第一临床医学院知识库

背景及目的  慢性阻塞性肺疾病(慢阻肺)肺泡巨噬细胞(AM)吞噬功能降低且与细胞骨架异常重排有关。Toll样受体(TLR)4可能通过调控磷脂酰肌醇3-激酶(PI3K)- Ras相关的C3肉毒素底物(Rac)1信号通路参与AM细胞骨架重排及吞噬过程，且国内外研究罕见。直接体内获取原代AM并进行传代较为困难，且不易进行RNA干扰(RNAi)等技术干预，小鼠巨噬细胞系RAW264.7保留巨噬细胞吞噬及粘附功能，可稳定传代并可进行RNAi等技术干预。故本研究选用RAW264.7细胞为研究对象，探讨TLR4-PI3K-Rac1信号通路在巨噬细胞骨架重排及吞噬功能中的作用。方法  RAW264.7细胞分为空白组、阴性对照组及TLR4-RNAi组。构建3个携带TLR4基因短发卡RNA(shRNA)的质粒及1个携带无意义shRNA序列的阴性对照质粒，采用脂质体转染法分别转染RAW264.7细胞。筛选转染效率最高的一条TLR4-shRNA，将TLR4-shRNA及无意义对照序列shRNA进行慢病毒包装，将携带TLR4-shRNA及携带无意义对照序列的慢病毒颗粒分别转染至TLR4-RNAi组及阴性对照组，空白组不进行转染。蛋白印迹法(Western blot)检测TLR4基因沉默效率。实时荧光定量PCR检测各组细胞PI3K、Rac1 mRNA的表达，Western blot测各组细胞PI3K、磷酸化Rac1(p-Rac1)、Rac1蛋白表达；激光共聚焦显微镜观察细胞骨架变化。流式细胞术检测各组细胞吞噬异硫氰酸荧光素标记的大肠杆菌(FITC-E.coli)的能力，用平均荧光强度(MFI)和吞噬细胞百分比(吞噬%)表示。结果  (1)转染效率及沉默效率检测：TLR4-shRNA质粒及慢病毒可成功转染RAW264.7细胞，转染效率大于80%，TLR4基因沉默效率为(63±4)%；(2)各组TLR4 mRNA和蛋白表达：TLR4-RNAi组TLR4 mRNA和蛋白表达(0.20±0.03、0.37±0.04)均低于空白组和阴性对照组[(1.00±0.00、1.00±0.05)和(0.98±0.09、0.97±0.07)](均P<0.01)；阴性对照组和空白组相比差异无统计学意义；(3)各组PI3K mRNA和蛋白表达：TLR4-RNAi组PI3K mRNA和蛋白表达(0.64±0.06、0.75±0.06)均低于空白组和阴性对照组[(1.00±0.00、1.00±0.08)和(0.98±0.05、1.00±0.09)](均P<0.01)；阴性对照组和空白组相比差异无统计学意义；(4) 各组Rac1 mRNA和蛋白表达：TLR4-RNAi组Rac1 mRNA、蛋白和p-Rac1蛋白表达(0.75±0.04、0.76±0.01、0.74±0.05)均低于空白组和阴性对照组[(1.00±0.00、1.02±0.05、1.00±0.06)和(0.96±0.10、1.07±0.09、1.00±0.06)](均P<0.01)，阴性对照组和空白组相比差异无统计学意义。(5)细胞骨架变化：TLR4-RNAi组RAW264.7细胞伪足伸出短少、僵硬，细胞内吞噬的FITC-E.coli较少，空白组和阴性对照组RAW264.7细胞伪足伸出良好，吞噬的FITC-E.coli较多。(6)各组细胞吞噬FITC-E.coli能力：TLR4-RNAi组MFI和吞噬%[(7453±564)、(70.20±2.27)%]均低于空白组和阴性对照组[(11733±935)、(79.97±1.07)%和(10983±954)、(79.60±1.17)%](均P<0.01)，阴性对照组MFI和吞噬%与空白组相比差异无统计学意义。(7)相关性分析：基础状态下及RNAi干预后TLR4 mRNA和蛋白与PI3K、Rac1 mRNA和蛋白及p-Rac1蛋白均呈正相关(均P<0.05)，基础状态下及RNAi干预后，PI3K mRNA、蛋白表达与Rac1 mRNA、蛋白及p-Rac1蛋白均呈正相关(均P<0.05)，基础状态下及RNAi干预后，TLR4、PI3K、Rac1 mRAN和蛋白及p-Rac1蛋白与MFI、吞噬%均呈正相关(均P<0.05)。

结论  TLR4-shRNA质粒和慢病毒可成功转染RAW264.7细胞，沉默TLR4基因；TLR4-PI3K-Rac1信号通路调控巨噬细胞吞噬功能；沉默TLR4基因可以抑制PI3K-Rac1信号通路，使巨噬细胞骨架排列紊乱、吞噬功能降低。

Background and Objective The phagocytosis of alveolar macrophages(AM) in chronic obstructive pulmonary disease(COPD) is declining and is associated with abnormal rearrangement of the cytoskeleton. Toll-like receptor (TLR) 4 may be involved in AM cytoskeleton rearrangement and phagocytosis by modulating phosphatidylinositol 3-kinase (PI3K)-Ras-associated C3 botulinum substrate (Rac) 1 signaling pathway.This kind of research was rarely reported. AM is difficult to obtain, it is difficult to subculture, and is not easy to use RNA interference (RNAi) and other technologies to intervene. The mouse macrophage cell line RAW264.7 retains macrophage phagocytosis and adhesion functions, can be stably passaged and can undergo technical interventions such as RNAi. Therefore, this study used RAW264.7 cells as a research object to investigate the effects of TLR4-PI3K-Rac1 signaling pathway on macrophage cytoskeletal rearrangement and phagocytosis.

