PTEN基因表达改变对人气道平滑肌迁移的影响

Alteration of PTEN gene expression affects the migration of human airway smooth muscle cells in vitro

目的通过腺病毒载体体外转染人气道平滑肌细胞(HASMC),使肿瘤抑制基因PTEN过表达和RNA干扰表达,观察其对HASMC迁移的影响,并探讨其作用机制。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HASMC细胞,并以感染空载腺病毒Ad-GFP和空白组(DMEM)作为对照,用流式细胞计数确立最佳转染复数后,通过Transwell趋化小室和细胞划痕实验检测HASMC跨膜迁移能力和横向迁移能力的变化。Western blotting检测PTEN蛋白的表达和AKT、ERK1/2通路的活化情况,并加入两通路的抑制剂作为阳性对照。结果腺病毒搭载的PTEN基因过表达和干扰载体能成功改变PTEN基因的表达。上调PTEN表达能显著抑制HASMC迁移;蛋白水平上抑制PI3K/AKT通路,而MAPK/ERK通路未见变化。下调PTEN表达不能明显促进HASMC迁移;但在蛋白水平能使PI3K/AKT通路激活,而MAPK/ERK通路却受到抑制。结论上调PTEN表达能有效抑制HASMC的迁移,可能主要是通过抑制PI3K/AKT通路起作用。

Objective To investigate the changes in human airway smooth muscle cell（HASMC） migration and related signaling pathway after interference with PTEN gene expression.Method HASMCs were infected with an adenovirus vector and RNA interference vector of human PTEN gene to establish the cell model with PTEN gene over-expression（Ad-GFP-PTEN-HASMC） and one with PTEN gene silencing（Ad-shPTEN-HASMC）,using Ad-GFP-infected and a blank cells as the negative controls and LY294002 as the positive control.Fluorescence microscopy and flow cytometric analysis were used to evaluate the transfection efficiency,and Western blotting was performed to examine the expression of PTEN and the activation of AKT and ERK1/2 signal pathway.Transwell assay and wound healing assay were used to assess the migration of HASMCs.Results The adenovirus over-expression vector and RNA interference vector significantly affected the expression of human PTEN gene.Up-regulation of PTEN gene resulted in a slow-down of the HASMC migration,an inhibition of PI3K/AKT signal pathway at the protein level but no changes in Ras-Raf-MEK1/2-ERK1/2 signal pathway.Down-regulated PTEN gene expression,however,was not associated with an enhancement of HASMC migration,but activated PI3K/AKT signal pathway and inhibited Ras-Raf-MEK1/2-ERK1/2 signal pathway.Conclusion Upregulation of PTEN gene can effectively inhibit airway smooth muscle cell migration,the effect of which is probably mediated by the PI3K/AKT pathway.