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中华妇幼临床医学杂志(电子版) ›› 2015, Vol. 11 ›› Issue (06) : 752 -756. doi: 10.3877/cma.j.issn.1673-5250.2015.06.015

所属专题: 文献

论著

应用干扰RNA慢病毒技术抑制人脑胶质瘤细胞中红细胞生成素受体mRNA的表达
兰海霞1, 呼格吉乐2,*,*()   
  1. 1. 010050 呼和浩特,解放军第253医院儿科
    2. 010050 呼和浩特,内蒙古医科大学附属医院检验科
  • 收稿日期:2015-04-07 修回日期:2015-09-15 出版日期:2015-12-01
  • 通信作者: 呼格吉乐

Silencing the expression of erythropoietin receptor in human glioma cells by interfering RNA expressing lentivirus

Haixia Lan1, Gejile Hu2()   

  1. 1. Department of Pediatrics, 253th Hospital of P. L.A, Hohhot 010050, Inner Mongolia Autonomous Region, China.
    2. Department of Laboratory Medicine, Affiliated Hospital of Inner Mongolia Modical University, Hohhot 010050, Inner Mongolia Autonomous Region, China
  • Received:2015-04-07 Revised:2015-09-15 Published:2015-12-01
  • Corresponding author: Gejile Hu
  • About author:
    Corresponding author: Hu Gejile, Email:
引用本文:

兰海霞, 呼格吉乐. 应用干扰RNA慢病毒技术抑制人脑胶质瘤细胞中红细胞生成素受体mRNA的表达[J]. 中华妇幼临床医学杂志(电子版), 2015, 11(06): 752-756.

Haixia Lan, Gejile Hu. Silencing the expression of erythropoietin receptor in human glioma cells by interfering RNA expressing lentivirus[J]. Chinese Journal of Obstetrics & Gynecology and Pediatrics(Electronic Edition), 2015, 11(06): 752-756.

目的

探讨重组红细胞生成素受体(EPOR)干扰RNA慢病毒抑制人脑胶质瘤细胞中EPOR mRNA的表达情况。

方法

选择人脑胶质瘤U251细胞株和人胚肾293T细胞株为研究对象。利用RNA干扰技术构建重组EPOR干扰RNA慢病毒表达载体,进行病毒包装、病毒收集、病毒滴度检测后,感染人脑胶质瘤U251细胞,选择最佳MOI值;采用实时聚合酶链式反应(RT-PCR)检测U251细胞和感染重组EPOR干扰RNA慢病毒的U251细胞EPOR mRNA相对表达量,并计算重组EPOR干扰RNA慢病毒对于U251细胞EPOR mRNA表达的干扰效率。

结果

①本研究成功构建重组EPOR干扰RNA慢病毒表达载体,并测定其病毒滴度为1×1014TU/L。②MOI值为100的重组EPOR干扰RNA慢病毒感染U251细胞的荧光蛋白表达量最高,MOI值为1的荧光蛋白表达量最低。③重组EPOR干扰RNA慢病毒对于U251细胞的干扰效率为69%,并使EPOR mRNA表达明显受到抑制。

结论

成功构建重组EPOR干扰RNA慢病毒表达载体,使U251细胞的EPOR mRNA相对表达量明显降低,为后续EPOR转录水平变化的相关研究奠定实验基础。

Objective

To discuss and construct the infected with the human U251 glioma cells, the lentivirus vector of siRNA interference specific of erythropoietin receptor (EPOR) interference RNA, aim at finding the ways for further research on EPOR and its role in glioma and provide valuable data.

Methods

Lentivirus expression vector of RNA interference specific of EPOR was selected and then detected by a drop degree test. Afterwards, the lentivirus vector was transfected with U251 cell lines. By the application of real-time polymerase chain reaction (RT-PCR), the expression levels of EPOR mRNA was detected. The best multiplicity of infection (MOI) values under different gradient were detected.

Results

Lentivirus vector of targets RNA interference specific for EPOR was properly constructed, which was resulted into LVEPOR. After a comparison and a statistical analysis of the PCR product's gray values in each group, the results showed that the expression of EPOR mRNA was significantly inhibited.

Conclusions

EPOR RNA interference lentivirus vector was successfully constructed. EPOR mRNA expression of U251 cells was noticeably reduced, and the screened MOI values of U251 cells was 100.

表1 GAPDH RT-PCR反应体系(μL)
表2 EPOR RT-PCR反应体系(μL)
图1 重组EPOR干扰RNA穿梭质粒靶序列测序图
图2 LV3穿梭质粒和包装质粒(pGag/Pol、pRev、pVSV-G)结构示意图(图2A:LV3穿梭质粒;图2B:pGag/Pol;图2C:pRev;图2D:pVSV-G)
图3 重组EPOR干扰RNA慢病毒感染人胚肾293T细胞图(中倍镜)
图4 不同MOI值重组EPOR干扰RNA慢病毒感染人脑胶质瘤U251细胞(图4A: MOI值为100;图4B: MOI值为10;图4C: MOI值为1)(高倍镜)
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