Methods RAW264.7 was divided into blank group, negative control group and TLR4-RNAi group. Three plasmids carrying the TLR4 short hairpin RNA (shRNA) and a negative control plasmid carrying non-significant shRNA sequences were constructed and transfected into RAW264.7 cells by lipofection. A TLR4-shRNA with the highest transfection efficiency was screened, the TLR4-shRNA and shRNA of non-significant control sequences were carried by lentivirus for subsequent transfection. The lentivirus carrying TLR4-shRNA and nonsense control sequence were transfected into TLR4-RNAi group and negative control group, respectively. The blank group was not transfected. The efficiency of silencing TLR4 gene was detected by Western blot. Real-time fluorescence quantitative PCR was used to detect the expression of PI3K and Rac1 mRNA in each group. The expressions of PI3K, p-Rac1 and Rac1 protein were detected by Western blot. Cytoskeleton was observed by laser scanning confocal microscopy. Mean fluorescence intensity (MFI) and the positive percent of RAW264.7 cells engulfing flurescein isothiocyanate-labeled Eseherichina coli (FITC-E.coli) (RAW264.7 cells％) were detected by flow cytometry.

Results  (1) Transfection Efficiency and Silence Efficiency Detection : TLR4-shRNA lentivirus can successfully transfected RAW264.7 cells and the transfection efficiency is more than 80%. The silencing efficiency of TLR4 gene was (63 ± 4)%.(2) TLR4 mRNA and protein expression in each group : TLR4 mRNA and protein expression in TLR4-RNAi group (0.20 ± 0.03, 0.37 ± 0.04) were lower than those in the blank group and the negative control group [(1.00 ± 0.00, 1.00 ± 0.05) and (0.98 ± 0.09, 0.97 ± 0.07)](all P<0.01), There was no significant difference between the negative control group and the blank group.(3) PI3K mRNA and protein expression in each group: The expression of PI3K mRNA and protein in TLR4-RNAi group (0.64 ± 0.06, 0.75 ± 0.06) were lower than those in the blank group and the negative control group [(1.00 ± 0.00, 1.00 ± 0.08) and (0.98 ± 0.05, 1.00 ± 0.09)](all P<0.01), There was no significant difference between the negative control group and the blank group.(4) Rac1 mRNA and protein expression in each group: The expression of Rac1 mRNA, protein and p-Rac1 protein in TLR4-RNAi group (0.75 ± 0.04, 0.76 ± 0.01, 0.74 ± 0.05) were lower than those in the blank group and the negative control group[(1.00 ± 0.00, 1.02 ± 0.05, 1.00 ± 0.06) and (0.96 ± 0.10, 1.07 ± 0.09, 1.00 ± 0.06)] (all P<0.01), There was no significant difference between the negative control group and the blank group.(5) Cytoskeleton of RAW264.7s: In the TLR4-RNAi group, the pseudopods of the TLR4-RNAi group were short and stiff, with the impaired capacity of engulfing FITC-E.coli. Blank group and negative control group RAW264.7 cells extend pseudopodia well, with strong capacity to engulfing FITC-E.coli.(6) The ability of each group of cells to engulf FITC-E.coli: The MFI and RAW264.7 cells% of TLR4-RNAi group[(7453 ± 564), (70.20±2.27)%] were lower than those in the blank group and the negative control group[(11733 ± 935), (79.97 ± 1.07)% and (10983 ± 954) , (79.60 ± 1.17)%](all P<0.01), MFI and phagocytosis% in the negative control group were not statistically different from the blank group.(7) Correlation analysis: In the basic state and after intervention with RNAi, TLR4 mRNA and protein were positively correlated with PI3K, Rac1 mRNA and protein, and p-Rac1 protein (all P<0.05), PI3K mRNA and protein expression was positively correlated with Rac1 mRNA, protein and p-Rac1 protein(all P<0.05), TLR4, PI3K, Rac1 mRAN and protein and p-Rac1 protein were positively correlated with MFI and RAW264.7%(all P<0.05).

Conclusion  TLR4-shRNA plasmid and lentivirus can successfully transfect RAW264.7 cells and silence the TLR4 gene. The TLR4-PI3K-Rac1 signaling pathway regulates macrophage phagocytosis. Silencing TLR4 gene can inhibit the PI3K-Rac1 signaling pathway, resulting in disordered arrangement of macrophages and impaired phagocytosis.