胸腺素β4基因沉默对膀胱移行细胞癌上皮间质转化的逆转作用  

The reverse effect of thymosin β4 gene silencing on epithelial-mesenchymal transition in human bladder transitional cell carcinoma

作　　者：王智宇[1] 曾甫清[1] 朱朝辉[1] 蒋国松[1] 吕磊[1] 邢诗安[2] 

机构地区：[1]华中科技大学同济医学院附属协和医院泌尿外科,武汉430022 [2]华中科技大学同济医学院附属协和医院中心实验室,武汉430022

出　　处：《中华实验外科杂志》2010年第4期501-503,F0003,共4页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金资助项目（30872561）

摘　　要：目的观察胸腺素β4（Tβ4）基因沉默对膀胱癌细胞上皮间质转化（EMT）的逆转，探讨其在肿瘤侵袭转移中的作用机制。方法使用针对Tβ4的慢病毒载体（Lenti—Tβ4）转染膀胱癌细胞株T24。采用定量逆转录-聚合酶链反应（RT—PCR）、Westernblot法检测Tβ4、整合素连接激酶（ILK）和上皮性标记基因E—cadherin、β-catenin的表达改变；Immunofluorescence法检测ILK、E—cadherin、β-catenin在转染后T24细胞中的表达改变；细胞划痕实验、Boyden小室体外侵袭实验、AO／EB荧光染色法反映细胞转移潜能和凋亡的变化。结果转染后48h的T24细胞，Tβ4、ILK、β-cateninmRNA或蛋白表达开始下降，以转染后96h最明显；而上皮标记基因E-cadhefin在转染96h后，表达显著增加（P〈0．05）；Immunofluorescence显示ILK和β-catenin在细胞质、细胞核中表达减弱，而E—cadherin在胞膜中表达增强，细胞形态向正常上皮细胞转化；转染后的T24细胞体外迁移能力与侵袭力下降[（10．4±1．2）％比（73．5±1．4）％]，细胞凋亡增多（12．3％比36．6％）。结论肿瘤细胞中的TB4表达下调可以逆转肿瘤细胞的间质表型，而向正常上皮表型转化，降低肿瘤转移潜能。Objective To investigate the reverse effect of epithelial-mesenchymal transition （EMT） by silencing human gene thymosin β4（Tβ4） , and further reseach for its role on invasion and metastasis of cancer. Methods Lentiviral shRNA vector encoding Tβ4 was transfected into T24 cell line. The expression of Tβ4, ILK and epithelial markers E-eadherin, β-catenin were detected by real-time reverse transcription-polymerase chain reaction （RT-PCR） and Western blotting; the expression of integrin-linked kinase （ILK）,E-cadherin and β-catenin in T24 cells were detected by immunofluorescence after transfection via lentiviral vector;the metastatic potential and apoptosis were examined by in vitro cell wound model, Boyden chamber invasion assay and acridine orange-ethidium bromide fluorescent staining. Results The expression of Tβ4, ILK and β-catenin were decreased in the T24 cells after transfected with Tβ4 shRNA, and the expression of E-eadherin was significantly up-regulated （ P 〈 0.05 ） ; the level of ILK in cytoplasm and level of β-eatenin in nuclear were obviously decreased by immunofluorescence analysis, and the higher level of E-cadherin was observed, the morphology of T24 Cells was transformed into a normal epithelial phenotype ; the motility, invation of T24 cells [ （ 10. 4 ± 1.2 ） % vs （ 73.5 ±1.4 ） % ] were inhibited and apoptosis （ 12. 3% vs 36. 6% ） was enhanced. Conclusion The silencing of Tβ4 may reverse fibroblastoid morphology into a normal epithelial phenotype, and suppress tumor metastatic potential.

关 键 词：上皮间质转化 胸腺素Β4 移行细胞癌 慢病毒 

分 类 号：R737.14[医药卫生—肿瘤]