中华耳鼻咽喉头颈外科杂志
中華耳鼻嚥喉頭頸外科雜誌
중화이비인후두경외과잡지
CHINESE JOURNAL OF OTORHINOLARYNGOLOGY HEAD AND NECK SURGERY
2009年
6期
504-508
,共5页
孟大为%暴继敏%马云鹏%李哲%李素洁%李丹
孟大為%暴繼敏%馬雲鵬%李哲%李素潔%李丹
맹대위%폭계민%마운붕%리철%리소길%리단
肿瘤细胞,培养的%鼻咽肿瘤%癌,鳞状细胞%Twist转录因子%紫杉酚%药物耐受性
腫瘤細胞,培養的%鼻嚥腫瘤%癌,鱗狀細胞%Twist轉錄因子%紫杉酚%藥物耐受性
종류세포,배양적%비인종류%암,린상세포%Twist전록인자%자삼분%약물내수성
Tumor cells,cultured%Nasopharyngeal neoplasms%Carcinoma,squamous cell%Twist transcription factor%Paclitaxel%Drug tolerance
目的 研究降低Twist1表达可否改变鼻咽癌细胞系HNE1对紫杉醇的敏感性.方法 用包含Twist1小分子干扰RNA(small interfering RNA,siRNA)序列的表达质粒转染HNE1细胞,以新霉素(400 μg/ml)筛选阳性克隆,采用反转录聚合酶链反应(RT-PCR)和蛋白印迹法鉴定Twist1蛋白的低表达.通过Annexin V-异硫氰酸荧光素(fluorescein isothiocyanate,FITC)-碘化丙啶(propidium iodide,PI)双标法和观察DNA降解的方法分析si-Twist1 HNE1细胞对紫杉醇的敏感性,采用实时PCR技术分析凋亡相关基因的表达,流式细胞周期分析和叫甲基偶氮唑蓝(MTT)方法分析降低Twist1表达是否影响HNE1细胞增殖.结果 Annexin V-FITC-PI双标法检测结果显示,10 ng/ml紫杉醇处理后,si-Twist1 HNE1细胞的凋亡率为40.2%,显著高于对照siRNA组的24.3%,差异有统计学意义(t=56.999,P=0.000);恢复Twist1蛋白的表达,紫杉醇引起的凋亡率在低Twist蛋白表达组为44.80%±4.80%(x±s,下同),在高表达组为27.00%±2.91%,差异有统计学意义(t=4.374,P=0.049).实时PCR实验结果显示,紫杉醇处理的HNEI细胞中si-Twist组凋亡相关蛋白B淋巴细胞(bcl)-2的相对表达量为0.28±0.05,显著低于对照siRNA组的0.57±0.08,差异有统计学意义(t=6.710,P=0.021);而bax和bcl-XL基因的转录水平没有明显改变(t值分别为2.000和1.502,P值均>0.05).MTT实验和流式细胞术检测显示Twist表达降低没有影响HNE1细胞增殖(P值均>0.05).结论 降低Twist1表达可能是通过诱导凋亡提高了细胞对化疗药物敏感性,提示Twist蛋白可望成为一项新的鼻咽癌治疗靶标.
目的 研究降低Twist1錶達可否改變鼻嚥癌細胞繫HNE1對紫杉醇的敏感性.方法 用包含Twist1小分子榦擾RNA(small interfering RNA,siRNA)序列的錶達質粒轉染HNE1細胞,以新黴素(400 μg/ml)篩選暘性剋隆,採用反轉錄聚閤酶鏈反應(RT-PCR)和蛋白印跡法鑒定Twist1蛋白的低錶達.通過Annexin V-異硫氰痠熒光素(fluorescein isothiocyanate,FITC)-碘化丙啶(propidium iodide,PI)雙標法和觀察DNA降解的方法分析si-Twist1 HNE1細胞對紫杉醇的敏感性,採用實時PCR技術分析凋亡相關基因的錶達,流式細胞週期分析和叫甲基偶氮唑藍(MTT)方法分析降低Twist1錶達是否影響HNE1細胞增殖.結果 Annexin V-FITC-PI雙標法檢測結果顯示,10 ng/ml紫杉醇處理後,si-Twist1 HNE1細胞的凋亡率為40.2%,顯著高于對照siRNA組的24.3%,差異有統計學意義(t=56.999,P=0.000);恢複Twist1蛋白的錶達,紫杉醇引起的凋亡率在低Twist蛋白錶達組為44.80%±4.80%(x±s,下同),在高錶達組為27.00%±2.91%,差異有統計學意義(t=4.374,P=0.049).實時PCR實驗結果顯示,紫杉醇處理的HNEI細胞中si-Twist組凋亡相關蛋白B淋巴細胞(bcl)-2的相對錶達量為0.28±0.05,顯著低于對照siRNA組的0.57±0.08,差異有統計學意義(t=6.710,P=0.021);而bax和bcl-XL基因的轉錄水平沒有明顯改變(t值分彆為2.000和1.502,P值均>0.05).MTT實驗和流式細胞術檢測顯示Twist錶達降低沒有影響HNE1細胞增殖(P值均>0.05).結論 降低Twist1錶達可能是通過誘導凋亡提高瞭細胞對化療藥物敏感性,提示Twist蛋白可望成為一項新的鼻嚥癌治療靶標.
