天津医药
天津醫藥
천진의약
TIANJIN MEDICAL JOURNAL
2014年
12期
1168-1171
,共4页
基因,p53%蛋白激酶类%细胞增殖%肿瘤侵润%真核细胞翻译启始因子2α
基因,p53%蛋白激酶類%細胞增殖%腫瘤侵潤%真覈細胞翻譯啟始因子2α
기인,p53%단백격매류%세포증식%종류침윤%진핵세포번역계시인자2α
genes,p53%protein kinases%cell proliferation%neoplasm invasiveness%αsubunit of eukaryotic initiation factor 2
目的：探讨P53蛋白对蛋白激酶R（PKR）表达和活性以及对宫颈癌HeLa细胞生物学行为的影响。方法构建过表达p53基因的重组质粒pEGFP-C1/p53，转染HeLa细胞，采用逆转录-聚合酶链反应(RT-PCR)法检测pEGFP-C1/p53转染组、空质粒pEGFP-C1转染组及空白对照组（仅加入转染试剂）p53及PKR mRNA的表达；采用Western Blot法检测上述3组中P53、PKR、磷酸化型PKR(p-PKR)，PKR下游底物真核细胞翻译启始因子2α（eIF2α）的磷酸化型p-eIF2α的表达；采用四甲基偶氮唑蓝（MTT）法检测HeLa细胞增殖活性变化，Transwell侵袭实验检测HeLa细胞侵袭能力变化。结果 pEGFP-C1/p53转染组p53及PKR mRNA的相对表达量高于pEGFP-C1转染组和空白对照组（均P＜0.05），pEGFP-C1转染组和空白对照组比较，差异无统计学意义；pEGFP-C1/p53转染组P53、PKR、p-PKR及p-eIF2α蛋白的相对表达量高于pEGFP-C1转染组和空白对照组（均P＜0.05），pEGFP-C1转染组和空白对照组比较，差异无统计学意义；pEGFP-C1/p53转染组HeLa细胞增殖活性及侵袭能力均显著低于pEGFP-C1转染组和空白对照组（均P＜0.05），pEGFP-C1转染组和空白对照组比较，差异无统计学意义。结论P53能上调PKR的表达及活性，激活PKR/eIF2α信号通路，抑制宫颈癌HeLa细胞增殖及侵袭。
目的：探討P53蛋白對蛋白激酶R（PKR）錶達和活性以及對宮頸癌HeLa細胞生物學行為的影響。方法構建過錶達p53基因的重組質粒pEGFP-C1/p53，轉染HeLa細胞，採用逆轉錄-聚閤酶鏈反應(RT-PCR)法檢測pEGFP-C1/p53轉染組、空質粒pEGFP-C1轉染組及空白對照組（僅加入轉染試劑）p53及PKR mRNA的錶達；採用Western Blot法檢測上述3組中P53、PKR、燐痠化型PKR(p-PKR)，PKR下遊底物真覈細胞翻譯啟始因子2α（eIF2α）的燐痠化型p-eIF2α的錶達；採用四甲基偶氮唑藍（MTT）法檢測HeLa細胞增殖活性變化，Transwell侵襲實驗檢測HeLa細胞侵襲能力變化。結果 pEGFP-C1/p53轉染組p53及PKR mRNA的相對錶達量高于pEGFP-C1轉染組和空白對照組（均P＜0.05），pEGFP-C1轉染組和空白對照組比較，差異無統計學意義；pEGFP-C1/p53轉染組P53、PKR、p-PKR及p-eIF2α蛋白的相對錶達量高于pEGFP-C1轉染組和空白對照組（均P＜0.05），pEGFP-C1轉染組和空白對照組比較，差異無統計學意義；pEGFP-C1/p53轉染組HeLa細胞增殖活性及侵襲能力均顯著低于pEGFP-C1轉染組和空白對照組（均P＜0.05），pEGFP-C1轉染組和空白對照組比較，差異無統計學意義。結論P53能上調PKR的錶達及活性，激活PKR/eIF2α信號通路，抑製宮頸癌HeLa細胞增殖及侵襲。
목적：탐토P53단백대단백격매R（PKR）표체화활성이급대궁경암HeLa세포생물학행위적영향。방법구건과표체p53기인적중조질립pEGFP-C1/p53，전염HeLa세포，채용역전록-취합매련반응(RT-PCR)법검측pEGFP-C1/p53전염조、공질립pEGFP-C1전염조급공백대조조（부가입전염시제）p53급PKR mRNA적표체；채용Western Blot법검측상술3조중P53、PKR、린산화형PKR(p-PKR)，PKR하유저물진핵세포번역계시인자2α（eIF2α）적린산화형p-eIF2α적표체；채용사갑기우담서람（MTT）법검측HeLa세포증식활성변화，Transwell침습실험검측HeLa세포침습능력변화。결과 pEGFP-C1/p53전염조p53급PKR mRNA적상대표체량고우pEGFP-C1전염조화공백대조조（균P＜0.05），pEGFP-C1전염조화공백대조조비교，차이무통계학의의；pEGFP-C1/p53전염조P53、PKR、p-PKR급p-eIF2α단백적상대표체량고우pEGFP-C1전염조화공백대조조（균P＜0.05），pEGFP-C1전염조화공백대조조비교，차이무통계학의의；pEGFP-C1/p53전염조HeLa세포증식활성급침습능력균현저저우pEGFP-C1전염조화공백대조조（균P＜0.05），pEGFP-C1전염조화공백대조조비교，차이무통계학의의。결론P53능상조PKR적표체급활성，격활PKR/eIF2α신호통로，억제궁경암HeLa세포증식급침습。
Objective To investigate the effects of p53 on expression and activity of protein kinase R (PKR) as well as biological characters of HeLa cells from cervical carcinoma patients. Methods Recombinant plasmid vector pEGFP-C1/p53 was constructed to over-express p53 then it was transfected into HeLa cells. Transcription levels of p53 and PKR mRNA were detected by reverse transcriptase polymerase chain reaction (RT-PCR) among pEGFP-C1/p53 transfection group, pEGFP-C1 transfection group and blank control group(only transfection reagent was added);Protein expression lev?els of p53, PKR, phosphated PKR(p-PKR)and phosphatedαsubunit of eukaryotic initiation factor 2(p-eIF2α)which is the downstream substrate of PKR were detected by Western Blot among three groups;Proliferation of HeLa cell were deter?mined by methyl thiazolyl tetrazolium(MTT)assay;Invasion of HeLa cell were determined by Transwell cell assay. Re?sults Recombinant plasmid vector pEGFP-C1/p53 was successfully constructed to overexpress p53;Transcription level of p53 and PKR mRNA in pEGFP-C1/p53 transfection group were higher than those in pEGFP-C1 transfection group and in blank control group (P<0.05),and there were no significant difference between their levels in pEGFP-C1 transfection group and in blank control group;Protein expression levels of p53, PKR, p-PKR andp-eIF2α in pEGFP-C1/p53 transfection group were higher than those in pEGFP-C1 transfection group and in blank control group (P<0.05),and there were no sig?nificant difference between those expression levels in pEGFP-C1 transfection group and in blank control group;MTT and Transwell cell results showed that proliferation and invasion of HeLa cells in pEGFP-C1/p53 transfection group were weaker than those in pEGFP-C1 transfection group and in blank control group (P<0.05),and there were no significant difference between proliferation and invasion of HeLa cells in pEGFP-C1 transfection group and in blank control group. Conclu?sion p53 can up-regulate the expression and activity of PKR, promote activation of PKR/eIF2αsignal transduction pas?sage and restrain cell proliferation and invasion of HeLa cells.
