中国临床康复
中國臨床康複
중국림상강복
CHINESE JOURNAL OF CLINICAL REHABILITATION
2003年
32期
4322-4324
,共3页
傅赞%赵翰林%王若宁%德伟%武正炎
傅讚%趙翰林%王若寧%德偉%武正炎
부찬%조한림%왕약저%덕위%무정염
基因%抑制%肿瘤%胆管癌%细胞周期
基因%抑製%腫瘤%膽管癌%細胞週期
기인%억제%종류%담관암%세포주기
背景 :对于胆管癌的治疗目前临床缺乏有效的诊治手段,抑癌基因替代疗法正逐步成为一种新型的、重要的抗肿瘤措施. P15作为一种重要的抑癌基因,发现可抑制肝癌细胞生长.但是对于其在胆管癌中的作用的相关研究尚在探索阶段 ,本研究为预防人胆管癌的发生提供一个理论框架. 目的 :通过构建稳定高表达抑癌基因 p15的人胆管癌细胞模型,研究 p15对胆管癌细胞增殖的影响. 设计:完全随机,设立实验对照组,开放性实验研究. 地点和对象 :实验地点在南京医科大学生化与分子生物学研究室 ,研究对象为人胆管癌细胞系. 干预:以稳定高表达 p15的人胆管癌细胞模型为实验组,稳定表达空载体的细胞模型为对照组.将抑癌基因 p15的 cDNA构建到高效真核表达的质粒载体 pcDNA3- neo中的 EcoRⅠ / XbaⅠ位点 ,构建成 p15真核表达质粒 pcDNA3p15;通过脂质体法将 pcDNA3p15质粒转染人胆管癌细胞系 QBC939,经 G- 418筛选 ,获得稳定高表达 p15的人胆管癌细胞模型及相应的表达空载体的细胞模型,并经 Western分子杂交分析证实. 主要观察指标:观察实验组和对照组细胞增殖速率、细胞克隆形成能力、细胞周期、 c- Fos蛋白表达水平的差异 结果:与对照组相比 ,p15高表达的人胆管癌细胞 QBC939的增殖受到明显抑制;克隆形成实验结果表明,高表达 p15人胆管癌细胞所形成的克隆明显少于对照组所形成的克隆 (P< 0.01);流式细胞光度术表明 p15同时阻遏细胞由 G1期向 S期及 G2期向 M期的转换; Western免疫印迹分析结果表明,高表达 p15抑癌基因的细胞癌基因 c- Fos的蛋白水平下降约为对照组的 40%. 结论: p15高表达明显抑制了人胆管癌细胞的生长 ,并且基因 c- myc的蛋白水平表达下降可能是 p15抑制细胞增殖的分子机理之一.
揹景 :對于膽管癌的治療目前臨床缺乏有效的診治手段,抑癌基因替代療法正逐步成為一種新型的、重要的抗腫瘤措施. P15作為一種重要的抑癌基因,髮現可抑製肝癌細胞生長.但是對于其在膽管癌中的作用的相關研究尚在探索階段 ,本研究為預防人膽管癌的髮生提供一箇理論框架. 目的 :通過構建穩定高錶達抑癌基因 p15的人膽管癌細胞模型,研究 p15對膽管癌細胞增殖的影響. 設計:完全隨機,設立實驗對照組,開放性實驗研究. 地點和對象 :實驗地點在南京醫科大學生化與分子生物學研究室 ,研究對象為人膽管癌細胞繫. 榦預:以穩定高錶達 p15的人膽管癌細胞模型為實驗組,穩定錶達空載體的細胞模型為對照組.將抑癌基因 p15的 cDNA構建到高效真覈錶達的質粒載體 pcDNA3- neo中的 EcoRⅠ / XbaⅠ位點 ,構建成 p15真覈錶達質粒 pcDNA3p15;通過脂質體法將 pcDNA3p15質粒轉染人膽管癌細胞繫 QBC939,經 G- 418篩選 ,穫得穩定高錶達 p15的人膽管癌細胞模型及相應的錶達空載體的細胞模型,併經 Western分子雜交分析證實. 主要觀察指標:觀察實驗組和對照組細胞增殖速率、細胞剋隆形成能力、細胞週期、 c- Fos蛋白錶達水平的差異 結果:與對照組相比 ,p15高錶達的人膽管癌細胞 QBC939的增殖受到明顯抑製;剋隆形成實驗結果錶明,高錶達 p15人膽管癌細胞所形成的剋隆明顯少于對照組所形成的剋隆 (P< 0.01);流式細胞光度術錶明 p15同時阻遏細胞由 G1期嚮 S期及 G2期嚮 M期的轉換; Western免疫印跡分析結果錶明,高錶達 p15抑癌基因的細胞癌基因 c- Fos的蛋白水平下降約為對照組的 40%. 結論: p15高錶達明顯抑製瞭人膽管癌細胞的生長 ,併且基因 c- myc的蛋白水平錶達下降可能是 p15抑製細胞增殖的分子機理之一.
