【作者】 崔志英；

【导师】 刘巍；

【作者基本信息】 河北医科大学 ， 肿瘤学， 2008， 硕士

【摘要】 目的:ERCC1(excision repair cross complementing1)和ERCC2(excision repair cross complementing2)即核苷酸切除修复交叉互补基因1和2,两者分别在DNA损伤修复中发挥受损DNA 5’端外切和双链解螺旋作用。大量文献和临床资料已经证实两者表达水平上调是铂类化疗药物耐药的重要分子基础。同时有资料显示ERCC2蛋白水平与抗有丝分裂剂紫杉醇敏感性呈显著负相关。尚无其mRNA水平与紫杉醇敏感性相关报道。顺铂和紫杉醇为经典化疗药物,在食管癌化疗中起着举足轻重的作用,但化疗并非对所有患者都有效。如何筛选出化疗敏感个体,减少陪疗人群,实现个体化化疗是目前研究热点。本研究通过测定6株常见食管鳞癌细胞株中ERCC1和ERCC2 mRNA水平表达,比较其表达水平与顺铂、紫杉醇药物敏感性的关系。并进一步研究了siRNA下调ERCC2表达后顺铂和紫杉醇敏感性的变化。以求在食管癌细胞株中证实ERCC1和ERCC2 mRNA水平与顺铂敏感性的关系,及探讨ERCC2 mRNA表达与紫杉醇敏感性的相关性。为临床筛选敏感个体和敏感药物,实现个体化化疗提供资料。方法:第一部分1培养国内常用食管鳞癌细胞株6株。RT-PCR法测定各细胞株ERCC1、ERCC2 mRNA表达及其表达相关性。2 MTT法测定浓度梯度的顺铂、紫杉醇作用24h后的IC50值(抑制率50%时的药物浓度)。比较两因子mRNA水平与顺铂、紫杉醇IC50值的相关性。第二部分1应用体外合成靶向ERCC2的siRNA(si-ERCC2),脂质体法瞬时转染ERCC2高表达细胞株KYSE150。同时设阴性对照、脂质体对照和空白对照组。RT-PCR和流式细胞术(FCM)分别检测各转染组细胞ERCC2 mRNA和蛋白水平的表达。2 MTT法检测各转染组细胞浓度梯度顺铂和紫杉醇的抑制率,计算IC50值。比较转染前后顺铂和紫杉醇IC50值的变化。结果:第一部分1 ERCC1和ERCC2 mRNA表达情况ERCC1表达:6株细胞均可测出ERCC1特异性条带,相对表达量在0.8214~0.1709间不等。表达最高的Eca109和最低的TE10表达量相差4.81倍。ERCC2表达:TE13未测出ERCC2条带,为不表达ERCC2细胞株。余5株细胞可测出ERCC2表达。以KYSE150表达量最高,为0.8712。余4株细胞表达量在0.0870~0.2033间不等。与表达最高的KYSE150表达量相差4~10倍。ERCC1和ERCC2表达相关系数Rs=0.258,P=0.076 >0.05。两因子表达存在相关趋势,但无统计学意义。2浓度梯度顺铂和紫杉醇作用24h后各细胞株IC50值及与ERCC1和ERCC2 mRNA表达的相关性顺铂IC50值在15.17±2.11ug/ml到42.84±3.82ug/ml间不等。其中KYSE150最高,TE13最低。两者相差2.82倍。紫杉醇IC50值在3.44±1.05ug/ml到12.85±2.17ug/ml间不等。其中KYSE150最高,TE1最低。两者相差3.74倍。ERCC1 mRNA水平与顺铂IC50值相关系数Rs=0.835,P<0.01。呈明显正相关。ERCC1 mRNA表达水平越高,顺铂IC50值越高,顺铂敏感性越低。ERCC2 mRNA水平与顺铂IC50值相关系数Rs=0.379,P<0.01。呈明显正相关。ERCC2 mRNA表达水平越高,顺铂IC50值越高,顺铂敏感性越低。ERCC1 mRNA水平与紫杉醇IC50值相关系数Rs= 0.180,P>0.05。两者之间无明显相关性。ERCC2 mRNA水平与紫杉醇IC50值相关系数Rs= 0.293,P<0.05。呈明显正相关。ERCC2 mRNA表达水平越高,紫杉醇IC50值越高,紫杉醇敏感性越低。第二部分1 siRNA转染KYSE150后ERCC2 mRNA及蛋白水平表达转染后ERCC2 mRNA水平表达:si-ERCC2组在转染后24、48、72h于相应部位均未测出ERCC2特异性条带。而三组对照三个时段均可见特异性条带,表达量间无明显差别(P>0.05)。说明si-ERCC2成功封闭了目的基因mRNA水平的表达。而三组对照对目的基因无干扰作用。转染后ERCC2蛋白水平表达:si-ERCC2组转染24、48、72h后ERCC2蛋白表达量分别为0.688±0.11,0.484±0.09和0.400±0.04。较对照组分别下调31.2%,51.6%和60.0%。而三组对照表达量无明显差别(P>0.05)。表明si-ERCC2成功下调了目的基因蛋白水平的表达,而三组对照对其无下调作用。2 siRNA转染KYSE150后顺铂和紫杉醇IC50值的变化si-ERCC2组顺铂IC50值为26.41±2.63ug/ml,较对照组40.32±3.44ug/ml降低34%。紫杉醇IC50值为6.32±0.87ug/ml,较对照组的12.66±1.69ug/ml降低49%。明显低于三组对照(P<0.01)。说明下调ERCC2表达降低了顺铂和紫杉醇IC50值,增加了两药敏感性。结论:1所测食管癌细胞株ERCC1、ERCC2有不同程度表达。2 ERCC1和ERCC2 mRNA水平均与顺铂敏感性呈负相关,ERCC2 mRNA水平与紫杉醇敏感性呈负相关。ERCC1 mRNA水平与紫杉醇敏感性无关。3转染siRNA成功下调了ERCC2 mRNA和蛋白水平的表达。4转染后细胞部分恢复了对顺铂和紫杉醇的敏感性,逆转了两药耐药,进一步说明ERCC2同时参与顺铂和紫杉醇耐药。更多还原

