天津医药

天津医药 ›› 2014, Vol. 42 ›› Issue (12): 1168-1171.doi: 10.3969/j.issn.0253-9896.2014.12.005

• 细胞与分子生物学 • 上一篇    下一篇

P53 对蛋白激酶 R 和宫颈癌 HeLa 细胞生物学行为的影响

罗远材,郭路   

  1. 天津市第一中心医院妇产科
  • 收稿日期:2014-02-13 修回日期:2014-09-05 出版日期:2014-12-15 发布日期:2014-12-15
  • 通讯作者: 罗远材 E-mail:lg050919@163.com
  • 基金资助:
    天津市卫生局科技基金重点项目

P53 Upregulated Protein Kinase R to Restrain Cervical Cancer HeLa Cell Proliferation and Invasion

LUO Yuancai,GUO Lu   

  1. Department of Gynecology and Obstetrics, Tianjin First Central Hospital
  • Received:2014-02-13 Revised:2014-09-05 Published:2014-12-15 Online:2014-12-15
  • Contact: LUO Yuancai E-mail:lg050919@163.com
  • About author:

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摘要：

【摘要】 目的 探讨 P53 蛋白对蛋白激酶 R（PKR）表达和活性以及对宫颈癌 HeLa 细胞生物学行为的影响。方法 构建过表达 p53 基因的重组质粒 pEGFP-C1/p53， 转染 HeLa 细胞， 采用逆转录-聚合酶链反应(RT-PCR)法检测 pEGFP-C1/p53 转染组、空质粒 pEGFP-C1 转染组及空白对照组（仅加入转染试剂）p53 及 PKR mRNA 的表达； 采用 Western Blot 法检测上述 3 组中 P53、 PKR、 磷酸化型 PKR(p-PKR)， PKR 下游底物真核细胞翻译启始因子 2α （eIF2α）的磷酸化型 p-eIF2α的表达； 采用四甲基偶氮唑蓝（MTT）法检测 HeLa 细胞增殖活性变化， Transwell 侵袭实验检测 HeLa 细胞侵袭能力变化。 结果 pEGFP-C1/p53 转染组 p53 及 PKR mRNA 的相对表达量高于 pEGFP-C1 转染组和空白对照组（均 P＜ 0.05）， pEGFP-C1 转染组和空白对照组比较， 差异无统计学意义； pEGFP-C1/p53 转染组 P53、PKR、p-PKR 及 p-eIF2α蛋白的相对表达量高于 pEGFP-C1 转染组和空白对照组（均 P＜ 0.05）， pEGFP-C1 转染组和空白对照组比较， 差异无统计学意义； pEGFP-C1/p53 转染组 HeLa 细胞增殖活性及侵袭能力均显著低于 pEGFP-C1 转染组和空白对照组（ 均 P＜ 0.05）， pEGFP-C1 转染组和空白对照组比较， 差异无统计学意义。 结论 P53 能上调 PKR 的表达及活性， 激活 PKR/eIF2α信号通路， 抑制宫颈癌 HeLa 细胞增殖及侵袭。

关键词: 基因, P53, 细胞增殖, 蛋白激酶类, 肿瘤侵润, 真核细胞翻译启始因子2α

Abstract:

[Abstract] Objective To investigate the effects of p53 on expression and activity of protein kinase R (PKR) as well as biological characters of HeLa cells from cervical carcinoma patients. Methods Recombinant plasmid vector pEGFPC1/p53 was constructed to over-express p53 then it was transfected into HeLa cells. Transcription levels of p53 and PKR mRNA were detected by reverse transcriptase polymerase chain reaction (RT-PCR) among pEGFP-C1/p53 transfection group, pEGFP-C1 transfection group and blank control group（only transfection reagent was added）； Protein expression lev? els of p53, PKR, phosphated PKR（p-PKR） and phosphated α subunit of eukaryotic initiation factor 2（p-eIF2α） which is the downstream substrate of PKR were detected by Western Blot among three groups； Proliferation of HeLa cell were deter? mined by methyl thiazolyl tetrazolium（MTT） assay； Invasion of HeLa cell were determined by Transwell cell assay. Results Recombinant plasmid vector pEGFP-C1/p53 was successfully constructed to overexpress p53； Transcription level of p53 and PKR mRNA in pEGFP-C1/p53 transfection group were higher than those in pEGFP-C1 transfection group and in blank control group (P＜ 0.05)， and there were no significant difference between their levels in pEGFP-C1 transfection group and in blank control group； Protein expression levels of p53, PKR, p-PKR andp-eIF2α in pEGFP-C1/p53 transfection group were higher than those in pEGFP-C1 transfection group and in blank control group (P＜ 0.05)， and there were no sig? nificant difference between those expression levels in pEGFP-C1 transfection group and in blank control group； MTT and Transwell cell results showed that proliferation and invasion of HeLa cells in pEGFP-C1/p53 transfection group were weaker than those in pEGFP-C1 transfection group and in blank control group (P＜ 0.05)， and there were no significant difference between proliferation and invasion of HeLa cells in pEGFP-C1 transfection group and in blank control group. Conclusion p53 can up-regulate the expression and activity of PKR, promote activation of PKR/eIF2α signal transduction pas? sage and restrain cell proliferation and invasion of HeLa cells.

Key words: genes, P53, cell proliferation, protein kinases, neoplasm invasiveness, &alpha, subunit of eukaryotic initiation factor 2