배경 :대우담관암적치료목전림상결핍유효적진치수단,억암기인체대요법정축보성위일충신형적、중요적항종류조시. P15작위일충중요적억암기인,발현가억제간암세포생장.단시대우기재담관암중적작용적상관연구상재탐색계단 ,본연구위예방인담관암적발생제공일개이론광가. 목적 :통과구건은정고표체억암기인 p15적인담관암세포모형,연구 p15대담관암세포증식적영향. 설계:완전수궤,설립실험대조조,개방성실험연구. 지점화대상 :실험지점재남경의과대학생화여분자생물학연구실 ,연구대상위인담관암세포계. 간예:이은정고표체 p15적인담관암세포모형위실험조,은정표체공재체적세포모형위대조조.장억암기인 p15적 cDNA구건도고효진핵표체적질립재체 pcDNA3- neo중적 EcoRⅠ / XbaⅠ위점 ,구건성 p15진핵표체질립 pcDNA3p15;통과지질체법장 pcDNA3p15질립전염인담관암세포계 QBC939,경 G- 418사선 ,획득은정고표체 p15적인담관암세포모형급상응적표체공재체적세포모형,병경 Western분자잡교분석증실. 주요관찰지표:관찰실험조화대조조세포증식속솔、세포극륭형성능력、세포주기、 c- Fos단백표체수평적차이 결과:여대조조상비 ,p15고표체적인담관암세포 QBC939적증식수도명현억제;극륭형성실험결과표명,고표체 p15인담관암세포소형성적극륭명현소우대조조소형성적극륭 (P< 0.01);류식세포광도술표명 p15동시조알세포유 G1기향 S기급 G2기향 M기적전환; Western면역인적분석결과표명,고표체 p15억암기인적세포암기인 c- Fos적단백수평하강약위대조조적 40%. 결론: p15고표체명현억제료인담관암세포적생장 ,병차기인 c- myc적단백수평표체하강가능시 p15억제세포증식적분자궤리지일.
BACKGROUND:At present,there is no effective method in treating cholangiocarcinoma.Anti- oncogene replacement therapy has gradually become an important new anticancer measure.As an important anti- oncogene,p15 can inhibit the growth of the hepatocarcinoma cell.But as to its role in the cholangiocarcinoma,more research is needed.This study provides a theory for preventing occurrence of human cholangiocarcinoma. OBJECTIVE:To study the inhibitory effect of anti- oncogene p15 on human cholangiocarcinoma cell line by establishing the cell model of the human cholangiocarcinoma that overexpresses p15 gene. DESIGN:Completely randomized controlled and open trials. SETTING AND PARTICIPANTS:Institute of Biochemistry and Molecular Biology,Nanjing Medical University.Hunman cholangiocarcinoma establisdhed cell line. INTERVENTIONS:The cell model of the human cholangiocarcinoma that overexpresses p15 gene was used as the trial group and the cell model that steadily expresses vector pcDNA3 was used as the control group.The cDNA of anti- oncogene p15 was constructed to the EcoRⅠ / XbaⅠ site of pcDNA3- neo,a plasmid carrier that showed a high effective eukaryon expression so as to construct pcDNA3p15,the plasmid that expresses eukaryon of p15.The pcDNA3p15 was transferred into the human cholangiocarcinoma cell line QBC939 using lipofectine.After screening by G- 418,we got the cell model of the human cholangiocarcinoma that steadily overexpresses p15 and we also got the corresponding control cell model that expresses no carrier,and this was confirmed by using Western molecular hybridization analysis. MAIN INDEXES OF OBSERVATION:The differences in cell proliferation velocity,the forming ability of cell clones,cell cycle and the expression of c- Fos protein were observed. RESULTS:Compared with the control group,the proliferation of QBC939 that overexpressed p15 was obviously inhibited.Cloning experimental results showed that the clones of the cholangiocarcinoma cells that overexpressed p15 were significantly fewer than those in control group(P< 0.01).The flow cytospectrophotometry showed that p15 repressed the conversion of both G1 to S and G2 to M.Western blot analysis showed that the expression level of c- Fos protein in cells that overexpressed p15 dropped to about 40% of that of the control group. CONCLUSION:The overexpressed p15 gene can significantly inhibit the growth of the cholangiocarcinoma cells, and the descenting of oncogene c- myc protein level may be one of the mechanisms that inhibit the proliferation of cancerous cells.
