CAJ | 학술논문

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(knti-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)、Western blot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124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48 h的T24细胞,Tp4、ILK、β-catenin mRNA或蛋白表达开始下降,以转染后96 h明显;而上皮标记基因E-cadherin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324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Tβ4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.
목적 관찰흉선소β4(Tβ4)기인침묵대방광암세포상피간질전화(EMT)적역전,탐토기재종류침습전이중적작용궤제.방법 사용침대Tβ4적만병독재체(knti-Tβ4)전염방광암세포주T24.채용정량역전록-취합매련반응(RT-PCR)、Western blot법검측Tβ4、정합소련접격매(ILK)화상피성표기기인E-cadherin、β-catenin적표체개변;Immunofluorescence법검측ILK、E-cadherin、β-catenin재전염후124세포중적표체개변;세포화흔실험、Boyden소실체외침습실험、AO/EB형광염색법반영세포전이잠능화조망적변화.결과 전염후48 h적T24세포,Tp4、ILK、β-catenin mRNA혹단백표체개시하강,이전염후96 h명현;이상피표기기인E-cadherin재전염96h후,표체현저증가(P<0.05);Immunofluorescence현시ILK화β-catenin재세포질、세포핵중표체감약,이E-cadherin재포막중표체증강,세포형태향정상상피세포전화;전염후적324세포체외천이능력여침습력하강[(10.4±1.2)%비(73.5±1.4)%],세포조망증다(12.3%비36.6%).결론 종류세포중적Tβ4표체하조가이역전종류세포적간질표형,이향정상상피표형전화,강저종류전이잠능.
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 me-tastasis of cancer. Methods Lentiviral shRNA vector encoding TIM was transfected into T24 cell hne. The expression of Tβ4, ILK and epithelial markers E-cadherin, β-cateniu 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 transfec-tion 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-cadherin was significantly up-regulated (P <0. 05 ) ;the level of ILK in cytoplasm and level of β-catenin 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 phe-notype ; the motility, invation of T24 cells [(10.4 ± 1.2) % vs (73.5 ± 1.4 ) %] were inhibited and apop-tosis (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.