中华消化外科杂志
中華消化外科雜誌
중화소화외과잡지
CHINESE JOURNAL OF DIGESTIVE SURGERY
2012年
1期
86-90
,共5页
马波%张汉超%徐新女%张飚%王金环
馬波%張漢超%徐新女%張飚%王金環
마파%장한초%서신녀%장표%왕금배
结肠肿瘤%人类免疫缺陷病毒1型%病毒蛋白r基因%细胞周期G2停滞%细胞凋亡
結腸腫瘤%人類免疫缺陷病毒1型%病毒蛋白r基因%細胞週期G2停滯%細胞凋亡
결장종류%인류면역결함병독1형%병독단백r기인%세포주기G2정체%세포조망
Colonic neoplasms%Human immunodeficiency virus 1%Viral protein r gene%G2/M arrest%Cell apoptosis
目的 探讨人类免疫缺陷病毒1型( HIV-1)病毒蛋白r(Vpr)对人结肠癌细胞HCT-8的抑制作用及其机制.方法 将细胞分为空白对照组、空载体转染组和Vpr转染组.空白对照组:细胞不予干预；空载体转染组:对数生长期的细胞加入不同感染复数(MOI)的空载体腺病毒；Vpr转染组:加入不同MOI的含有HIV1-Vpr基因的重组腺病毒(Adv-Vpr).应用MTT比色法检测细胞增殖活性,流式细胞仪检测细胞周期、细胞凋亡及线粒体膜电位,Western blot法检测细胞凋亡相关蛋白的表达.多组间均数比较采用单因素方差分析,两两比较采用q检验；或采用双因素方差分析,组内比较采用t检验.结果 Vpr显著抑制人结肠癌细胞HCT-8增殖,Adv-Vpr转染72 h后(MOI=200),Vpr转染组细胞MTT值为1.03±0.04,与空载体转染组(2.46 +0.15)及空白对照组(2.51±0.14)比较,差异有统计学意义(F=144.6,P＜0.05)；Adv-Vpr转染48 h后(MOI=200),Vpr转染组处于G2/M期的细胞比例及线粒体膜电位下降的细胞比例增加,分别为37.31％±5.90％和32.07％±5.64％,与空载体转染组(18.30％±6.04％、3.32％±0.79％)及空白对照组916.66％±3.51％、2.76％±1.43％)比较,差异有统计学意义(F=10.08,64.45,P＜0.05).Adv-Vpr转染72 h后(MOI=200),Vpr转染组细胞凋亡率为37.62％±6.48％,与空载体转染组(3.44％±1.11％)及空白对照组(2.93％±1.07％)比较,差异有统计学意义(F=122.4,P＜0.05).Adv-Vpr处理48 h后(MOI=200),Westem blot检测发现Vpr导致了Caspase-9、Caspase-3剪切为活性片段,p-Chk1-S345磷酸化增加,而Fas、Fas-L、ERK1及ERK2表达未见上调.结论 Vpr在体外能够有效抑制结肠癌细胞株HCT-8增殖,并诱导其细胞周期G2期阻滞及凋亡,其机制分别与DNA损伤信号通路激活及线粒体凋亡通路启动有关.Vpr在结肠癌的治疗中具有潜在的应用前景.
目的 探討人類免疫缺陷病毒1型( HIV-1)病毒蛋白r(Vpr)對人結腸癌細胞HCT-8的抑製作用及其機製.方法 將細胞分為空白對照組、空載體轉染組和Vpr轉染組.空白對照組:細胞不予榦預；空載體轉染組:對數生長期的細胞加入不同感染複數(MOI)的空載體腺病毒；Vpr轉染組:加入不同MOI的含有HIV1-Vpr基因的重組腺病毒(Adv-Vpr).應用MTT比色法檢測細胞增殖活性,流式細胞儀檢測細胞週期、細胞凋亡及線粒體膜電位,Western blot法檢測細胞凋亡相關蛋白的錶達.多組間均數比較採用單因素方差分析,兩兩比較採用q檢驗；或採用雙因素方差分析,組內比較採用t檢驗.結果 Vpr顯著抑製人結腸癌細胞HCT-8增殖,Adv-Vpr轉染72 h後(MOI=200),Vpr轉染組細胞MTT值為1.03±0.04,與空載體轉染組(2.46 +0.15)及空白對照組(2.51±0.14)比較,差異有統計學意義(F=144.6,P＜0.05)；Adv-Vpr轉染48 h後(MOI=200),Vpr轉染組處于G2/M期的細胞比例及線粒體膜電位下降的細胞比例增加,分彆為37.31％±5.90％和32.07％±5.64％,與空載體轉染組(18.30％±6.04％、3.32％±0.79％)及空白對照組916.66％±3.51％、2.76％±1.43％)比較,差異有統計學意義(F=10.08,64.45,P＜0.05).Adv-Vpr轉染72 h後(MOI=200),Vpr轉染組細胞凋亡率為37.62％±6.48％,與空載體轉染組(3.44％±1.11％)及空白對照組(2.93％±1.07％)比較,差異有統計學意義(F=122.4,P＜0.05).Adv-Vpr處理48 h後(MOI=200),Westem blot檢測髮現Vpr導緻瞭Caspase-9、Caspase-3剪切為活性片段,p-Chk1-S345燐痠化增加,而Fas、Fas-L、ERK1及ERK2錶達未見上調.結論 Vpr在體外能夠有效抑製結腸癌細胞株HCT-8增殖,併誘導其細胞週期G2期阻滯及凋亡,其機製分彆與DNA損傷信號通路激活及線粒體凋亡通路啟動有關.Vpr在結腸癌的治療中具有潛在的應用前景.
