中华医学杂志
中華醫學雜誌
중화의학잡지
National Medical Journal of China
2008年
43期
3080-3084
,共5页
廖书杰%邓东锐%夏曦%韩凌斐%胡晓继%白向阳%王薇%卢运萍%王世宣%马丁
廖書傑%鄧東銳%夏晞%韓凌斐%鬍曉繼%白嚮暘%王薇%盧運萍%王世宣%馬丁
료서걸%산동예%하희%한릉비%호효계%백향양%왕미%로운평%왕세선%마정
人乳头瘤病毒%宫颈癌%机制
人乳頭瘤病毒%宮頸癌%機製
인유두류병독%궁경암%궤제
Human papillomavirus%Cervical cancer%Mechanism
目的 通过构建和筛选持续表达HPV16E5的细胞SiHa/16E5,探讨HPV16E5对宫颈癌SiHa细胞的作用及机理.方法 利用pEGFP-C1构建HPV16型E5的正义全长真核表达载体并稳定转染SiHa细胞;利用RT-PCR和Western印迹技术检测转染前后E5和P21基因的mRNA和GFP+E5和P21蛋白的变化;利用MTT法检测SiHa细胞稳定转染后的增殖活性;利用软琼脂克隆形成和裸鼠成瘤实验分别在体内外验证了SiHa/16E5细胞的致瘤情况.结果 稳定转染携带.HPV16全长E5基因的质粒后,SiHa细胞中E5基因的mRNA和相应蛋白表达明显升高,而P21的表达则明显下调;MTT结果 显示稳定转染后细胞的增殖活性与转染空载体细胞和未转染细胞比较明显增高(P<0.05);软琼脂克隆形成结果 示:SiHa/16ES细胞组的可隆形成数为33.4±1.6,与空质粒对照组细胞(15.1±3.1)及未转染组细胞(16.3±2.5)比较差异有统计学意义(P<0.05);裸鼠成瘤实验结果 显示,4个实验组在共20 d的观察中,SiHa/16E5组裸鼠成瘤明显快于其他3个对照组(P<0.05).结论 HPV16 E5可降低宫颈癌SiHa细胞中P21的表达,加强宫颈癌SiHa细胞增殖和成瘤效应.
目的 通過構建和篩選持續錶達HPV16E5的細胞SiHa/16E5,探討HPV16E5對宮頸癌SiHa細胞的作用及機理.方法 利用pEGFP-C1構建HPV16型E5的正義全長真覈錶達載體併穩定轉染SiHa細胞;利用RT-PCR和Western印跡技術檢測轉染前後E5和P21基因的mRNA和GFP+E5和P21蛋白的變化;利用MTT法檢測SiHa細胞穩定轉染後的增殖活性;利用軟瓊脂剋隆形成和裸鼠成瘤實驗分彆在體內外驗證瞭SiHa/16E5細胞的緻瘤情況.結果 穩定轉染攜帶.HPV16全長E5基因的質粒後,SiHa細胞中E5基因的mRNA和相應蛋白錶達明顯升高,而P21的錶達則明顯下調;MTT結果 顯示穩定轉染後細胞的增殖活性與轉染空載體細胞和未轉染細胞比較明顯增高(P<0.05);軟瓊脂剋隆形成結果 示:SiHa/16ES細胞組的可隆形成數為33.4±1.6,與空質粒對照組細胞(15.1±3.1)及未轉染組細胞(16.3±2.5)比較差異有統計學意義(P<0.05);裸鼠成瘤實驗結果 顯示,4箇實驗組在共20 d的觀察中,SiHa/16E5組裸鼠成瘤明顯快于其他3箇對照組(P<0.05).結論 HPV16 E5可降低宮頸癌SiHa細胞中P21的錶達,加彊宮頸癌SiHa細胞增殖和成瘤效應.
목적 통과구건화사선지속표체HPV16E5적세포SiHa/16E5,탐토HPV16E5대궁경암SiHa세포적작용급궤리.방법 이용pEGFP-C1구건HPV16형E5적정의전장진핵표체재체병은정전염SiHa세포;이용RT-PCR화Western인적기술검측전염전후E5화P21기인적mRNA화GFP+E5화P21단백적변화;이용MTT법검측SiHa세포은정전염후적증식활성;이용연경지극륭형성화라서성류실험분별재체내외험증료SiHa/16E5세포적치류정황.결과 은정전염휴대.HPV16전장E5기인적질립후,SiHa세포중E5기인적mRNA화상응단백표체명현승고,이P21적표체칙명현하조;MTT결과 현시은정전염후세포적증식활성여전염공재체세포화미전염세포비교명현증고(P<0.05);연경지극륭형성결과 시:SiHa/16ES세포조적가륭형성수위33.4±1.6,여공질립대조조세포(15.1±3.1)급미전염조세포(16.3±2.5)비교차이유통계학의의(P<0.05);라서성류실험결과 현시,4개실험조재공20 d적관찰중,SiHa/16E5조라서성류명현쾌우기타3개대조조(P<0.05).결론 HPV16 E5가강저궁경암SiHa세포중P21적표체,가강궁경암SiHa세포증식화성류효응.
Objective The human papillomavirns type 16 (HPV16) early gene (ES) could stimulate cell proliferation and transformation in different ways, and complement or enhancement the function of E6 and E7. It is closely sociated with the carcinogenes of cervical cancer. This study was to investigate the effects of HPVI6 E5 on the human cervical cancer cell line SiHa. Methods The sense sequence of HPV16 E5 was cloned into eukaryotic expression vector pEGFP-C1 to prepare recombinant plasmid, which was stable transfected into SiHa cells. The mRNA and protein levels of E5 and p21 gene were detected by Western blot and reverse transcription-polymerase chain reaction (RT-PCR). Cell proliferation after stable transfection was evaluated by MTr assay. The tumorigenesis of SiHa/16E5 cells was assessed both in vitro and in vivo by soft agar clone form test and naked mouse form test. Results After stable transfection of HPV16 E5 sense DNA, the mRNA and protein levels of HPV16 E5 in SiHa cells were obviously increased, but that of P21 were decreased. The proliferation activity of SiHa/16E5 cells was significantly higher than that of SiHa /pEGFP-C1 and SiHa cells (P<0.05) .The clone numbers of SiHa/16E5 cells, were significantly more than SiHa / pEGFP-C1 and SiHa cells [(33.4±1.6) vs (15.1±3.1),(16.3±2.5), P<0.05] in vitro. The growth of tumors on naked mouse was much faster in SiHa/16E5 group than those in the SiHa /pEGFP-C1 and SiHa cells groups (P<0. 05). Conclusion HPV16 E5 decreased the expression of P21 in SiHa cells, and enhancement the proliferation of SiHa cells and the ability of tumorigenesis.
