【作者】 田分；

【导师】 涂亚庭；

【作者基本信息】 华中科技大学 ， 皮肤病与性病学， 2010， 博士

【摘要】 第一部分c-FLIP在黑素瘤组织中的表达及其与临床病理特征的关系目的检测c-FLIP在黑素瘤组织及细胞系中的表达,探究其与临床病理特征之间的关系。方法免疫组化方法检测c-FLIP在77例黑素瘤组织及23例色素痣组织中的表达；并检测其在34例CA和16例正常包皮组织中的表达,同时用western blotting和RT-PCR检测了其分别在CA和正常包皮组织中的蛋白表达和mRNA水平。western blotting和流式细胞术检测c-FLIP在A375, A875, SK-Mel-1和SK-Mel-28黑素瘤细胞系中的表达。结果c-FLIP在恶性黑素瘤组织中的表达明显高于在色素痣中的表达,其高表达与组织病理学分型相关并与黑素瘤的Clark’s分级相关。与Ki-67标记指数(KI)高度相关。四株黑素瘤细胞中,A875细胞的c-FLIP的表达最高。c-FLIP在CA中的蛋白和mRNA水平均较正常包皮组织中的明显增高。结论c-FLIP除了具有抗凋亡功能外,它还具有促增殖作用,在恶性黑素瘤的侵袭性生物学特性中可能具有重要的作用,对恶性黑素瘤的预后具有重要意义。c-FLIP在CA中的高表达可能与角质形成细胞的过度增殖有关。第二部分c-FLIPshRNA载体的构建及鉴定目的构建针对c-FLIP不同亚型的shRNA真核表达载体c-FLIPT/L/S shRNA,鉴定其正确性,并转染恶性黑素瘤A875细胞系,证实其在体外培养的恶性黑素瘤细胞中对c-FLIP具有干扰作用。方法①人工合成互补并编码相应短发夹状c-FLIPT/L/S shRNAs的寡核苷酸链,将其插入到Pgenesil-1载体中,经酶切和测序鉴定所构建的重组体是否正确；②采用逆转录聚合酶链反应(RT-PCR)、Western blot和流式细胞术分别检测转染的A875细胞中c-FLIP mRNA和蛋白的表达变化。③在人恶性黑素瘤细胞系A875中,用G418筛选沉默效果最佳的c-FLIPshRNAs,用流式细胞术(FCM)检测GFP的表达,鉴定稳定转染的单克隆细胞系。结果①经酶切和测序证明c-FLIPT/L/S shRNAs序列正确；②A875细胞系中转染c-FLIPT/L/S shRNAs载体后,c-FLIPT1shRNA和c-FLIPT1shRNA沉默效果最佳。③荧光显微镜下观察到了G418筛选稳定表达c-FLIPT1shRNA的A875细胞,FCM检测GFP表达均在96%以上。结论测序结果表明发卡样的c-FLIPT/L/S shRNAs真核表达载体构建成功,转染A875细胞后获得稳定表达,并可特异性封闭c-FLIPT/L/S的表达,筛选和鉴定出稳定转染单克隆细胞系A875,为后续实验及进一步研究c-FLIPT/L/S shRNAs载体在恶性黑素瘤治疗中的作用提供了理论基础。第三部分c-FLIP的下调诱导恶性黑素瘤A875细胞系JNK信号途径的活化目的观察c-FLIP不同亚型shRNA对恶性黑素瘤A875细胞系JNK信号途径的影响。方法western blot检测c-FLIPT/L/S shRNAs真核表达载体稳转的A875细胞系中p-JNK表达水平。结果与未转染质粒的A875细胞和稳定转染c-FLIPC shRNA真核表达载体的A875细胞相比,c-FLIPT/LshRNAs真核表达载体稳定转染的A875细胞系中p-JNK表达水平明显升高,而c-FLIPS-shRNA真核表达载体稳转的A875细胞系中p-JNK表达水平无明显变化。结论在黑素瘤细胞系中下调c-FLIP (c-FLIPL)的表达可以诱导JNK信号途径的活化。第四部分c-FLIP不同亚型shRNA对恶性黑素瘤细胞生物学特性的影响目的观察c-FLIP各亚型shRNA对A875细胞的生长、增殖及凋亡的影响。方法在c-FLIPT/L/S/C shRNA稳定转染A875细胞中,运用绝对细胞计数对c-FLIP各亚型shRNA稳定转染细胞绘制生长曲线,比较其与对照组的差异；用MTT法检测各稳定转染细胞增殖能力的差异；用Annexin v/PI双染细胞,检测各组细胞凋亡率的差异。结果稳定转染c-FLIPT/L/sshRNAA875细胞生长速度较未转染质粒的A875细胞和稳定转染c-FLIPC shRNA A875细胞的生长速度慢,进入对数生长期的时间延长且平台降低。与未转染质粒的A875细胞和稳定转染c-FLIPC shRNA真核表达载体的A875细胞相比,稳定转染c-FLIPT/L/S shRNA真核表达载体的A875细胞光密度值明显降低。运用Annexin v/PI双染,流式细胞术检测细胞凋亡显示与未转染质粒的A875细胞和稳定转染c-FLIPc shRNA真核表达载体的A875细胞相比,稳定转染c-FLIPT/L/S shRNA真核表达载体的A875细胞凋亡率明显增加。结论靶向c-FLIP基因不同亚型的shRNA不仅对黑素瘤细胞的生长和增殖具有抑制作用,而且能够促进黑素瘤的凋亡。更多还原

