中华消化杂志
中華消化雜誌
중화소화잡지
Chinese Journal of Digestion
2012年
11期
744-749
,共6页
吴平平%吴鹏%龙启强%李楠%金治%田晓强%黄培林
吳平平%吳鵬%龍啟彊%李楠%金治%田曉彊%黃培林
오평평%오붕%룡계강%리남%금치%전효강%황배림
结肠肿瘤%质粒%密码子,起动%RhoGTP酶激活蛋白%细胞增殖
結腸腫瘤%質粒%密碼子,起動%RhoGTP酶激活蛋白%細胞增殖
결장종류%질립%밀마자,기동%RhoGTP매격활단백%세포증식
Colonic neoplasms%Plasmids%Codon,initiator%Rho GTPase-activating protein%Cell proliferation
目的 探讨肝癌缺失基因-1(DLC-1)基因主要结构域在调控人结肠癌HT29细胞增殖中的作用.方法 分别构建DLC-1基因Rho蛋白GTP酶活化蛋白(RhoGAP)、SAM、START结构域敲除的亚克隆重组质粒,并转染至人结肠癌HT29细胞,另设野生型HT29细胞组(对照组)、pcDNA3.1-HT29细胞组(空载体组)和pcDNA3.1-HT29-DLC-1细胞组(全长转染组)作为对照.应用四甲基偶氮唑盐(MTT)实验、平板克隆实验检测细胞增殖的改变；流式细胞术检测细胞凋亡；pull-down方法分析RhoA蛋白活性.组间差异采用方差分析.结果 转染成功48 h后,与对照组及空载体组相比,全长转染组细胞增殖(F=146.36)及RhoA蛋白活性(F＝698.08)明显抑制(P均＜0.05),细胞早期凋亡增加(F=294.08,P＜0.05).敲除RhoGAP转染组细胞的增殖能力(F＝0.99)、细胞凋亡(F=0.049)及RhoA蛋白活性(F=5.769)与对照组及空载体组相比差异均无统计学意义(P均＞0.05)；敲除SAM转染组细胞比DLC-1基因全长转染组更显著地抑制了细胞增殖(F=31.00,P＜0.05),使RhoA蛋白活性下调(F=92.57,P＜0.05),凋亡增加(F=130.44,P＜0.05)；敲除START转染组相对于DLC-1基因全长转染组,细胞增殖能力增强(F＝15.47,P＜0.05),凋亡细胞减少(F=110.23,P＜0.05),RhoA蛋白活性上调(F＝199.39,P＜0.05).结论 DLC-1基因抑制HT29细胞增殖具有RhoGAP依赖性,SAM结构域可能是内源性RhoGAP活性的自身抑制区域,START结构域可能协助RhoGAP结构域而起作用.
目的 探討肝癌缺失基因-1(DLC-1)基因主要結構域在調控人結腸癌HT29細胞增殖中的作用.方法 分彆構建DLC-1基因Rho蛋白GTP酶活化蛋白(RhoGAP)、SAM、START結構域敲除的亞剋隆重組質粒,併轉染至人結腸癌HT29細胞,另設野生型HT29細胞組(對照組)、pcDNA3.1-HT29細胞組(空載體組)和pcDNA3.1-HT29-DLC-1細胞組(全長轉染組)作為對照.應用四甲基偶氮唑鹽(MTT)實驗、平闆剋隆實驗檢測細胞增殖的改變；流式細胞術檢測細胞凋亡；pull-down方法分析RhoA蛋白活性.組間差異採用方差分析.結果 轉染成功48 h後,與對照組及空載體組相比,全長轉染組細胞增殖(F=146.36)及RhoA蛋白活性(F＝698.08)明顯抑製(P均＜0.05),細胞早期凋亡增加(F=294.08,P＜0.05).敲除RhoGAP轉染組細胞的增殖能力(F＝0.99)、細胞凋亡(F=0.049)及RhoA蛋白活性(F=5.769)與對照組及空載體組相比差異均無統計學意義(P均＞0.05)；敲除SAM轉染組細胞比DLC-1基因全長轉染組更顯著地抑製瞭細胞增殖(F=31.00,P＜0.05),使RhoA蛋白活性下調(F=92.57,P＜0.05),凋亡增加(F=130.44,P＜0.05)；敲除START轉染組相對于DLC-1基因全長轉染組,細胞增殖能力增彊(F＝15.47,P＜0.05),凋亡細胞減少(F=110.23,P＜0.05),RhoA蛋白活性上調(F＝199.39,P＜0.05).結論 DLC-1基因抑製HT29細胞增殖具有RhoGAP依賴性,SAM結構域可能是內源性RhoGAP活性的自身抑製區域,START結構域可能協助RhoGAP結構域而起作用.
