CAJ | 학술논문

目的探讨沉默survivin对喉癌细胞Hep-2生长的影响.方法将重组质粒(psi-survivin)和阴性对照质粒(psi-scramble)用脂质体包裹转染人喉癌细胞株Hep-2,分别用反转录聚合酶链反应(RT-PCR)和蛋白免疫印迹(Western blot)从mRNA和蛋白水平检测survivin的表达.四甲基偶氮唑蓝(MTT)观察喉癌细胞增殖,流式细胞仪检测细胞凋亡.建立裸鼠喉癌移植瘤模型,将psi-survivin和psi-scramble注射于肿瘤周围,观察其抗肿瘤的效果.免疫组化法和Western blot法检测重组质粒对肿瘤Survivin蛋白表达的影响.末端脱氧核苷酸转移酶介导的d-UTP缺口末端标记技术(Tunel)观察肿瘤细胞的凋亡.结果 psi-survivin不仅在mRNA水平(抑制率为54.4%),而且蛋白水平也抑制Survivin表达,抑制率为37.0%.MTT证实psi-survivin明显抑制喉癌细胞的增殖,抑制率达71.7%.流式细胞仪结果显示psi-survivin组细胞凋亡明显增加,凋亡率达(13.05±0.56)%((-x)±s,下同).体内实验表明,质粒注射后第32天,盐水组瘤体积为(1443.9±230.5)mm3,psi-scramble组为(1348.5±198.4)Rim3,而psi-survivin组为(624.6±188.4)mm3,与对照组相比,差异有统计学意义(t=-5.917,P＜0.01).psi-survivin能明显抑制肿瘤survivin的表达,抑制率为41.8%,与对照组相比,差异有统计学意义(t=-80.343,P＜0.01).Tunel染色结果显示psi-survivin组肿瘤组织中出现较多凋亡细胞,而对照组未见凋亡细胞.结论沉默survivin能明显抑制喉癌细胞Hep-2的生长.
목적 탐토침묵survivin대후암세포Hep-2생장적영향.방법 장중조질립(psi-survivin)화음성대조질립(psi-scramble)용지질체포과전염인후암세포주Hep-2,분별용반전록취합매련반응(RT-PCR)화단백면역인적(Western blot)종mRNA화단백수평검측survivin적표체.사갑기우담서람(MTT)관찰후암세포증식,류식세포의검측세포조망.건립라서후암이식류모형,장psi-survivin화psi-scramble주사우종류주위,관찰기항종류적효과.면역조화법화Western blot법검측중조질립대종류Survivin단백표체적영향.말단탈양핵감산전이매개도적d-UTP결구말단표기기술(Tunel)관찰종류세포적조망.결과 psi-survivin불부재mRNA수평(억제솔위54.4%),이차단백수평야억제Survivin표체,억제솔위37.0%.MTT증실psi-survivin명현억제후암세포적증식,억제솔체71.7%.류식세포의결과현시psi-survivin조세포조망명현증가,조망솔체(13.05±0.56)%((-x)±s,하동).체내실험표명,질립주사후제32천,염수조류체적위(1443.9±230.5)mm3,psi-scramble조위(1348.5±198.4)Rim3,이psi-survivin조위(624.6±188.4)mm3,여대조조상비,차이유통계학의의(t=-5.917,P＜0.01).psi-survivin능명현억제종류survivin적표체,억제솔위41.8%,여대조조상비,차이유통계학의의(t=-80.343,P＜0.01).Tunel염색결과현시psi-survivin조종류조직중출현교다조망세포,이대조조미견조망세포.결론 침묵survivin능명현억제후암세포Hep-2적생장.
Objective To study the effect of silencing survivin on the growth of Hep-2 human laryngeal cancer cells in vitro and in vivo. Methods Hep-2 cells were transfected with pGCsilencer-siRNA-survivin(psi-survivin)by Lipofectamine 2000. The mRNA and protein expressions of survivin were detected by semi-quantitative RT-PCR and Western blot, respectively. Cell proliferation activity was measured by MTT assay. Apoptosis was assessed by flow cytometry. The implanted tumors were formed from injected Hep-2 cells in nude mice. After the tumor formation, psi-survivin was injected into peritumor tissues. The growth of tumor were observed. The tumor volume was calculated and the tumor growth curve was plotted. The expression of survivin in tumor tissues was detected by Western blot. The tumor cell apoptosis was observed by Tunel stainning. Results The sequence-specific siRNA of survivin inhibited the expressions of survivin mRNA and protein. The inhibition rates of survivin mRNA and protein expression were 54.4% and 37.0%respectively. Also the growth of Hep-2 cells was inhibited significanly,with a decrease by 71.7%. By the day 32 of tumor growth, the mean tumor volumes were ( 1443.9 ± 230.5 ) mm3 ( (-x) ± s ) in saline control group, ( 1348.5 ± 198.4) mm3 in plasmid-negative control group, and (624. 6 ± 188.4) mm3 in psi-survivin group,respectively (t = -5.917 ,P ＜0.01 ). In the implanted tumors injected with psi-survivin, survivin protein expression was down-regulated significantly,with a inhibition rate of 41.8%. Tunel stainning showed the apoptosis occurred in the implanted tunors. Conclusion Silencing survivin could significantly inhibit the growth of Hep-2 human laryngeal cancer cells in vitro and in vivo.