人剪切修复基因XPD转染对人肝癌细胞SMMC-7721的生物学影响
인전절수복기인XPD전염대인간암세포SMMC-7721적생물학영향
Biological effects of SMMC-7721 hepatoma cells transfected by XPD in viro
  • 万方数据
    国际肿瘤学杂志 국제종류학잡지
    JOURNAL OF INTERNATIONAL ONCOLOGY
    2008年 11期 869-873 ,共5页

    饶建锋%陈建勇%罗忠金%张吉翔

    요건봉%진건용%라충금%장길상

    癌,肝细胞%DNA修复 癌,肝細胞%DNA脩複
    암,간세포%DNA수복
    Carcinoma,hepatocellular%DNA repair
    目的 将人剪切修复基因XPD稳定转染人SMMC-7721肝癌细胞,观察转染后细胞内野生型p53、XPD、周期素依赖性蛋白激酶(CDK)7、c-myc等基因表达的变化以及对细胞生长的影响,探讨野生型XPD基因与p53、CDK7、c-myc的相互作用及细胞凋亡机制.方法 将表达绿色荧光蛋白并含有人类全长野生型XPD的pEGFP-N2-XPD重组体质粒稳定转染人SMMC-7721肝癌细胞中,选择培养基筛选单克隆稳定转染重组质粒的人SMMC-7721肝癌细胞(SMMC-7721-pEGFP-N2-XPD)和稳定转染空载质粒的人SMMC-7721肝癌细胞(SMMC.7721-pEGFP-N2),并将人SMMC-7721肝癌细胞作为空白对照,利用荧光显微镜观测绿色荧光蛋白表达,用逆转录-聚合酶链反应(RT-PCR)、Westem blot法检测转染XPD基因后细胞内XPD、p53、CDK7、c-myc的表达量变化,并用细胞增殖力检测(MTT)法及流式细胞仪分别检测细胞增殖及凋亡和细胞周期变化.结果 ①免疫荧光显微镜下,SMMC.7721.pEGFP.N2.XPD和SMMC-7721-pEGFP-N2细胞中观察到绿色荧光蛋白表达,说明pEGFP-N2-XPD重组质粒和pEGFP-N2空载质粒成功转染.②RT-PCR检测:SMMC-7721-pEGFP-N2-XPD中p53 mRNA、XPD mR-NA表达量与SMMC-7721-pEGFP-N2和SMMC-7721相比均明显增高(P<0.01),CDK7 mRNA、c-myc mRNA在SMMC-7721-pEGFP-N2-XPD的表达量比两对照组明显降低(P<0.01),而两对照组各个基因的表达没有明显差异(P>0.05).③Western blot检测:SMMC-7721-pEGFP-N2-XPD细胞的p53、XPD蛋白表达最较两对照组升高(P<0.01),CDK7、c-myc的蛋白相对表达量比两对照组降低(P<0.01),两对照组差异没有统计学意义(P>0.05).④M1Tr检测:SMMC-7721-pEGFP-N2-XPD的细胞增殖力较对照组减弱(P<0.05),两对照组筹异没有统计学意义(P>0.05).⑤流式细胞仪检测:SMMC-7721-pEGFP-N2-XPD细胞进入s期出现障碍,停滞在G1期的细胞增多.结论 将XPD成功稳定转染到人SMMC-7721肝癌细胞中,XPD、p53在转录和蛋白水平的表达明显升高,CDK7、c-myc表达明显降低,野生型XPD基因的过表达可能抑制CDK7、c-myc表达,改变细胞周期,并促进p53抑制肝癌细胞增长,促进细胞凋亡.
    목적 장인전절수복기인XPD은정전염인SMMC-7721간암세포,관찰전염후세포내야생형p53、XPD、주기소의뢰성단백격매(CDK)7、c-myc등기인표체적변화이급대세포생장적영향,탐토야생형XPD기인여p53、CDK7、c-myc적상호작용급세포조망궤제.방법 장표체록색형광단백병함유인류전장야생형XPD적pEGFP-N2-XPD중조체질립은정전염인SMMC-7721간암세포중,선택배양기사선단극륭은정전염중조질립적인SMMC-7721간암세포(SMMC-7721-pEGFP-N2-XPD)화은정전염공재질립적인SMMC-7721간암세포(SMMC.7721-pEGFP-N2),병장인SMMC-7721간암세포작위공백대조,이용형광현미경관측록색형광단백표체,용역전록-취합매련반응(RT-PCR)、Westem blot법검측전염XPD기인후세포내XPD、p53、CDK7、c-myc적표체량변화,병용세포증식력검측(MTT)법급류식세포의분별검측세포증식급조망화세포주기변화.결과 ①면역형광현미경하,SMMC.7721.pEGFP.N2.XPD화SMMC-7721-pEGFP-N2세포중관찰도록색형광단백표체,설명pEGFP-N2-XPD중조질립화pEGFP-N2공재질립성공전염.②RT-PCR검측:SMMC-7721-pEGFP-N2-XPD중p53 mRNA、XPD mR-NA표체량여SMMC-7721-pEGFP-N2화SMMC-7721상비균명현증고(P<0.01),CDK7 mRNA、c-myc mRNA재SMMC-7721-pEGFP-N2-XPD적표체량비량대조조명현강저(P<0.01),이량대조조각개기인적표체몰유명현차이(P>0.05).③Western blot검측:SMMC-7721-pEGFP-N2-XPD세포적p53、XPD단백표체최교량대조조승고(P<0.01),CDK7、c-myc적단백상대표체량비량대조조강저(P<0.01),량대조조차이몰유통계학의의(P>0.05).④M1Tr검측:SMMC-7721-pEGFP-N2-XPD적세포증식력교대조조감약(P<0.05),량대조조주이몰유통계학의의(P>0.05).⑤류식세포의검측:SMMC-7721-pEGFP-N2-XPD세포진입s기출현장애,정체재G1기적세포증다.결론 장XPD성공은정전염도인SMMC-7721간암세포중,XPD、p53재전록화단백수평적표체명현승고,CDK7、c-myc표체명현강저,야생형XPD기인적과표체가능억제CDK7、c-myc표체,개변세포주기,병촉진p53억제간암세포증장,촉진세포조망.
