with the NS goup,the expression of CD105 in each group were significantly different(P<0.01).Conclusion:1.TPL exert anti tumor activity by promote secrete IL-2 and inhibiton secretion of TNF-a.2.TPL play an anti tumor activity by improve peripheral blood CD4~+/CD8~+ratio,regulating the balance between immune cell subsets.3.TPL inhibit the formation of tumor blood vessels and prevent the invasion and migration of tumor cells by down regulating the expression of VEGF. |
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