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目的:通过基因过表达和RNA干扰(RNAi)技术改变PTEN基因的表达并观察其对人支气管平滑肌细胞(BSMC)增殖和相关信号通路的影响。方法:利用携带野生型PTEN基因的重组腺病毒(Ad-GFP-PTEN)和针对PTEN基因的腺病毒短发夹状RNA(shRNA)载体(Ad-GFP-shRNA-PTEN),转染BSMC细胞,建立PTEN基因过表达细胞模型Ad-GFP-PTEN-BSMC和PTEN基因沉默细胞模型Ad-GFP-shPTEN-BSMC,并以感染Ad-GFP和空白组作为对照。通过荧光倒置显微镜检测转染效率;Westernblot法检测PTEN蛋白的表达和AKT、ERK1/2通路的活化情况;MTS/PMS法检测细胞生长的变化;PI单染流式细胞术检测细胞周期的变化。结果:腺病毒介导的过表达载体和shRNA载体都能有效改变PTEN基因的表达。PTEN基因表达上调后,BSMC细胞生长受到抑制,G0-G1期细胞增多,S期细胞和G2/M期细胞减少,PI3K/AKT通路受到抑制,而MAPK/ERK通路未见变化;PTEN基因表达下调后,BSMC细胞生长受到促进,S期细胞增多,G0-G1期细胞和G2/M期细胞减少,PI3K/AKT通路激活,但MAPK/ERK通路却受到抑制。结论:PTEN基因可能主要通过调节PI3K/AKT通路影响人支气管平滑肌细胞的生长。
OBJECTIVE: To study the effect of overexpression and RNA interference (RNAi) on the expression of PTEN gene in human bronchial smooth muscle cells (BSMC) and its related signaling pathways. Methods: BSMC cells were transfected with recombinant adenovirus (Ad-GFP-PTEN) carrying wild type PTEN gene and adenovirus short hairpin RNA (shRNA) vector targeting PTEN gene. Ad-GFP-PTEN-BSMC and PTEN gene silencing cell model Ad-GFP-shPTEN-BSMC were constructed, and infected with Ad-GFP and blank group as a control. The transfection efficiency was detected by fluorescence inverted microscope. The expression of PTEN protein and the activation of AKT and ERK1 / 2 pathways were detected by Western blot. The cell growth was detected by MTS / PMS and the cell cycle was detected by PI single flow cytometry. Results: Both adenovirus-mediated overexpression vector and shRNA vector could effectively change the expression of PTEN gene. After up-regulated PTEN gene expression, the growth of BSMC cells was inhibited, the number of cells in G0-G1 phase increased, the number of S phase cells and G2 / M phase cells decreased, and the PI3K / AKT pathway was inhibited, while the MAPK / ERK pathway was not changed; The growth of BSMC cells was promoted. The number of cells in S phase increased, the number of G0-G1 phase cells and G2 / M phase cells decreased, while the activation of PI3K / AKT pathway, but the MAPK / ERK pathway was inhibited. Conclusion: The PTEN gene may affect the growth of human bronchial smooth muscle cells mainly through the regulation of PI3K / AKT pathway.