목적 탐토간암결실기인-1(DLC-1)기인주요결구역재조공인결장암HT29세포증식중적작용.방법 분별구건DLC-1기인Rho단백GTP매활화단백(RhoGAP)、SAM、START결구역고제적아극륭중조질립,병전염지인결장암HT29세포,령설야생형HT29세포조(대조조)、pcDNA3.1-HT29세포조(공재체조)화pcDNA3.1-HT29-DLC-1세포조(전장전염조)작위대조.응용사갑기우담서염(MTT)실험、평판극륭실험검측세포증식적개변；류식세포술검측세포조망；pull-down방법분석RhoA단백활성.조간차이채용방차분석.결과 전염성공48 h후,여대조조급공재체조상비,전장전염조세포증식(F=146.36)급RhoA단백활성(F＝698.08)명현억제(P균＜0.05),세포조기조망증가(F=294.08,P＜0.05).고제RhoGAP전염조세포적증식능력(F＝0.99)、세포조망(F=0.049)급RhoA단백활성(F=5.769)여대조조급공재체조상비차이균무통계학의의(P균＞0.05)；고제SAM전염조세포비DLC-1기인전장전염조경현저지억제료세포증식(F=31.00,P＜0.05),사RhoA단백활성하조(F=92.57,P＜0.05),조망증가(F=130.44,P＜0.05)；고제START전염조상대우DLC-1기인전장전염조,세포증식능력증강(F＝15.47,P＜0.05),조망세포감소(F=110.23,P＜0.05),RhoA단백활성상조(F＝199.39,P＜0.05).결론 DLC-1기인억제HT29세포증식구유RhoGAP의뢰성,SAM결구역가능시내원성RhoGAP활성적자신억제구역,START결구역가능협조RhoGAP결구역이기작용.
Objective To study the role of deleted in liver cancer-1 (DLC-1) gene main domains on the regulation of human colon cancer HT29 cell proliferation.Methods Subcloning recombinant plasmid vectors with Rho GTPase activating protein (RhoGAP),sterile alpha motif (SAM) or steroidogenic acute regulatory-related lipid-transfer (START) domains of DLC-1 gene knockout were constructed and transfected into human colon cancer cell HT29.Wild HT29 cell group (control group),pcDNA3.1-HT29 cell group (vector group) and pcDNA3.1-HT29-DLC-1 cell group (whole DLC-1 gene transfected group) were set as control.The change of cell proliferation was detected by methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay and colony formation test.The cell apoptosis was analyzed by flow cytometry.The activity of RhoA protein was detected by pull-down assay.The differences between the groups were analyzed by the analysis of variance.Results At 48 hours after the successful transfection,compared with control group and vector group,cells proliferation and the activity of RhoA protein were significantly suppressed in whole DLC-1 gene transfected group (F=146.36,698.08,both P＜0.05) and early cell apoptosis increased (F=294.08,P＜0.05).Compared with control group and vector group,there was no significant difference in cell proliferation ability,cell apoptosis and the activity of RhoA protein activity in RhoGAP knockout transfected cells (F=0.99,0.049,5.769,all P＞0.05).Compared with whole DLC-1 gene transfected group,the suppression of cell proliferation was more significant in SAM knockout transfected cells (F=31.00,P＜0.05),the activity of RhoA protein down regulated (F=92.57,P＜0.05) and apoptosis increased (F=130.44,P＜0.05).Compared with whole DLC-1 gene transfected group,the ability of cell proliferation increased (F=15.47,P＜0.05),apoptosis cell decreased (F＝110.23,P＜0.05) and the activity of RhoA protein up regulated (F=199.39,P＜0.05) in START knockout transfected cells.Conclusions The role of DLC-1 gene in the suppression of cell proliferation in HT29 cells was RhoGAP-dependent.SAM domain may be the self suppression domain for endogenous RhoGAP activity.START domain may take effect through enhancing RhoGAP domain.
