【作者】 刘海英；

【导师】 魏素菊；

【作者基本信息】 河北医科大学 ， 肿瘤学， 2008， 硕士

【摘要】 目的:卵巢癌患者对化疗产生耐药性是影响疗效的主要原因之一。传统的逆转剂因副作用大而使得临床应用受到限制,因此寻找高效、低毒的逆转剂是目前研究方向及热点之一。重组改构人肿瘤坏死因子(recombinant mutant human tumor necrosis factor, rmh-TNF)是由国内学者通过蛋白质工程技术改造天然TNF的结构而研制出的一种高效、低毒的TNF突变体。本研究通过体外实验观察rmh-TNF对人卵巢癌多药耐药细胞系SKOV3/DDP耐药性的影响,并对可能存在的机制进行初步的探讨,以期寻找一种新的药物帮助解决卵巢癌治疗中出现的多药耐药问题。方法:体外培养人卵巢癌多药耐药细胞株SKOV3/DDP,采用四氮唑噻蓝盐(MTT)比色法检测rmh-TNF对SKOV3/DDP耐药株的细胞毒作用,选择杀伤率<10%的浓度作为该药物的无毒剂量,即本实验的逆转剂量。同法测定逆转剂量的rmh-TNF干预后细胞株SKOV3/DDP对顺铂耐药性的变化;应用流式细胞术(FCM)检测rmh-TNF干预不同时间段SKOV3/DDP细胞株中GST-π蛋白、BCL-2蛋白的表达,应用逆转录酶链反应(RT-PCR)分析rmh-TNF处理SKOV3/DDP细胞前后mdr1 mRNA、BCL-2 mRNA的表达水平。结果:1 MTT结果显示: rmh-TNF在(50-3200)U/ml以内能抑制SKOV3/DDP细胞的体外增殖,且随着浓度增加抑制率相应上升。回归分析得出当rmh-TNF终浓度<122.34U/ml,其对细胞生长无明显抑制作用(细胞存活率均﹥90%)因此本实验选用100U/ml作为逆转剂量。100U/ml rmh-TNF作用SKOV3/DDP 24、48、72h的逆转倍数分别为1.19倍、2.20倍、2.64倍。2流式细胞术检测结果显示rmh-TNF处理前SKOV3、SKOV3/DDP两组细胞GST-π蛋白表达量的相对值(FI)分别是1、2.33±0.30,两株细胞GST-π蛋白的表达具有极显著性差异(P<0.01),100U/ml rmh-TNF作用细胞SKOV3/DDP 24、48、72h后GST-π蛋白表达量的相对值随作用时间的延长而逐渐降低,FI值分别为1.82±0.13、1.73±0.28、1.31±0.17,作用前后比较均有显著性差异(P<0.05);rmh-TNF处理前SKOV3、SKOV3/DDP两株细胞BCL-2蛋白表达量的相对值(FI)分别是1、2.62±0.15,两株细胞BCL-2蛋白的表达具有极显著性差异(P<0.01),100U/ml rmh-TNF作用细胞SKOV3/DDP 24、48、72h后BCL-2蛋白表达量的相对值随作用时间的延长而逐渐降低,FI值分别为2.24±0.20、1.71±0.19、1.30±0.15,作用前后比较均有显著性差异(P<0.05)。3 RT-PCR法检测结果显示rmh-TNF处理前SKOV3细胞株不表达mdr1 mRNA, SKOV3/DDP细胞株mdr1 mRNA表达量的相对值(mdr1/β-actin)是0.76±0.01。100U/ml rmh-TNF作用细胞株SKOV3/DDP 24、48、72,mdr1 mRNA表达量的相对值随作用时间的延长而逐渐降低,mdr1/β-actin值分别为0.66±0.05、0.52±0.04、0.45±0.03,作用前后差异均有显著性。(P<0.05);rmh-TNF处理前SKOV3、SKOV3/DDP两株细胞BCL-2 mRNA表达量的相对值(BCL-2/β-actin)分别是0.09±0.15、0.47±0.01,两株细胞BCL-2 mRNA的表达具有极显著性差异(P<0.01),100U/ml rmh-TNF作用细胞SKOV3/DDP 24、48、72h后BCL-2 mRNA表达量的相对值随作用时间的延长而逐渐降低,BCL-2/β-actin值分别为0.45±0.01、0.31±0.01、0.27±0.01,作用前后比较均有极显著性差异(P<0.01)结论:1重组改构人肿瘤坏死因子(rmh-TNF)在(50-3200)U/ml浓度范围内,对SKOV3/DDP细胞有生长抑制作用,且呈浓度依赖性。2逆转剂量的重组改构人肿瘤坏死因子(rmh-TNF)对SKOV3/DDP细胞耐顺铂效应有一定的逆转作用,且逆转效应存在时间-效应关系。3逆转机制可能为:①逆转剂量的重组改构人肿瘤坏死因子(rmh-TNF)可能通过下调耐药相关基因mdr1 mRNA表达,进而降低SKOV3/DDP细胞的耐药性实现逆转耐药。②逆转剂量的重组改构人肿瘤坏死因子(rmh-TNF)可在转录和蛋白翻译水平降低BCL-2表达,进而降低BCL-2抑制凋亡的作用,使耐药细胞株凋亡增加,发挥对细胞耐顺铂效应的逆转作用。③逆转剂量的重组改构人肿瘤坏死因子(rmh-TNF)可能通过下调GSTπ表达,减少细胞毒性药物代谢产物的外排从而实现耐药性的逆转。更多还原

