【作者】 臧凤琳；

【导师】 孙保存；

【作者基本信息】 天津医科大学 ， 肿瘤学， 2009， 博士

【摘要】 研究目的1．观察c-FLIP(L)蛋白及TRAIL凋亡通路受体DR4／DR5在乳腺癌临床标本及体外培养细胞系中的表达，结合临床病理资料加以分析，初步探讨c-FLIP(L)蛋白、DR4／DR5受体在乳腺癌中的存在意义。2．在乳腺癌组织和细胞系中，观察c-FLIP(L)蛋白与细胞凋亡敏感性的相关性，明确c-FLIP(L)蛋白对乳腺癌TRAIL凋亡通路的调节作用。3．以凋亡信号转导中caspases蛋白酶级联活化程序为切入点，通过研究c-FLIP(L)蛋白和caspase-8分子的相互作用，初步探讨c-FLIP(L)促进乳腺癌细胞TRAIL凋亡通路转导的作用机理。研究方法1．选取乳腺浸润性导管癌和配对的癌旁乳腺组织各150例，应用免疫组织化学方法检测c-FLIP(L)蛋白的表达，搜集患者的临床病理参数，以DR4、DR5的染色评价TRAIL通路受体表达水平，所得结果进行统计学分析。2．体外培养乳腺癌细胞系，应用半定量RT-PCR和Western blot技术从mRNA和蛋白两个水平检测c-FLIP(L)表达水平，通过间接免疫荧光流式技术检测DR4、DR5受体的阳性率，比较不同细胞间上述指标的差异。3．通过TUNEL技术和对Ki-67的免疫组化检测，评价乳腺癌细胞的凋亡指数和增殖指数，分析c-FLIP(L)蛋白表达水平与二者的相关性。4．在乳腺癌细胞系MDA-MB-231细胞中，转染特异性表达质粒，应用RT-PCR和Western blot技术检测c-FLIP(L)mRNA和蛋白的表达水平。在c-FLIP(L)高表达的前提下，外源给予TRAIL诱导细胞凋亡，MTT法绘制TRAIL作用的剂量曲线和时间曲线，选择最佳的诱导条件。在TRAIL处理下，应用Annexin V流式细胞分析术、DNA Ladder和相差显微镜技术观察MDA-MB-231细胞在高表达c-FLIP(L)后凋亡敏感性的变化。5．以转染c-FLIP(L)表达质粒的乳腺癌细胞系MDA-MB-231为研究对象，通过caspases蛋白酶活性分析，观察c-FLIP(L)蛋白对酶活性的影响。应用Westem blot技术，检测c-FLIP(L)蛋白与caspase-8分子的酶切水解产物，推测它们的活化过程，并进一步应用免疫共沉淀技术分析二者的相互作用。研究结果1．c-FLIP(L)蛋白在乳腺浸润性导管癌组织中呈低～中等程度表达(阳性率为36．36％)，在癌旁乳腺组织中呈高表达(阳性率为75％)；其中强阳性染色病例在乳腺癌组织中占14．29％，在癌旁乳腺组织中占57．58％；两组比较有显著性差别(P＜0．01)。统计分析结果显示，c-FLIP(L)蛋白高表达与患者年龄(＜35岁)、绝经前、未发生远处转移、较早的pTNM分期(Ⅰ-Ⅱ期)密切相关以及ER、PR蛋白的表达水平相关(P＜0．05)。2．在体外培养的不同乳腺癌细胞系中，均可检测到c-FLIP(L)mRNA和蛋白的表达，但c-FLIP(L)在不同的细胞间存在表达差异。TRAIL通路受体DR4、DR5在不同的乳腺癌细胞中表达水平不同，相对于每一株细胞来说，二者的阳性率呈现一致性。3．免疫组化染色发现，c-FLIP(L)蛋白的表达与DR4受体的表达呈正相关，二者之间关系有统计学意义(r_s=0．452，P=0．035)；对于DR5受体，虽然呈现出c-FLIP(L)蛋白表达越高，DR5受体表达越高的趋势，但二者之间无明显相关性(r_s=0．419，P=0．066)。此外，c-FLIP(L)蛋白的表达与凋亡指数正相关(r_s=0．554，P=0．005)；该蛋白表达升高增殖指数有所下降，但二者之间无显著相关性。4．转染特异性表达质粒发现，转染后24h，c-FLIP(L)mRNA和蛋白含量出现升高；转染后48h和72h mRNA含量仍能维持较高水平，蛋白的表达量有所下降。在此基础上，TRAIL诱导细胞4h，抑制率出现变化，随时间延长呈现逐渐升高的趋势，其中处理24h可达到较高水平；每个观察时间点下TRAIL浓度为10ng/ml时的抑制率均较高，随浓度加大仍能维持。5．TRAIL诱导后，在高表达c-FLIP(L)的MDA-MB-231细胞中，凋亡细胞比例可升至51．65％；经电泳分析，其DNA断裂呈规律的阶梯状；此时大部分细胞失去原有形态，胞体圆缩，间隙增大，折光性减弱，分裂像少见。6．应用TRAIL诱导凋亡后，在高表达c-FLIP(L)的MDA-MB-231细胞中，caspase-8活性提升5倍，caspase-3活性提升3倍，caspase-9、caspase-2活性变化不明显。7．与转染空载体细胞相比，TRAIL可诱导c-FLIP(L)高表达细胞内的caspase-8蛋白发生酶切降解，全长的p55 caspase-8含量减少，胞浆中出现p43、p18和p10亚片段；同时c-FLIP(L)p55片段含量亦出现降低，p43和p12片段含量增多。通过免疫共沉淀实验发现，c-FLIP(L)蛋白与caspase-8蛋白能够发生相互作用，此外作用复合物上尚可检测到二者的p43片段。研究结论1．通过检测c-FLIP(L)在癌和癌旁组织中的表达、明确该蛋白调节细胞凋亡的作用机制，从体内和体外不同角度证实，c-FLIP(L)在乳腺癌中具有促进TRAIL凋亡通路的作用，其阳性表达提示较为良好的临床预后。2．c-FLIP(L)作为天然存在的蛋白酶类似物，在不同条件下可能存在不同的表达水平、具有截然不同的凋亡调节功能，具体机制仍需要进一步的研究。3．鉴于c-FLIP(L)在不同类型的肿瘤组织和不同个体间存在表达差异，在将其开发为有意义的预测因子和治疗靶点之前，要充分考虑这一特点，注意诊治的个体化。更多还原

