第一临床医学院知识库

背景和目的：慢性阻塞性肺疾病（简称慢阻肺）发病率和死亡率呈逐年上升趋势，疾病负担重，但其具体发病机制不清。研究发现，肺泡巨噬细胞（AM）吞噬功能下降是慢阻肺急性加重的重要原因之一，AM吞噬过程主要与细胞骨架重排有关；另有研究发现，磷脂酰肌醇3-激酶（PI3K）-哺乳动物雷帕霉素靶蛋白（mTOR）-Ras同源基因家族成员A（RhoA）信号通路参与调控细胞骨架重排，但PI3K-mTOR-RhoA信号通路调控细胞骨架重排与巨噬细胞吞噬能力的研究鲜见报道，故本研究采用基因干扰（RNAi）技术沉默该通路的PI3K、mTOR蛋白，探讨PI3K-mTOR-RhoA信号通路调控细胞骨架重排与巨噬细胞吞噬能力的关系及其具体机制。方法：培养小鼠巨噬细胞系RAW264.7细胞，实验分为4组：空白组（不进行转染）、阴性对照组（用含随机序列的短发夹RNA，即shRNA转染细胞）、沉默PI3K基因组（PI3K-RNAi组）和沉默mTOR基因组（mTOR-RNAi组）。蛋白印迹法（Western blot）检测基因沉默效率；实时荧光定量多聚核苷酸链式反应检测PI3K、mTOR、RhoA的mRNA水平；Western blot检测PI3K、mTOR、RhoA和磷酸化RhoA（p-RhoA）蛋白的表达；激光共聚焦显微镜下观察细胞骨架形态；流式细胞术检测细胞吞噬异硫氰酸酯荧光素标记大肠杆菌（FITC-E.coli）的能力，平均荧光强度（MFI）和吞噬阳性细胞百分比（吞噬%）代表吞噬能力的强弱。结果：1.基因沉默效率：PI3K和mTOR的基因沉默效率分别为（62±7）%、（61±8）%。2.各组PI3K、mTOR、RhoA的mRNA和蛋白及磷酸化RhoA（p-RhoA）蛋白表达：（1）PI3K-RNAi组PI3K、mTOR、RhoA的mRNA、蛋白表达和p-RhoA [（0.29±0.07、0.38±0.02）、（0.54±0.13、0.72±0.02）、（0.59±0.06、0.74±0.03）和0.68±0.02]均低于空白组[（1.00±0.00、1.00±0.04）、（1.00±0.00、1.00±0.04）、（1.00±0.00、1.00±0.04）和1.00±0.06]和阴性对照组[（0.98±0.05、1.00±0.04）、（1.01±0.11、1.01±0.08）、（0.99±1.03、1.00±0.04）和1.03±0.07]（均Р<0.01）； （2）mTOR-RNAi组mTOR的mRNA、蛋白表达（0.30±0.08 、0.39±0.02）均较空白组、阴性对照组减低（均Р<0.01），RhoA的 mRNA、蛋白和p-RhoA蛋白表达 [（1.40±0.21、1.54±0.04）和1.33±0.01]较空白组和阴性对照组升高（均Р<0.01）。3.各组细胞骨架形态变化：PI3K-RNAi组细胞形态僵硬，丝状伪足伸出短小、杂乱，应力纤维数量减少；mTOR-RNAi组细胞变形更为明显，丝状伪足伸出更细长而密集，应力纤维数量明显增加；空白组和阴性对照组细胞变形较明显，丝状伪足伸出较长，应力纤维数量较多。4．各组细胞吞噬FITC-E.coli的能力：PI3K-RNAi组MFI及吞噬%[7435±705、（70.73±2.66）%]均较空白组[11733±935、（79.97±1.07）%]和阴性对照组[10983±954、（79.60±1.17）%]减低（均Р<0.01）；mTOR-RNAi组MFI及吞噬%[18583±1090 、（87.72±1.58）%]均较空白组和阴性对照组增加（均Р<0.01）；空白组和阴性对照组MFI及吞噬%无差异（Р>0.01）。5.相关性分析：RNAi沉默前后，各组细胞PI3KmRNA、蛋白表达与mTORmRNA、蛋白表达呈正相关（均Р<0.05）; PI3KmRNA、蛋白表达与RhoAmRNA、蛋白、p-RhoA蛋白表达呈正相关（均Р<0.05）； mTORmRNA、蛋白表达与RhoAmRNA、蛋白、p-RhoA蛋白表达呈负相关（均Р<0.05）；PI3K、RhoA 的mRNA和蛋白表达和p-RhoA与MFI和吞噬%均呈正相关（均Р<0.05）；mTOR mRNA和蛋白与MFI和吞噬%均呈负相关（均Р<0.05）。结论：1．慢病毒转染法可以成功沉默PI3K、mTOR基因；2．细胞骨架重排与巨噬细胞吞噬能力改变有关；3．PI3K-mTOR-RhoA信号通路调控细胞骨架重排；4.  PI3K正性调控mTOR-RhoA通路，促进细胞骨架恰当重排，增强巨噬细胞吞噬能力；mTOR负性调控该通路，抑制细胞骨架恰当重排，减弱巨噬细胞吞噬能力。

Background and objective: Chronic obstructive pulmonary disease (COPD) has a high incidence and mortality, and the burden of disease is high, but the specific pathogenesis of COPD is unclear. Some studies have found that decreased phagocytic function of alveolar macrophages (AM) is one of the causes of acute exacerbation of COPD, and phagocytosis is mainly related to cytoskeleton rearrangement,another study also found that phosphatidylinositol 3-kinase (PI3K)-mammalian target of rapamycin (mTOR)-Ras homologue family member A (RhoA) signaling pathway is involved in the regulation of cytoskeleton rearrangements, but the relationship between PI3K-mTOR-RhoA signaling pathway regulateing cytoskeleton rearrangement and macrophage phagocytosis is reported rarely, Our study used RNA interference technology (RNAi) to make the PI3K and mTOR gene silenced which belong to the PI3K-mTOR-RhoA signaling pathway and to investigate the relationship between PI3K-mTOR-RhoA signaling pathway regulating cytoskeleton rearrangement and macrophage phagocytosis and the specific mechanisms.Methods: The mouse macrophage cell line RAW264.7 cells were cultured and divided into four groups: blank group(cells were not transfected), negative control group(cells were transfected with random sequence small interference RNA，shRNA),PI3K geneing silence group(PI3K-RNAi group), mTOR geneing silence group(mTOR-RNAi group). The gene silencing efficiency were detected