【Abstract】 Objective: ERCC1 (excision repair cross complementing 1) and ERCC2 (excision repair cross complementing 2) are leading members in DNA repair pathway. ERCC1 can execute the 5’incision and ERCC2 can open the damaged DNA double strand. Preclinical studies and clinical trials have confirmed that both genes were related to cisplatin (DDP) resistance. And a few data suggested that ERCC2 protein level might affect paclitaxel (PTX) sensitivity. There is no report about the relationship between ERCC2 mRNA and PTX up to now. DDP and PTX are effective agents and commonly used in esophageal carcinoma. But chemotherapy is not efftctive in all patients. How to select the chemosensitive individual and sensitivity drug is of great importance. This article detect the ERCC1 and ERCC2mRNA expression in 6 common esophageal carcinoma cell lines, and the IC50 after DDP and PTX treated for 24h. We evaluat the relationship between gene expression and DDP/PTX sensitivity. Moreover, we transfect KYSE150 using ERCC2 siRNA(small interfering RNA) cell, which harbored the highest ERCC2 mRNA. To observe the impact of ERCC2 siRNA transfecting on drug sensitivity. Methods:Section 11 ERCC1 and ERCC2 mRNA of six esophageal carcinoma cell lines are detected by Reverse transcription polymerase (RT-PCR) .To observe the gene expression correlation.2 MTT are used to detect IC50 after DDP and PTX treated for 24h. To observe the correlation between IC50 and gene expression.Section 21 Lipofectmine2000 are used to transfect siRNA into the cell KYSE150. The negative-control, lipofectmine 2000 and bl- ank control group are established.2 ERCC2 mRNA and protein expression are assayed by RT-PCR and FCM after transfection 24h,48h,72h respectively.3 IC50 of DDP and PTX are detected by MTT after transfection.Results:Section 11 ERCC1 mRNA is detected in all the 6 cell lines. The high-est expression cell line is Eca109 (0.8214), and the lowest is TE10 (0.1709). The highest is 4.81 times higher than the lowest.ERCC2 mRNA is detected in 5 cell lines except TE13. The highest expression cell line is KYSE150(0.8712), and the lowest is KYSE450 (0.0870). The highest is 4~10 times higher than the lowest. TE13 is under detectable line. There is no significant correlation between the expression of ERCC1 and ERCC2 (P=0.076).2 DDP IC50(ug/ml) is from 15.17±2.11 to 42.84±3.82 in 6 cells, while KYSE150 is the highest and TE13 is the lowest. The highest is 2.82 times than the lowest.PTX IC50(ug/ml) is from 3.44±1.05 to 12.85±2.17 in 6 cells, while KYSE150 is the highest and TE1 is the lowest. The highest is 3.74 times than the lowest.There is a positive correlation between ERCC1, ERCC2 mRNA and DDP IC50(P<0.01).A positive correlation between ERCC2 mRNA and DDP IC50(P<0.01) is found. And the correlation between ERCC1 mRNA and PTX IC50 is no statistical significance (P>0.05).Section 21 ERCC2 mRNA is not detectable in KYSE150 by RT- PCR after transfection for 24h, 48h and 72h in si-ERCC2 group. As for 3 control groups, ERCC2 level was no significant differ -ence compared with baseline (P>0.05).ERCC2 protein expression are 0.688±0.11, 0.484±0.09 and 0.400±0.04 after transfection for 24h,48h,72h. Compared with baseline, ERCC2 proein are descent 31.2%,51.6%,60.0% resp- ectively. Control groups are no any significance campared with the baseline(P>0.05). It indicates that siRNA could down- regulate ERCC2 in mRNA and protein level.2 IC50 (ug/ml) for DDP is 26.41±2.63 after transfection, which decrease 34%. PTX IC50 is 6.32±0.87 after transfection, is 49% decreased.si-ERCC2 IC50 is much lower than control groups (P<0.01). There is no any significance among control groups (P>0.05). The result suggests that DDP and PTX sensi- tivity increased after transfection.Conclusions:1 ERCC1 and ERCC2 is express in esophageal carcinoma cell lines at different levels. ERCC1 and ERCC2 expression are relate to DDP sensitivity, and ERCC2 mRNA are also correlate with PTX sensitivity.2 ERCC2 siRNA can dramaticlly down-regulate the expre- ssion of ERCC2 mRNA and protein.3 After transfection of ERCC2 siRNA, the cell partially revers DDP and PTX resistance. Futher support the presume that ERCC2 is not only involved in cisplatin resistance,but also make a contribution to Paclixetal resistance.更多还原