【Abstract】 Objective1.To study the expression of c-FLIP(L) and TRAIL-DR4/DR5 in human breastcancer tissue and cell lines and analysis the correlation between c-FLIP(L) expressionand clinicopathologic parameters.2.To observe the association of c-FLIP(L) expression and apoptotic sensitivity inbreast cancer tissue and cell lines and primarily confirm the exact regulation ofc-FLIP(L) in TRAIL pathway in breast cancer.3.To study the co-operation between c-FLIP(L) and caspase-8 and make sure themechanism of c-FLIP(L) in TRAIL pathway in breast cancer primarily.Methods1.Use the technique of IHC to detect the expression of c-FLIP(L) and DR4/DR5 inbreast cancer tissue and its counterparts (150 cases each).Analysis the association ofthese factors and the correlation between c-FLIP(L) and clinicopathologic parameters.2.Detect c-FLIP(L) in mRNA and protein level by RT-PCR and Western blot andevaluate the difference of DR4/DR5 by indirect immunofluorescence FCM in breastcell lines.3.Calculate the apoptosis index and proliferate index by TUNEL and Ki-67immunohistochemistry and evaluate their correlation.4.Establish c-FLIP(L) high-expressed MDA-MB-231 breast cancer cell line bytransfecting c-FLIP(L) plasmids.Under TRAIL inducing,map out dose-dependentand time-dependent curve and select the perfect condition by MTT analysis.Detectapoptosis by Annexin V-FITC,DNA Ladder and phase contrast microscope inc-FLIP(L) high-expressed MDA-MB-231 cells.5.Use caspases assay plates,Western blot and Co-immunoprecipitation (Co-IP) toanalysis the co-operation between c-FLIP(L) and caspase-8.Results1.c-FLIP(L) is low or moderate in breast invasive ductal cancer tissue (36.36%),while it is high-expressed in normal counterparts (75%) (P＜0.01).The positive rate ofexpression level of+++ is 14.29% in malignant and 57.58% in normal breast tissue (P＜0.01).c-FLIP(L) expression is positively associated with age,menopausal status,distant metastasis and pathologic stage (P＜0.05).Still c-FLIP(L) is positive in the ERand/or PR positive cases and it has no correlation with Her-2 expression.2.c-FLIP(L) and DR4/DR5 are present in four breast cancer cell lines.Although thereare different levels of these factors,DR4 and DR5 are consistent in single cell line.3.DR4 is high-expressed in the cases which has high level of c-FLIP(L) and thecorrelation of them is 0.452 (P=0.035).As to DR5 and c-FLIP(L),the association hasno statistic significance (r_s=0.419,P=0.066).There is positive correlation betweenc-FLIP(L) expression and apoptotic index (r_s=0.554,P=0.005).When c-FLIP(L) ishigh-expressed,proliferate index changed into low or moderate and they are notstatistic correlated.4.The mRNA and protein level of c-FLIP(L) are increased after transfecting plasmids24h,and the mRNA level is still after 48h and 72h,while protein level is decreasedslightly.The growth inhibition rate begins to change after TRAIL induction 4h and ishigh after 24h.Under each time point,the rate increases obviously when TRAIL is10ng/ml compared with other doses.5.After TRAIL inducing,apoptotic rate increases to 51.56% in c-FLIP(L)high-expressed MDA-MB-231 cells.DNA Ladder can be detected and apoptoticmorphologic changes can be observed by phase contrast microscope.6.Under TRAIL inducing,the caspase-8 activity increases to 5-folds and caspase-3to 3-folds in c-FLIP(L) high-expressed MDA-MB-231 cell line,while the activitiesofcaspase-2 and caspase-9 do not change obviously.7.Compared with cells transfected with pEGFP-N1,there are some changes in thecells with c-FLIP(L) plasmids:the p55 segment of caspase-8 decreases and the p43,p 18,and p 10 increase;the p55 of c-FLIP(L) also decreases and p43 and p 12 increase.c-FLIP(L) can co-operate with caspase-8 by Co-IP analysis,and the p43 segment ofthese two factors can be detected in their complex.Conclusion1.We confirm that c-FLIP(L) protein can promote TRAIL pathway in breast cancerby ditecting the expression of c-FLIP(L) in cancer and its counterparts and observingthe exact regulation of c-FLIP(L) in apoptosis.We also report that the low-expressed c-FLIP(L) indicats good prognosis in breast cancer.2.c-FLIP(L) may have different expression level and apoptotic modulation underdifferent conditions,which needs further research to confirm.3.We should consider the different of c-FLIP(L) in diverse cancer types and distinctindividuals before use it as a significative target for prognosis and therapy.更多还原