by Western blot, The mRNA of PI3K , mTOR and RhoA were measured by real-time polymerase chain reaction ,and the protein expression of PI3K and mTOR and RhoA were detected by Western blot. Mean fluorescence intensity(MFI)and percentage of RAW264.7 cells engulfing fluoresceine isothiocyanate-labled escheruchia coli (FITC-E.coli) were detected by flow cytometry. The changes of cytoskeleton were observed by laser scaning confocal microscopy.Results:1. The gene silencing efficiency of PI3K and mTOR is (62±7)% and  (61±8)% .2.The mRNA and protein expression of PI3K and mTOR and RhoA: The mRNA, protein expression of PI3K, mTOR, RhoA and p-RhoA of PI3K-RNAi group  [(0.29±0.07,0.38±0.02),(0.54±0.13,0.72±0.02),(0.59±0.06,0.74±0.03),0.68±0.02]weresignificantly lower than those in blank group[(1.00±0.00,1.00±0.04),(1.00±0.00, 1.00±0.04),(1.00±0.00,1.00±0.04),1.00±0.06]and negative control group[(0.98±0.05、1.00±0.04),(1.01±0.11、1.01±0.08),(0.99±1.0、1.00±0.04),1.03±0.07)](all Р<0.01),The mRNA, protein of mTOR in mTOR-RNAi group(0.30±0.08,0.39±0.02)were lower than those in blank group and negative control group(all Р<0.01), while The mRNA, protein of RhoA and p-RhoA(1.40±0.21,1.54±0.04,1.33±0.01)were higher than those in blank group and negative control group(all Р<0.01).3. Morphological changes of cytoskeleton: The cell morphology of PI3K-RNAi group was stiff, the filopodia were short and messy, the number of stress fibers was decreased compared with the blank group; the cell deformation of mTOR-RNAi group was observed more obviously, more slender and more densely, the number of stress fibers were increased significantly compared with the blank group; The blank group and the negative control group cells deformed obviously, extended longer pseudopodia and more stress fibers.4. The cell phagocytic ability of FITC-E.coli: the MFI and RAW264.7 cells% of the PI3K-RNAi group[7435±705,(70.73±2.66)]% were lower than those  in blank group[11733±935,(79.97±1.07)%]and negative control group[10983±954、(79.60±1.17)%](all Р<0.01),while those parameters in mTOR-RNAi group[18583±1090, (87.72±1.58)%] were higher than the blank group and negative control group(all Р<0.01).5. Correlation analysis: Before or after transfection, positive correlations were existed between mRNA and protein of PI3K and mTOR(all P<0.05),positive correlations were also existed between mRNA and protein of PI3K and RhoA and p-RhoA(all P<0.05),while negative correlations were existed between mRNA and protein of mTOR and RhoA and p-RhoA(all Р<0.05). positive correlations were  existed between PI3K mRNA，protein and MFI and phagocytic% (all Р<0.05), positive correlations were also existed between RhoA mRNA，protein, p-RhoA protein and MFI and phagocytic% (all Р<0.05). while negative correlations were existed between mTOR mRNA, protein and MFI and phagocytic% (all Р<0.05).Conclusion:1. The method of lentivirus transfection can successfully make gene of PI3K and mTOR been silenced;2. The cytoskeleton rearrangements was associated with the phagocytic ability of macrophages;3. PI3K-mTOR-RhoA signaling pathway regulating the cytoskeleton rearrangements.4. PI3K positively regulates the mTOR-RhoA pathway, promotes cytoskeleton rearrangement correctly and enhances macrophage ability of phagocytosis. While mTOR negatively regulates the pathway, impedes cytoskeleton rearrangement and attenuates macrophage ability of phagocytosis.