【Abstract】 PartⅠExpression of c-FLIP in malignant melanoma and itsrelation to the clinicopathologic featuresObjective:The aim of this work was to examine the expression patterns of c-FLIP in MM in vivo and explore the relationship of its expression with the clinicopathologic featuresMethods:Immunohistochemical staining with anti-c-FLIP antibody was performed in 77 tissue samples obtained from MM and 23 tissue samples from naevi. Immunoperoxidase staining method was applied to analyze the location of c-FLIP expressions in 34 CA and 16 normal foreskin tissues. Real time quantitative reverse transcriptase-polymerase chain reactions (RT-PCR) and western blotting were performed to further identify the expression of c-FLIP in CA. c-FLIP expression in A375, A875, SK-Mel-land SK-Mel-28 cell lines were examined by WB and FCM.Results:The expression of c-FLIP was increased in MM tissue compared with the detectable levels in the matched pigmented naevi lesions. It was significantly associated with the histological type and Clark’s level of malignant melanoma. In four MM cell lines, c-FLIP expression was the highest in A875 cell lines. c-FLIP expression at either its mRNA or protein level was significantly higher in CA than that in normal foreskin.Conclusion:Exprect the role of anti-apoptosis, c-FLIP could promote the proliferation of cells. It might play an important role in the obtaining of aggressive biologic behaviors and be useful in predicting prognosis of patients with MM. Overexpression of c-FLIP might be involved in the hyperproliferation of keratinocytes in CA. PartⅡConstruction and Identification of c-FLIP shRNAsObjectives:Short interfering RNA (siRNA) eukaryotic expression vector for c-FLIPT/L/S shRNA was constructed and transfected into MM A875 cell lines. To verify its effection of interference for c-FLIP in MM cell lines in vitro.Methods:①c-FLIPT/L/S shRNA targeting human c-FLIP isoforms common sequence was synthesized and it was inserted into Bam HI-HindⅢlinearized Pgenesile-1 vector. The sequence of c-FLIPT/L/S shRNA plasmid was analyzed by DNA sequencer and restrict endonuclease cutting.②To screen the best silencing effect c-FLIPT/L/S shRNA, the alteration of c-FLIP mRNA and protein was checked by Rt-PCR, western blot and FCM after c-FLIPT/L/S shRNAs were transfected in MM cell lines.③The monoclone A875 cells with stable expression of best effect c-FLIPshRNAs were obtained by G418 selection and were identified with checking GFP expression by FCM.Results:①It was verified that the sequence of constructed recombinant plasmids were correct by DNA sequencing and restrict endonuclease cutting.②Among the c-FLIPT/L/S shRNAs we tested, c-FLIPT1shRNA和c-FLIPL1shRNA possessed the strongest inhibitory effect against c-FLIP.③We screened and obtained A875 cell lines with stable expressions of c-FLIPT/L/S shRNAs as A875 stable clone could observed under Fluorescence microscope and 96% A875 stable clone expressed GFP detected by FCM.Conclusions:It indicated that hairpin siRNA eukaryotic expression vector for c-FLIP isoforms would be successfully establishedand. It played a specific inhibitory role in A875 cell lines. This study laid theoretical foundation for further research of the therapy of c-FLIPT/L/S shRNAs vector in MM. PartⅢDownregulation of c-FLIP induce JNK activion in malignant melanoma cell linesObjective:To observe the influence of difference isoforms of c-FLIP shRNA on JNK pathway in MM A875 cell lines.Methods:To detecte the expression of p-JNK protein in stable clone A875 transfected with c-FLIPT/L/S shRNAs by western blot.Results:Comparing with untranstected and stable transtected with c-FLIPC shRNA A875 cell lines, p-JNK protein was increased significantly in A875 cell lines which were transtected with c-FLIPT/LshRNA. And in transtected with c-FLIPS shRNA A875 cell lines, p-JNK protein was unchanged.Couclusions:Downregulation of c-FLIP can induce the activion of JNK pathway in MM A875 cell lines. PartⅣThe influence of difference isoforms of c-FLIP shRNA on bionomics of MM cell linesObjective:To observe the influence of difference isoforms of c-FLIP shRNA on bionomics of MM A875 cell lines such as growth, proliferation and apoptosis.Methods:To count cells of A875cell lines which were transtected with c-FLIPT/L/S/C shRNA and obtain the cells’growth curve, and then to compare the difference among those type of cells. To detecet the proliferational activity of those cells by MTT; Using annexin V and propidium iodide(PI) double staining, we observe the rate of apoptosis by Flow cytometry(FCM).Results:Comparing with untranstected and stable transtected with c-FLIPc shRNA A875 cell lines, the growth were slower in A875 cell lines which were transtected with c-FLIPT/LshRNA. And the time they enter the log phase prolonged and platform were cut down. And optical density decreased significantly. Using annexin V and propidium iodide(PI) double staining, we observed that the rate of apoptosis of A875 cell lines which were transtected with c-FLIPT/LshRNA comparing with untranstected and stable transtected with c-FLIPc shRNA A875 cell lines by FCMConclusions:Difference isoforms of c-FLIP shRNA can not only suppress the growth and proliferation of MM A875 cell lines but also promote their apoptosis.更多还原