| 羌伟光,吴琴琴,杨垒,等.siRNA抑制TPP1对人骨肉瘤端粒酶阴性细胞U2OS放射敏感性的影响[J].中华放射医学与防护杂志,2013,33(6):579-583.QIANG Wei-guang,WU Qin-qin,YANG Lei,et al.Influence of TPP1 siRNA on the radiosensitivity of telomerase-negative human osteosarcoma cells U2OS[J].Chin J Radiol Med Prot,2013,33(6):579-583 |
| siRNA抑制TPP1对人骨肉瘤端粒酶阴性细胞U2OS放射敏感性的影响 |
| Influence of TPP1 siRNA on the radiosensitivity of telomerase-negative human osteosarcoma cells U2OS |
| 投稿时间：2013-03-06 |
| DOI：10.3760/cma.j.issn.0254-5098.2013.06.003 |
| 中文关键词: RNA干扰 TPP1 端粒 端粒长度 放射敏感性 |
| 英文关键词:RNA interference TPP1 Telomere Telomere length Radiosensitivity |
| 基金项目:国家自然科学基金（81071825） |
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| 中文摘要: |
| 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TPP1 mRNA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。 |
| 英文摘要: |
| 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. |
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