中华口腔医学杂志
中華口腔醫學雜誌
중화구강의학잡지
Chinese Journal of Stomatology
2010年
9期
525-530
,共6页
齐红%郭建%张引成%李淑薇%杨荔琳
齊紅%郭建%張引成%李淑薇%楊荔琳
제홍%곽건%장인성%리숙미%양려림
癌,黏液表皮样%寡核糖核苷酸类,反义%细胞凋亡%生存素
癌,黏液錶皮樣%寡覈糖覈苷痠類,反義%細胞凋亡%生存素
암,점액표피양%과핵당핵감산류,반의%세포조망%생존소
Carcinoma,mucoepidermoid%Oligoribonucleotides,antisense%Apoptosis%Survivin
目的 研究生存素(survivin)反义寡核苷酸(antisense oligodeoxynucleotide,ASODN)对人黏液表皮样癌高转移细胞株Mc3细胞的生长抑制效应及可能的作用机制,探讨生存素基因作为黏液表皮样癌基因治疗靶点的可行性.方法 设计合成靶向生存素特异性ASODN,转染Mc3细胞.将转染的Mc3细胞分为4组:空白对照组、脂质体转染组、生存素正义寡核苷酸(sense oligodeoxynucleotide,SODN)转染组、生存素ASODN转染组.转染后24、48及72 h倒置显微镜观察Mc3细胞形态学变化,甲基噻唑基四唑(MTT)法观察转染前后对细胞增殖的影响,流式细胞仪技术检测细胞凋亡率,脱氧核糖核苷酸末端转移酶介导的原位缺口末端标记法分析细胞凋亡指数,半定量反转录聚合酶链反应分别检测生存素mRNA的表达.结果 生存素ASODN转染组的Mc3细胞数量明显少于其他组,脱壁悬浮细胞明显增多,可见典型凋亡改变,并呈时间依赖性.生存素ASODN转染组Mc3细胞G1～G0期前出现明显的亚二倍体凋亡峰,Mc3细胞的凋亡率在转染后24、48及72 h分别为12.96%、14.43%及22.69%,明显高于其他组(P＜0.01),并呈时间依赖性(P＜0.05).生存素ASODN对Mc3细胞增殖抑制率分别为22.35%、39.04%、43.46%,明显高于其他组(P＜0.01),并呈时间依赖性(P＜0.05).生存素ASODN转染组在细胞转染后24、48及72 h Mc3的凋亡指数分别为11.038%、12.172%及18.900%,明显高于其他组(P＜0.01),并呈时间依赖性(P＜0.05).生存素ASODN转染组Mc3细胞生存素mRNA在24、48及72 h的相对表达量分别为0.739±0.008、0.668±0.007、0.500±0.006,相对抑制率分别为18.21%、26.06%、44.82%,明显低于其他组(P＜0.01),并呈时间依赖性(P＜0.01).结论 生存素ASODN可抑制人黏液表皮样癌高转移细胞株Mc3的生长增殖,诱导其凋亡,同时亦可抑制Mc3细胞生存素mRNA的表达,并呈时间依赖性.生存素可作为黏液表皮样癌基因治疗研究的一个靶点.
目的 研究生存素(survivin)反義寡覈苷痠(antisense oligodeoxynucleotide,ASODN)對人黏液錶皮樣癌高轉移細胞株Mc3細胞的生長抑製效應及可能的作用機製,探討生存素基因作為黏液錶皮樣癌基因治療靶點的可行性.方法 設計閤成靶嚮生存素特異性ASODN,轉染Mc3細胞.將轉染的Mc3細胞分為4組:空白對照組、脂質體轉染組、生存素正義寡覈苷痠(sense oligodeoxynucleotide,SODN)轉染組、生存素ASODN轉染組.轉染後24、48及72 h倒置顯微鏡觀察Mc3細胞形態學變化,甲基噻唑基四唑(MTT)法觀察轉染前後對細胞增殖的影響,流式細胞儀技術檢測細胞凋亡率,脫氧覈糖覈苷痠末耑轉移酶介導的原位缺口末耑標記法分析細胞凋亡指數,半定量反轉錄聚閤酶鏈反應分彆檢測生存素mRNA的錶達.結果 生存素ASODN轉染組的Mc3細胞數量明顯少于其他組,脫壁懸浮細胞明顯增多,可見典型凋亡改變,併呈時間依賴性.生存素ASODN轉染組Mc3細胞G1～G0期前齣現明顯的亞二倍體凋亡峰,Mc3細胞的凋亡率在轉染後24、48及72 h分彆為12.96%、14.43%及22.69%,明顯高于其他組(P＜0.01),併呈時間依賴性(P＜0.05).生存素ASODN對Mc3細胞增殖抑製率分彆為22.35%、39.04%、43.46%,明顯高于其他組(P＜0.01),併呈時間依賴性(P＜0.05).生存素ASODN轉染組在細胞轉染後24、48及72 h Mc3的凋亡指數分彆為11.038%、12.172%及18.900%,明顯高于其他組(P＜0.01),併呈時間依賴性(P＜0.05).生存素ASODN轉染組Mc3細胞生存素mRNA在24、48及72 h的相對錶達量分彆為0.739±0.008、0.668±0.007、0.500±0.006,相對抑製率分彆為18.21%、26.06%、44.82%,明顯低于其他組(P＜0.01),併呈時間依賴性(P＜0.01).結論 生存素ASODN可抑製人黏液錶皮樣癌高轉移細胞株Mc3的生長增殖,誘導其凋亡,同時亦可抑製Mc3細胞生存素mRNA的錶達,併呈時間依賴性.生存素可作為黏液錶皮樣癌基因治療研究的一箇靶點.
