14-3-3γ表达载体的构建及其对子宫肌瘤细胞增殖和凋亡的影响

Construction of a 14-3-3γ expression vector and its effect on proliferation and apoptosis of uterine leiomyoma cells

目的:构建一种稳定、高表达的14-3-3γ基因真核表达质粒载体,并探索其抗子宫肌瘤的作用。方法:提取子宫肌瘤细胞总RNA,反转录后通过RT-PCR扩增14-3-3γ,并将扩增的目的基因片段插入pCMVN-Flag真核表达质粒,构建重组质粒14-3-3γ-pCMV-N-Flag,重组体经限制性内切酶酶切分析及测序鉴定后,用脂质体转染技术导入子宫肌瘤细胞。应用Western Blot法检测14-3-3γ蛋白的表达,应用CCK-8检测细胞增殖变化,应用Annexin V-FITC/PI双染色法经流式细胞术检测细胞凋亡。结果:①构建的14-3-3γ-pCMV-N-Flag真核表达载体经酶切后电泳和DNA测序,显示载体构建正确;②子宫肌瘤细胞转染重组体后的14-3-3γ表达明显高于转染前(P<0.05);③子宫肌瘤细胞转染重组体后,细胞增殖抑制率为52.90%(P<0.05);④转染重组体后肌瘤细胞的凋亡率明显高于转染前(P<0.05)。结论:本研究成功构建14-3-3γ-pCMV-N-Flag真核表达质粒载体,14-3-3γ在子宫肌瘤细胞内高表达后可抑制细胞的增殖和促进细胞凋亡。

Objective： To construct stably and highly expressing 14-3-3γ gene eukaryotic expression plas-mid vector and to explore its antitumor effects on uterine leiomyoma cells in vitro. Methods： The total RNA was isolated from the uterine leiomyoma cells. After reverse transcription reaction, 14-3-3γ was amplified by RT- PCR. The PCR product was cloned into the eukaryote expression vector pCMV-N-Flag, the resulted recombinant was designated as 14-3-3γ-pCMV-N-Flag. After sequence determination, the recombinant vector was transfected into uterine leiomyoma cells by lipofectin. The expression of the interesting protein was confirmed by Western Blot analysis. Cell proliferation was assessed by cell counting kit-8 assay and the percentage of apoptotic cells detected by annexinV-FITC/PI double-staining was determined by flow cytometry. Results： ① The 14-3-3γ, gene eukaryotic expression vector was successfully constructed and identified by sequencing. ②The expression level of 14-3-3γ protein was increased obviously after transfection （P〈0.05）. ③ The inhibitory effect on the rate of cell proliferation was 52.90% after transfection （P〈0.05）. ④ After transfection, the percentage of apoptotic cells was increased obviously （P〈0.05）. Conclusion： The 14-3-3γ -pCMV-N-Flag gene eukaryotic expression plasmid vector is successfully constructed which could suppress proliferation and induce apoptosis of uterine leiomyoma cells.