목적 탐토인류면역결함병독1형( HIV-1)병독단백r(Vpr)대인결장암세포HCT-8적억제작용급기궤제.방법 장세포분위공백대조조、공재체전염조화Vpr전염조.공백대조조:세포불여간예；공재체전염조:대수생장기적세포가입불동감염복수(MOI)적공재체선병독；Vpr전염조:가입불동MOI적함유HIV1-Vpr기인적중조선병독(Adv-Vpr).응용MTT비색법검측세포증식활성,류식세포의검측세포주기、세포조망급선립체막전위,Western blot법검측세포조망상관단백적표체.다조간균수비교채용단인소방차분석,량량비교채용q검험；혹채용쌍인소방차분석,조내비교채용t검험.결과 Vpr현저억제인결장암세포HCT-8증식,Adv-Vpr전염72 h후(MOI=200),Vpr전염조세포MTT치위1.03±0.04,여공재체전염조(2.46 +0.15)급공백대조조(2.51±0.14)비교,차이유통계학의의(F=144.6,P＜0.05)；Adv-Vpr전염48 h후(MOI=200),Vpr전염조처우G2/M기적세포비례급선립체막전위하강적세포비례증가,분별위37.31％±5.90％화32.07％±5.64％,여공재체전염조(18.30％±6.04％、3.32％±0.79％)급공백대조조916.66％±3.51％、2.76％±1.43％)비교,차이유통계학의의(F=10.08,64.45,P＜0.05).Adv-Vpr전염72 h후(MOI=200),Vpr전염조세포조망솔위37.62％±6.48％,여공재체전염조(3.44％±1.11％)급공백대조조(2.93％±1.07％)비교,차이유통계학의의(F=122.4,P＜0.05).Adv-Vpr처리48 h후(MOI=200),Westem blot검측발현Vpr도치료Caspase-9、Caspase-3전절위활성편단,p-Chk1-S345린산화증가,이Fas、Fas-L、ERK1급ERK2표체미견상조.결론 Vpr재체외능구유효억제결장암세포주HCT-8증식,병유도기세포주기G2기조체급조망,기궤제분별여DNA손상신호통로격활급선립체조망통로계동유관.Vpr재결장암적치료중구유잠재적응용전경.
Objective To investigate the inhibitive effects of viral protein r (Vpr) of human immunodeficiency virus 1 ( HIV-1 ) on human colorectal cancer cell line HCT-8,and to find the possible mechanisms.Methods The HCT-8 cells were divided into the control group,adv group and adv-Vpr group.HCT-8 cells were not treated in the control group; HCT-8 cells were treated with Adv or Adv-Vpr at different multiplicity of infection (MOI) in the Adv group or Adv-Vpr group,respectively.Cell proliferation was detected by MTT assay.Cell cycle,apoptosis and mitochondrial membrane potential were detected by flow cytometry.The expression of apoptosisrelated proteins was detected by Western blot.All data were analyzed by using the q test,t test and one-way or two-way analysis of variance.Results The proliferation of HCT-8 cells was significantly inhibited by Vpr.The MTT value of HCT- 8 cells in the Adv-Vpr group was 1.03 ± 0.04,which was significantly lower than 2.46 ± 0.15 in the Adv group and 2.51 ± 0.14 in the control group at 72 hours after Adv-Vpr transfection ( MOI =200) ( F =144.6,P ＜ 0.05).The ratio of HCT-8 cells in the G2/M phase was 37.31％ ± 5.90％ in the Adv-Vpr group,which was significantly higher than 18.30％ ± 6.04％ in the Adv group and 16.66％ ± 3.51％ in the control group ( F =10.08,P ＜ 0.05 ).The ratio of HCT-8 cells with decreased mitochondrial membrane potential was 32.07％ ±5.64％ in the Adv-Vpr group,which was significantly higher than 3.32％ ±0.79％ in the Adv group and 2.76％ ±1.43 ％ in the control group at 48 hours after Adv-Vpr transfection ( MOI =200) ( F =64.45,P ＜ 0.05).The apoptosis rate of HCT-8 cells was 37.62％ ±6.48％ in the Adv-Vpr group,which was significantly higher than 3.44％ ± 1.11％ in the Adv group and 2.93％ ± 1.07％ in the control group at 72 hours after Adv-Vpr transfection ( MOI =200) ( F =122.4,P ＜ 0.05 ).The results of Western blot showed that Vpr induced cleavage and activation of Caspase-9 and Caspase-3 and phosphorylation of Chk1-S345,while the expression levels of Fas,Fas-L,ERK1,ERK2 remained the same at 48 hours after Adv-Vpr treatment ( MOI =200).Conclusions Vpr inhibits the proliferation of the HCT-8 cells in vitro through G2/M phase arrest and apoptosis.Vpr plays its role by activating DNA damaging pathway and initiating mitochondria apoptotic pathway.Vpr is a potential therapeutic agent for colorectal cancer.
