中华放射医学与防护杂志
中華放射醫學與防護雜誌
중화방사의학여방호잡지
Chinese Journal of Radiological Medicine and Protection
2013年
6期
579-583
,共5页
羌伟光%吴琴琴%杨垒%柳正春%江换钢%周福祥%谢丛华%周云峰
羌偉光%吳琴琴%楊壘%柳正春%江換鋼%週福祥%謝叢華%週雲峰
강위광%오금금%양루%류정춘%강환강%주복상%사총화%주운봉
RNA干扰%TPP1%端粒%端粒长度%放射敏感性
RNA榦擾%TPP1%耑粒%耑粒長度%放射敏感性
RNA간우%TPP1%단립%단립장도%방사민감성
RNA interference%TPP1%Telomere%Telomere length%Radiosensitivity
目的 观察人骨肉瘤细胞U2OS中TPP1表达受抑制后其端粒长度和放射敏感性变化,探讨在端粒酶阴性肿瘤细胞中TPP1与端粒长度及放射敏感性之间的关系.方法 根据TPP1mRNA编码序列设计并合成干扰siRNA序列,瞬时转染U2OS细胞;使用RT-PCR和Western blot分别在基因和蛋白水平检测干扰前后TPP1表达量的变化;利用Real-time PCR检测细胞端粒长度的变化;运用流式细胞术(Annexin V-FITC法)检测细胞凋亡情况;采用克隆形成实验分析放射敏感性的变化.结果 siRNA转染U2OS细胞后,siRNA干扰组的mRNA基因表达抑制率为(65.70±2.10)％,蛋白表达抑制率为(74.80 ±3.20)％;siRNA干扰组相对端粒长度(0.99 ±0.07)明显短于阴性对照组(1.64 ±0.05)及空白组(1.55±0.05) (F=113.68,P＜0.01),而阴性对照组与空白组间端粒长度差异无统计学意义;siRNA干扰组细胞凋亡率为(13.67±0.85)％,高于阴性对照组(8.59±0.38)％及空白组(8.18 ±0.21)％ (F=92.32,P＜0.01);siRNA干扰组细胞株SF2值(0.6074 ±0.0115)较空白组(0.8062±0.0056)降低显著(F=246.45,P＜0.01),放射增敏比为1.33.结论 在端粒酶阴性细胞U2OS中,特异性地沉默TPP1能够引起端粒长度的缩短,并且增加了细胞的放射敏感性,推测干扰TPP1引起端粒缩短很可能是其放射增敏的机制之一.
目的 觀察人骨肉瘤細胞U2OS中TPP1錶達受抑製後其耑粒長度和放射敏感性變化,探討在耑粒酶陰性腫瘤細胞中TPP1與耑粒長度及放射敏感性之間的關繫.方法 根據TPP1mRNA編碼序列設計併閤成榦擾siRNA序列,瞬時轉染U2OS細胞;使用RT-PCR和Western blot分彆在基因和蛋白水平檢測榦擾前後TPP1錶達量的變化;利用Real-time PCR檢測細胞耑粒長度的變化;運用流式細胞術(Annexin V-FITC法)檢測細胞凋亡情況;採用剋隆形成實驗分析放射敏感性的變化.結果 siRNA轉染U2OS細胞後,siRNA榦擾組的mRNA基因錶達抑製率為(65.70±2.10)％,蛋白錶達抑製率為(74.80 ±3.20)％;siRNA榦擾組相對耑粒長度(0.99 ±0.07)明顯短于陰性對照組(1.64 ±0.05)及空白組(1.55±0.05) (F=113.68,P＜0.01),而陰性對照組與空白組間耑粒長度差異無統計學意義;siRNA榦擾組細胞凋亡率為(13.67±0.85)％,高于陰性對照組(8.59±0.38)％及空白組(8.18 ±0.21)％ (F=92.32,P＜0.01);siRNA榦擾組細胞株SF2值(0.6074 ±0.0115)較空白組(0.8062±0.0056)降低顯著(F=246.45,P＜0.01),放射增敏比為1.33.結論 在耑粒酶陰性細胞U2OS中,特異性地沉默TPP1能夠引起耑粒長度的縮短,併且增加瞭細胞的放射敏感性,推測榦擾TPP1引起耑粒縮短很可能是其放射增敏的機製之一.
목적 관찰인골육류세포U2OS중TPP1표체수억제후기단립장도화방사민감성변화,탐토재단립매음성종류세포중TPP1여단립장도급방사민감성지간적관계.방법 근거TPP1mRNA편마서렬설계병합성간우siRNA서렬,순시전염U2OS세포;사용RT-PCR화Western blot분별재기인화단백수평검측간우전후TPP1표체량적변화;이용Real-time PCR검측세포단립장도적변화;운용류식세포술(Annexin V-FITC법)검측세포조망정황;채용극륭형성실험분석방사민감성적변화.결과 siRNA전염U2OS세포후,siRNA간우조적mRNA기인표체억제솔위(65.70±2.10)％,단백표체억제솔위(74.80 ±3.20)％;siRNA간우조상대단립장도(0.99 ±0.07)명현단우음성대조조(1.64 ±0.05)급공백조(1.55±0.05) (F=113.68,P＜0.01),이음성대조조여공백조간단립장도차이무통계학의의;siRNA간우조세포조망솔위(13.67±0.85)％,고우음성대조조(8.59±0.38)％급공백조(8.18 ±0.21)％ (F=92.32,P＜0.01);siRNA간우조세포주SF2치(0.6074 ±0.0115)교공백조(0.8062±0.0056)강저현저(F=246.45,P＜0.01),방사증민비위1.33.결론 재단립매음성세포U2OS중,특이성지침묵TPP1능구인기단립장도적축단,병차증가료세포적방사민감성,추측간우TPP1인기단립축단흔가능시기방사증민적궤제지일.
Objective To observe the changes of telomere length and radiosensitivity of U2OS cells after its TPP1 gene is suppressed by small interfering RNA (siRNA).Methods According to the coding sequence of TPP1 mRNA,the interference sequence of TPP1-siRNA was designed.The U2OS cells were transfected with TPP1-siRNA by lipofectamine.The expression of TPP1 was detected by RT-PCR and Western blot,the telomere length was measured by Real-time PCR.The cell apoptosis was analyzed by flow cytometry,and the cell radiosensitivity was determined by clone formation assay.Results The inhibition rates of TPP1 gene and protein expressions in the TPP1-siRNA interference group were (65.70 ± 2.10)％ and (74.80±3.20)％,respectively.The results of Real-time PCR showed that the telomere length of the TPP1-siRNA interference group (0.99 ± 0.07) was significantly shorter than that of the negative group (1.64 ±0.05) and the blank control group (1.55 ±0.05) (F=113.68,P＜0.01).Compared with the negative group,the telomere length of the control group didn't change significantly (P =0.09).The apoptosis rate of TPP1-siRNA interference group (13.67 ± 0.85)％ was higher than those of negative control group (8.59 ±0.38)％ and blank control group (8.18 ±0.21)％ (F=92.32,P ＜ 0.01).U2OS cells with silenced TPP1 (0.6074 ± 0.0115) had a SF2 lower than that of control group (0.8062 ±0.0056) (F=246.45,P＜0.01),and the SER was 1.33.Conclusions Silencing of TPP1 in U2OS cells could shorten the telomere length and enhance the radiosensitivity.The telomere shortening caused by TPP1-siRNA is likely to be one of the reasons of radiosensitization.