    Objective To investigate the role of wild-type XPD in SMMC-7721 hepatoma cells,its re-lationship with wild-type p53.CDK7 and c-myc.And the apoptosis mechanism of SMMC-7721 hepatoma cells.Methotis We inserted the human length XPD into the pEGFP-N2 plasmid vector which expresses the green fluorescence protein(GFP).And the pEGFP-N2 and pEGFP-N2-XPD were transfected into SMMC-7721 hepa-toma cell lines stably.Cel lines for omparison were matched on the sanle genetic background and passage.The expression of wild-type XPD,CDK7,p53,c-myc waft detected by RT-PCR,Westem blot.Cell growth wag detet-ed by MTT FCM wag employed for examining the cell cycle and apoptosis of the transfected SMMC-7721 hepa-toms cells.Results ①SMMC-7721-pEGFP-N2-xPD and SMMC-7721-pEGFP-N2 express the green fluores-cence protein which could be detected under the immunoffuorescence microscope.②RT-PCR The relative ex-\pression of p53 mRNA,XPD mRNA in the SMMC-7721,SMMC-7721-pEGFP-N2 and SMMC-7721-pEGFP-N2-XPD,the relative expression of p53 mRNA,XPD mRNA in the SMMC-7721-pEGFP-N2-XPD was signifiantly higher than other two oontrols(P<0.01).Othewise,the relative expression of CDK7 mRNA,c-myc mRNA Wag signifi-antlv lower than other two oontrols(P<0.01),the difference of two controls was not signifiant(P>0.05). ③Western blot The relative expression of p53,XPD protein was signifiantly higher than other two oontrols (P<0.01).Othewise,the relative expression of CDK7,c-Myc protein was signifiantly lower than other two oontrols(P<O.01),the difference of two controls Wag not signifiant(P>0.05).④MTT The ressuh showedthat the proliferative ability of SMMC-7721-pEGFP-N2-XPD was much more descreaged than other two oontrols (P<0.05),the other two oontrols were not different(P>0.05).⑤FCM Wild-type XPD gene could change the cell8 and induce apoptitise in vitro though the result of FCM.Conclusions The pEGFP-N2 and pEGFP-N2-XPD were transfected into SMMC-7721hepatoma cell lines stably,wild-type XPD could decreaSe the expres-sion of CDK7,c-myc and increase the expression of p53.Wild-type.XPD could inhibit the activity of cell growth and change cell cycle,as well as induce cell apoptosis in SMMC-7721 hepatoma cell line in vitro.
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