下调c-flip在恶性黑素瘤细胞系中的表达诱导jnk信号途径活化的研究-down - regulation of c - flip expression in malignant melanoma cell lines induces activation of jnk signaling pathway.docx

华中科技大学博士学位论文 华 中 科 技 大 学 博 士 学 位 论 文 PAGE 10 PAGE 10 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/SshRNA A875 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 Annexinⅴ/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。 关键词 MM；c-FLIPT/L/S/shRNA； 增殖；凋亡；生长曲线； MTT Downregulation of c-FLIP induce JNK activion in malignant melanoma cell line Abstract PartⅠ Expression of c-FLIP in malignant melanoma and itsrelation to the clinicopathologic features Objective: 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 features Methods: 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-1and 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 prot