大鼠CB1基因真核表达载体的构建及其对CaSki细胞凋亡的影响

发布时间：2018-01-21 20:07

本文关键词： CB基因 HEK细胞 Ca Ski细胞细胞凋亡　出处：《中国实验动物学报》2015年02期 　论文类型：期刊论文

【摘要】：目的构建大鼠CB1(r CB1)基因真核表达载体,检测r CB1基因在细胞中的表达,研究r CB1对人宫颈癌Ca Ski细胞凋亡的影响。方法从大鼠脑组织中提取总RNA,RT-PCR扩增r CB1基因,通过酶切、纯化、连接PCR纯化产物与p CDNA 3.1(+)质粒,构建pc DNA3.1(+)-r CB1。脂质体法将其转染到HEK293和Ca Ski细胞,Western blot及细胞免疫荧光联合激光扫描共聚焦方法检测r CB1的表达及定位,流式细胞仪检测细胞凋亡率,Western blot与实时荧光定量PCR检测r CB1、Bcl-2、Bax、Bad表达。结果酶切重组质粒获得5300 bp的载体片段和1500 bp的目的片段,测序结果和r CB1基因序列(NM_012784.4)一致。转染HEK293细胞后,r CB1在HEK293细胞细胞膜和细胞质表达。转染Ca Ski细胞后,r CB1使细胞凋亡率增加(P0.05);r CB1基因上调Bax、Bad的表达,同时抑制Bcl-2的表达,与空白组比较,其差异具有显著性(P0.05)。结论成功构建了pc DNA3.1(+)-r CB1真核表达载体,r CB1表达于细胞膜和细胞质,r CB1可以明显促进宫颈癌Ca Ski细胞凋亡,其机制是上调Bax、Bad和抑制Bcl-2表达。
[Abstract]:Objective to construct the eukaryotic expression vector of rat CB1(r CB1 gene and detect the expression of r CB1 gene in cells. To study the effect of r CB1 on apoptosis of human cervical cancer Ca Ski cells. Methods the r CB1 gene was amplified by RT-PCR from rat brain tissue and purified by enzyme digestion. The purified product of PCR was ligated with p CDNA 3.1 () plasmid. PcDNA3.1 was constructed and transfected into HEK293 and Ca Ski cells by liposome method. The expression and localization of r CB1 were detected by Western blot and immunofluorescence combined with laser scanning confocal method, and the apoptosis rate was detected by flow cytometry. Western blot and real-time fluorescence quantitative PCR were used to detect rCB1, Bcl-2 and Bax. Results 5300bp vector fragment and 1500bp target fragment were obtained by restriction endonuclease digestion. The results of sequencing were consistent with the sequence of r CB1 gene nmsp. 012784.4). After transfection of HEK293 cells. R CB1 was expressed in the cell membrane and cytoplasm of HEK293 cells. After transfection of Ca Ski cells, r CB1 increased the apoptosis rate of HEK293 cells. R CB1 gene upregulated the expression of Baxon Bad and inhibited the expression of Bcl-2, compared with the control group. Conclusion the eukaryotic expression vector of pc DNA3.1 (pc-r CB1) was successfully constructed and expressed in cell membrane and cytoplasm. R CB1 can obviously promote apoptosis of Ca Ski cells in cervical cancer by up-regulating Bad and inhibiting Bcl-2 expression.
【作者单位】：重庆医科大学实验动物中心;重庆三峡医药高等专科学校;重庆市啮齿类实验动物工程技术研究中心;
【基金】：重庆市教委自然科学基金项目(KJ111802) 重庆市卫生局医学科研项目(2012-1-096) 重庆市高等教育教学改革研究重点项目(132128、133309) 重庆万州区科技计划项目(201203055) 重庆市应用开发计划项目(cstc2013yykf C10005)
【分类号】：R965
【正文快照】： 。3.重庆市啮齿类实验动物工程技术研究中心,重庆400016)ting the pc DNA3.1(+)-r CB1.The result of sequencing was in agreement with the sequence of r CB1 gene(NM_大麻素是从印度大麻(Cannabis sativa)里发现的一组萜酚类化合物,存在于动物神经系统和免疫系统里[1,2]。

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