中华实验外科杂志
中華實驗外科雜誌
중화실험외과잡지
CHINESE JOURNAL OF EXPERIMENTAL SURGERY
2014年
2期
353-355
,共3页
张士龙%丁德刚%张祥生%单磊%刘中华
張士龍%丁德剛%張祥生%單磊%劉中華
장사룡%정덕강%장상생%단뢰%류중화
细胞周期%S期激酶相关蛋白2%移行细胞癌%腺病毒
細胞週期%S期激酶相關蛋白2%移行細胞癌%腺病毒
세포주기%S기격매상관단백2%이행세포암%선병독
Cell cycle%S-phase kinase-associated protein 2%Transitional cell carcinoma%Adenovirius
目的 观察沉默S期激酶相关蛋白2(Skp2)基因对细胞周期蛋白依赖性激酶抑制蛋白27(P27KiP1)、第10号染色体缺失的磷酸酶及张力蛋白同源物基因(PTEN)的表达调控及对膀胱癌细胞生长活性的影响,探讨其在肿瘤侵袭、凋亡中的作用机制.方法 采用Western blot法检测经腺病毒载体(Ad-Skp2)转染后膀胱癌细胞株EJ的Skp2、P27kip1和PTEN的表达改变;用细胞划痕实验、Boyden小室体外侵袭实验、吖啶橙/溴化乙锭(AO/EB)荧光染色法、噻唑蓝(MTT)比色法反映转染后细胞迁移力、外侵袭力、凋亡及生长活性的变化.结果 转染后48 h的膀胱癌EJ细胞,Skp2蛋白表达开始下降,以转染后96 h最明显;而P27kiP1和PTEN在转染96 h后,表达显著增加(P<0.05);转染后的EJ细胞体外侵袭力下降[(11.7±1.2)%比(69.5±1.4)%,P<0.05],迁移能力减弱(P<0.05),细胞凋亡增多(11.8%比45.6%,P<0.05),生长活性降低(P<0.05).结论 膀胱癌细胞中的Skp2表达下调可能通过负向调控P27kipl、PTEN而影响癌细胞的分裂周期,从而使癌细胞衰老,最终诱导凋亡.
目的 觀察沉默S期激酶相關蛋白2(Skp2)基因對細胞週期蛋白依賴性激酶抑製蛋白27(P27KiP1)、第10號染色體缺失的燐痠酶及張力蛋白同源物基因(PTEN)的錶達調控及對膀胱癌細胞生長活性的影響,探討其在腫瘤侵襲、凋亡中的作用機製.方法 採用Western blot法檢測經腺病毒載體(Ad-Skp2)轉染後膀胱癌細胞株EJ的Skp2、P27kip1和PTEN的錶達改變;用細胞劃痕實驗、Boyden小室體外侵襲實驗、吖啶橙/溴化乙錠(AO/EB)熒光染色法、噻唑藍(MTT)比色法反映轉染後細胞遷移力、外侵襲力、凋亡及生長活性的變化.結果 轉染後48 h的膀胱癌EJ細胞,Skp2蛋白錶達開始下降,以轉染後96 h最明顯;而P27kiP1和PTEN在轉染96 h後,錶達顯著增加(P<0.05);轉染後的EJ細胞體外侵襲力下降[(11.7±1.2)%比(69.5±1.4)%,P<0.05],遷移能力減弱(P<0.05),細胞凋亡增多(11.8%比45.6%,P<0.05),生長活性降低(P<0.05).結論 膀胱癌細胞中的Skp2錶達下調可能通過負嚮調控P27kipl、PTEN而影響癌細胞的分裂週期,從而使癌細胞衰老,最終誘導凋亡.
목적 관찰침묵S기격매상관단백2(Skp2)기인대세포주기단백의뢰성격매억제단백27(P27KiP1)、제10호염색체결실적린산매급장력단백동원물기인(PTEN)적표체조공급대방광암세포생장활성적영향,탐토기재종류침습、조망중적작용궤제.방법 채용Western blot법검측경선병독재체(Ad-Skp2)전염후방광암세포주EJ적Skp2、P27kip1화PTEN적표체개변;용세포화흔실험、Boyden소실체외침습실험、아정등/추화을정(AO/EB)형광염색법、새서람(MTT)비색법반영전염후세포천이력、외침습력、조망급생장활성적변화.결과 전염후48 h적방광암EJ세포,Skp2단백표체개시하강,이전염후96 h최명현;이P27kiP1화PTEN재전염96 h후,표체현저증가(P<0.05);전염후적EJ세포체외침습력하강[(11.7±1.2)%비(69.5±1.4)%,P<0.05],천이능력감약(P<0.05),세포조망증다(11.8%비45.6%,P<0.05),생장활성강저(P<0.05).결론 방광암세포중적Skp2표체하조가능통과부향조공P27kipl、PTEN이영향암세포적분렬주기,종이사암세포쇠로,최종유도조망.
Objective To investigate the regulatory effect of cyclin dependent kinase inhibitor protein 27 (P27KiP1),phosphatase and tensin homolog deleted on chromosome ten (PTEN) expression and cell viability by silencing human gene S-phase kinase-associated protein 2 (Skp2),and further reseach for its role on invasion and apoptosis of cancer.Methods Adenoviral shRNA vector encoding Skp2 was transfected into EJ cell line.The expression of Skp2,P27kip1 and PTEN were detected by Western blotting assay; 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 Skp2 was decreased in the EJ cells after transfected with Skp2 shRNA,and the expression of P27kip1,PTEN were significantly up-regulated (P < 0.05) ; the motility,invation of EJ cells [(11.7 ± 1.2) % vs.(69.5 ±1.4) %] were inhibited and apoptosis (11.8% vs.45.6%) was enhanced.Conclusion The silencing of Skp2 may take an important role in cell cycle of bladder cancer cells by regulating P27kip1 and PTEN,which resulted in cell aging and lastly death.