목적 연구생존소(survivin)반의과핵감산(antisense oligodeoxynucleotide,ASODN)대인점액표피양암고전이세포주Mc3세포적생장억제효응급가능적작용궤제,탐토생존소기인작위점액표피양암기인치료파점적가행성.방법 설계합성파향생존소특이성ASODN,전염Mc3세포.장전염적Mc3세포분위4조:공백대조조、지질체전염조、생존소정의과핵감산(sense oligodeoxynucleotide,SODN)전염조、생존소ASODN전염조.전염후24、48급72 h도치현미경관찰Mc3세포형태학변화,갑기새서기사서(MTT)법관찰전염전후대세포증식적영향,류식세포의기술검측세포조망솔,탈양핵당핵감산말단전이매개도적원위결구말단표기법분석세포조망지수,반정량반전록취합매련반응분별검측생존소mRNA적표체.결과 생존소ASODN전염조적Mc3세포수량명현소우기타조,탈벽현부세포명현증다,가견전형조망개변,병정시간의뢰성.생존소ASODN전염조Mc3세포G1～G0기전출현명현적아이배체조망봉,Mc3세포적조망솔재전염후24、48급72 h분별위12.96%、14.43%급22.69%,명현고우기타조(P＜0.01),병정시간의뢰성(P＜0.05).생존소ASODN대Mc3세포증식억제솔분별위22.35%、39.04%、43.46%,명현고우기타조(P＜0.01),병정시간의뢰성(P＜0.05).생존소ASODN전염조재세포전염후24、48급72 h Mc3적조망지수분별위11.038%、12.172%급18.900%,명현고우기타조(P＜0.01),병정시간의뢰성(P＜0.05).생존소ASODN전염조Mc3세포생존소mRNA재24、48급72 h적상대표체량분별위0.739±0.008、0.668±0.007、0.500±0.006,상대억제솔분별위18.21%、26.06%、44.82%,명현저우기타조(P＜0.01),병정시간의뢰성(P＜0.01).결론 생존소ASODN가억제인점액표피양암고전이세포주Mc3적생장증식,유도기조망,동시역가억제Mc3세포생존소mRNA적표체,병정시간의뢰성.생존소가작위점액표피양암기인치료연구적일개파점.
Objective To determine the effects of survivin antisense oligonucleotide (ASODN) on the expression levels of survivin mRNA and human mucoepidermoid carcinoma cell line (highly metastatic Mc3) apoptosis and to explore the feasibility of survivin gene as the mucoepidermoid carcinoma therapeutic targets. Methods The survivin ASODN was designed and synthesized and then respectively transfected into Mc3 cells. The morphological changes of the Mc3 cells were observed 24, 48 and 72 h after transfection by inverted microscope and the apoptosis rate detected by flow cytometry. Methyl thiazolyl tetrazolium (MTT) assay was used to detect the effect of the transfection on cell poliferation, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) method for analysis of apoptotic index, and semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) to detect the expression of survivin. Results In survivin ASODN transfection group, there was less Mc3 cells than in other groups. The suspended cells dropping from the wall increased and showed typical apoptotic changes and timedependent. Mc3 cell apoptosis rate in survivin ASODN transfection group transfected for 24, 48, 72 h was 12.96% , 14. 43% , 22. 69%,respectively, which were significantly higher than in other groups (P <0. 01) and time-dependent (P < 0.05). The inhibitory rate of Mc3 cells in survivin ASODN transfection group was 22.35% ,39.04% ,43.46% , which were significantly higher than other groups (P < 0. 01) and timedependent(P<0. 05). The apoptosis index (AI) of Mc3 cells in survivin ASODN transfection group was 11.038% , 12. 172% , 18.900% , significantly higher than other groups (P < 0. 01) and time-dependent (P < 0. 05). The survivin mRNA levels in Mc3 cells were 0. 739 ± 0. 008, 0.668 ± 0. 007, 0. 500 ± 0. 006, and the relative inhibition rate in these cells was 18. 21% , 26. 06% , 44. 82% , significantly lower than other groups (P < 0.01) and time-dependent manner (P < 0. 01). Conclusions Survivin ASODN could inhibit the proliferation of Mc3 cells and induce the apoptosis of Mc3 cells. It also can inhibit the expression of survivin mRNA. Survivin can be used as a gene therapy targets for mucoepidermoid carcinoma .