【Abstract】 Object:Multidrug resistance (MDR) of Ovarian cancer cells is one of the major reasons that lead to chemothrapy falure, therefor how to overcome MDR to increase the curative effect of chemotherapy has become the focus. Rcombinant mutant human tumor necrosis factor(rmh-TNF) is a high- efficiency low-toxicity mutation developed by chinese scholar. This experiment aimed to investigate the reversing effect and its possible mechanism of rmh-TNF on Cisplatin-resistant Human Ovarian cell lines in vitro.Methods:Choosing Human multidrug-resistant Ovarian cell lines SKOV3/DDP ,Detect rmh-TNF’s cell toxicity to SKOV3/DDP to select nontoxic dose and examine the vary of Cisplatin-resistant by MTT assay. The expression of BCL-2 protein and GST-πprotein were measured by flow cytometry. The expression of BCL-2 mRNA and mdr1 mRNA were studied by RT-PCR in SKOV3/DDP cells.Results:1 MTT assay results: Within the concentration of ( 50-3200 ) U/ml, rmh-TNF can inhibit proliferation of SKOV3/DDP cells in vitro. The inhibition ration was stepping up as the concentration increasing, When rmh-TNF’s concentration was low than 122.34U/ml , rmh-TNF could not obviously proliferation inhibiting by regression analysis, and the experiment choose 100 U/ml for nontoxic dose. After being treated with 100U/ml rmh-TNF for 24、48、72h,the RF are 1.19、2.06 and 2.64 .2 FCM assay results: The FI-value of GST-πprotein expression in SKOV3 was 1,while 2.33±0.30 in SKOV3/DDP, it was significant difference in the expression of GST-πprotein (P<0.01).After being treated with rmh-TNF for 24、48、72h,GST-πprotein expression in SKOV3/DDP cell became reducing as action time increasing, and the FI-value are 1.82±0.13 1.73±0.28 and 1.31±0.17 , There was significantly difference in the GST-πprotein expression between before and after rmh-TNF action. (P<0.05) ;The FI-value of BCL-2 protein expression in SKOV3 was 1,while 2.62±0.15 in SKOV3/DDP, their BCL-2 protein expression was significant difference (P<0.01). After being treated with rmh-TNF for 24h、48h、72h,BCL-2 protein expression in SKOV3/DDP cell became reducing as action time increasing, and the FI-value are 2.24±0.20、1.71±0.19 and 1.30±0.15, There was significant difference of BCL-2 protein expression between before and after rmh-TNF action. (P<0.05) 3 RT-PCR detection results: Before being treated with rmh-TNF , The gene mdr1 mRNA was expressed in SKOV3/DDP cell, and hardly espressed in SKOV3 cell.After being treated by rmh-TNF for 24、48、72h mdr1 mRNA expression in SKOV3/DDP cell became reduing as action time increasing, the weakest expression was in the group with 72h. The mdr1/β-actin value are 0.66±0.05、0.52±0.04 and 0.45±0.03,rmh-TNF could inhibiting the expression of mdr1 mRNA in a certain extent. There was significant difference of mdr1 mRNA expression between before and after rmh-TNF action. (P<0.05); The value of BCL-2 mRNA expression in SKOV3 was 0.09±0.15,while 0.47±0.01 in SKOV3/DDP, their BCL-2 mRNA expression was significant difference (P<0.01). After being treated with rmh-TNF for 24h、48h、72h BCL-2 mRNA expression in SKOV3/DDP cell became reduing as action time increasing,the weakest expression was in the group with 72h. The mdr1/β-actin value are 2.24±0.20、1.71±0.19、1.30±0.15, rmh-TNF could inhibiting the expression of BCL-2 mRNA in a certain extent. There was significantly difference of BCL-2 mRNA expression between before and after rmh-TNF action.Conclusion: 1 Within the concentration of (50-3200)U/ml, rmh-TNF might inhibit proliferation of SKOV3/DDP cells in vitro 2 Nontoxic doses rmh-TNF could partly reverse the Cisplatin-resistant of SKOV3/DDP cells. 3 The mechanisnm of reverse might conclude three sides:①Within a certain drug concentration , rmh-TNF might reverse multidrug-resistance by inhibiting expression of mdr1 mRNA.②rmh-TNF could reduce the apoptosis-inhibition action by inhibition the expression of BCL-2 mRNA and BCL-2 protein to promote cells apoptosis.③Within a certain drug concentration, rmh-TNF might reduce the drainage of celltoxicitive drug’s metabolizable producetion by inhibiting expression of GST-πprotein.更多还原