中华实验外科杂志
中華實驗外科雜誌
중화실험외과잡지
CHINESE JOURNAL OF EXPERIMENTAL SURGERY
2013年
3期
487-489
,共3页
刘洋%卢秀波%耿祖仕%贾勐%申林林
劉洋%盧秀波%耿祖仕%賈勐%申林林
류양%로수파%경조사%가맹%신림림
pEZX%微小RNA-21%甲状腺乳头状癌%转染
pEZX%微小RNA-21%甲狀腺乳頭狀癌%轉染
pEZX%미소RNA-21%갑상선유두상암%전염
pEZX%MicroRNA-21%Papillary thyroid carcinoma%Transfection
目的 构建微小RNA (miR)-21的真核表达载体pEZX-eGFP-microRNA-21.将构建成功的pEZX-eGFP-microRNA-21瞬时转染至甲状腺乳头状癌细胞株TPC-1中,探讨转染前后细胞蛋白程序性细胞凋亡4(PDCD4)蛋白的表达差异及其机制.方法 人工合成microRNA-21基因序列,构建成重组质粒pEZX-eGFP-microRNA-21并转染TPC-1细胞.将TPC-1细胞分为转染组、空载组、空白组,每组20个复孔.实时定量聚合酶链反应(Real-time PCR)、Western blot分别检测转染后miR-21及PDCD4蛋白在各组细胞中的表达.结果 经酶切和测序分析鉴定后证明重组质粒pEZX-eGFP-microRNA-21构建成功.转染组TPC-1细胞miR-21的表达水平明显高于对照组:加尾法、茎环法中分别是对照组的6.39倍(P＜0.01)、7.58倍(P＜0.01).Western blot显示转染组细胞PDCD4表达量明显降低(0.24±0.03,P＜0.05).结论 成功构建miR-21基因真核表达载体pEZX-eGFP-microRNA-21.miR-21在甲状腺乳头状癌细胞TPC-1中高表达可能导致PDCD4蛋白低表达.
目的 構建微小RNA (miR)-21的真覈錶達載體pEZX-eGFP-microRNA-21.將構建成功的pEZX-eGFP-microRNA-21瞬時轉染至甲狀腺乳頭狀癌細胞株TPC-1中,探討轉染前後細胞蛋白程序性細胞凋亡4(PDCD4)蛋白的錶達差異及其機製.方法 人工閤成microRNA-21基因序列,構建成重組質粒pEZX-eGFP-microRNA-21併轉染TPC-1細胞.將TPC-1細胞分為轉染組、空載組、空白組,每組20箇複孔.實時定量聚閤酶鏈反應(Real-time PCR)、Western blot分彆檢測轉染後miR-21及PDCD4蛋白在各組細胞中的錶達.結果 經酶切和測序分析鑒定後證明重組質粒pEZX-eGFP-microRNA-21構建成功.轉染組TPC-1細胞miR-21的錶達水平明顯高于對照組:加尾法、莖環法中分彆是對照組的6.39倍(P＜0.01)、7.58倍(P＜0.01).Western blot顯示轉染組細胞PDCD4錶達量明顯降低(0.24±0.03,P＜0.05).結論 成功構建miR-21基因真覈錶達載體pEZX-eGFP-microRNA-21.miR-21在甲狀腺乳頭狀癌細胞TPC-1中高錶達可能導緻PDCD4蛋白低錶達.
목적 구건미소RNA (miR)-21적진핵표체재체pEZX-eGFP-microRNA-21.장구건성공적pEZX-eGFP-microRNA-21순시전염지갑상선유두상암세포주TPC-1중,탐토전염전후세포단백정서성세포조망4(PDCD4)단백적표체차이급기궤제.방법 인공합성microRNA-21기인서렬,구건성중조질립pEZX-eGFP-microRNA-21병전염TPC-1세포.장TPC-1세포분위전염조、공재조、공백조,매조20개복공.실시정량취합매련반응(Real-time PCR)、Western blot분별검측전염후miR-21급PDCD4단백재각조세포중적표체.결과 경매절화측서분석감정후증명중조질립pEZX-eGFP-microRNA-21구건성공.전염조TPC-1세포miR-21적표체수평명현고우대조조:가미법、경배법중분별시대조조적6.39배(P＜0.01)、7.58배(P＜0.01).Western blot현시전염조세포PDCD4표체량명현강저(0.24±0.03,P＜0.05).결론 성공구건miR-21기인진핵표체재체pEZX-eGFP-microRNA-21.miR-21재갑상선유두상암세포TPC-1중고표체가능도치PDCD4단백저표체.
Objective To construct microRNA-21 eukaryotic expression vector pEZX-eGFP-microRNA-21,and explore the differences of programmed cell death 4 (PDCD4) protein expression and the mechanisms after transient transfection of pEZX-eGFP-microRNA-21 into papillary thyroid cancer cell line TPC-1.Methods miR-21 sequence was synthesized and cloned into pEZX-eGFP to construct recombinant plasmid pEZX-eGFP-microRNA-21.TPC-1 cells were divided into transfected group,the no-load group,blank group (n =20 each).Transcription level of microRNA-21 was detected by using real-time PCR.Western blotting was used to detect the differences of PDCD4 protein expression.Results Recombinant plasmid pEZX-eGFP-microRNA-21 was successfully constructed.The microRNA-21 expression level in transfected group was significantly higher than in no-load group and blank group.Tailing method revealed the expression level in transfected group was 6.39 times that of no-load and blank groups (P ＜0.01),and stem-loop method 7.58 times (P ＜0.01).Western blotting showed that PDCD4 expression level in transfected group was significantly lower than other groups (0.24 ±0.03,P ＜0.05).Conclusion miR-21 expression vector was constructed successfully.The high expression of miR-21 in TPC-1 cells may lead to the low expression of PDCD4 protein.