목적 연구강저Twist1표체가부개변비인암세포계HNE1대자삼순적민감성.방법 용포함Twist1소분자간우RNA(small interfering RNA,siRNA)서렬적표체질립전염HNE1세포,이신매소(400 μg/ml)사선양성극륭,채용반전록취합매련반응(RT-PCR)화단백인적법감정Twist1단백적저표체.통과Annexin V-이류청산형광소(fluorescein isothiocyanate,FITC)-전화병정(propidium iodide,PI)쌍표법화관찰DNA강해적방법분석si-Twist1 HNE1세포대자삼순적민감성,채용실시PCR기술분석조망상관기인적표체,류식세포주기분석화규갑기우담서람(MTT)방법분석강저Twist1표체시부영향HNE1세포증식.결과 Annexin V-FITC-PI쌍표법검측결과현시,10 ng/ml자삼순처리후,si-Twist1 HNE1세포적조망솔위40.2%,현저고우대조siRNA조적24.3%,차이유통계학의의(t=56.999,P=0.000);회복Twist1단백적표체,자삼순인기적조망솔재저Twist단백표체조위44.80%±4.80%(x±s,하동),재고표체조위27.00%±2.91%,차이유통계학의의(t=4.374,P=0.049).실시PCR실험결과현시,자삼순처리적HNEI세포중si-Twist조조망상관단백B림파세포(bcl)-2적상대표체량위0.28±0.05,현저저우대조siRNA조적0.57±0.08,차이유통계학의의(t=6.710,P=0.021);이bax화bcl-XL기인적전록수평몰유명현개변(t치분별위2.000화1.502,P치균>0.05).MTT실험화류식세포술검측현시Twist표체강저몰유영향HNE1세포증식(P치균>0.05).결론 강저Twist1표체가능시통과유도조망제고료세포대화료약물민감성,제시Twist단백가망성위일항신적비인암치료파표.
Objective To investigate whether down-regulation of Twist1 could change sensitivity of nasopharyngeal carcinoma cell line HNE1 to taxol. Methods HNE1 cells were transfected with the small interfering RNA (siRNA) expression vector pSuppressor-Retro-Si-Twist, containing the short hairpin RNA (shRNA) sequence targeting the Twist gene-coding region by Fugene 6. Positive clones were then selected in Neomycin (400 μg/ml) for 21 days. The low expressions of Twist1 were examined by real-time reverse-transcription polymerase chain reaction (RT-PCR) and Western blot. Drug sensitivity of si-Twist1 HNE1 to taxol was determined by Annexin V-fluorescein isothiocyanate( FITC)/prepidium Iodide(PI) double-labeled flow cytometry and detection of DNA ladder. The Effect of Twist1 inactivation on HNE1 cell proliferation was observed by MTT assay and flow cytometry. Results Annexin V- FITC-PI assay showed that apoptosis ratio was 40.2% in si-Twist HNE1 after treated with 10 ng/ml taxol, significantly higher than that in the control siRNA group 24. 3%. The deference had statistic meaning. After the re-expression of HNE1, apoptosis ratio was 44. 80% ± 4. 80% (x±s) in low Twist1 protein expression group and that was 27. 00%±2. 91% in high expression group. The deference had statistic meaning ( t = 4. 374, P = 0. 049). Real time PCR test revealed apoptosis protein bcl-2 expression in ai-Twist HNE1 was 0. 28±0. 05, significantly lower than that in the control siRNA HNE1 (0. 57 ± 0. 08,t = 6. 710, P = 0. 021 ), nevertheless, significant bax and bcl-XL changes were not observed (t = 2. 000, P = 0. 184 and t = 1. 502, P = 0. 272 ). MTT and FCM showed that down-regulation of Twist1 did not alter cell proliferation rate ( P > 0. 05 ). Conclusions Down-regulation of Twist1 could increase drug sensitivity of nasopharyngeal carcinoma cell line HNE1 to taxol by inducing apoptosis. These results suggested that Twist1 may be a promising treatment target for nasopharyngeal carcinoma therapy.
