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目的 探讨小分子RNA干扰雷帕霉素靶蛋白(mTOR)基因表达对百草枯致大鼠肺纤维化的影响.方法 体外培养人胚肾细胞HEK-293,构建mTOR小干扰RNA(mTOR-siRNA)表达质粒转染慢病毒,并以与mTOR基因无同源性的非特异性序列质粒作为对照.将72只健康雄性SD大鼠按随机数字表法分为生理盐水(NS)对照组、百草枯模型组、mTOR无关序列组、mTOR-siRNA组,每组18只.经腹腔注射20%百草枯溶液15 mg/kg制备百草枯中毒动物模型,NS对照组腹腔注射等量NS;mTOR无关序列组和mTOR-siRNA组大鼠分别经气道内注入滴度为1×109 TU/mL的慢病毒溶液50μL,NS对照组和百草枯模型组注入等量NS.各组分别于7、14、28 d处死6只大鼠取肺组织,光镜下观察病理学改变及纤维化情况;采用碱水解法检测肺组织羟脯氨酸(HYP)水平;采用反转录-聚合酶链反应(RT-PCR)和蛋白质免疫印迹试验(Western Blot)分别检测肺组织mTOR的mRNA和蛋白表达.结果 光镜下显示,NS对照组肺组织无明显病理学改变;百草枯模型组和mTOR无关序列组肺组织结构破坏,有大量炎性细胞浸润,并出现大量基质胶原及纤维组织增生,且随时间延长逐渐加重,符合百草枯致肺组织纤维化过程;而mTOR-siRNA组沉默mTOR基因后肺组织病理学及纤维化改变明显减轻.百草枯模型组和mTOR无关序列组肺组织HYP水平以及mTOR mRNA和mTOR蛋白表达量均呈时间依赖性持续升高,且明显高于NS对照组相应时间点;而mTOR无关序列组与百草枯模型组无明显差异.mTOR-siRNA组沉默mTOR基因后能抑制百草枯中毒导致肺组织中HYP的生成,以及mTOR mRNA和mTOR蛋白的表达量升高,其值接近NS对照组水平,在7 d或14 d时与百草枯模型组出现统计学差异,并持续至28 d〔7 d:HYP(μg/mg)为1.13±0.06比1.25±0.07;14 d:HYP(μg/mg)为1.19±0.09比1.29±0.12,mTOR mRNA(2-ΔΔCt)为0.99±0.11比1.94±0.12,mTOR蛋白(灰度值)为0.39±0.08比0.75±0.09;28 d:HYP(μg/mg)为1.28±0.06比1.40±0.05,mTOR mRNA(2-ΔΔCt)为1.15±0.13比2.85±0.15,mTOR蛋白(灰度值)为0.45±0.10比0.86±0.12,均P<0.05〕.结论 慢病毒介导的mTOR-siRNA可有效抑制百草枯中毒大鼠肺组织中mTOR表达,减轻百草枯致肺组织损伤和纤维化程度.“,”Objective To investigate the effects of small RNA interference targeting mammalian target of rapamycin (mTOR) expression on paraquat-induced pulmonary fibrosis in rats.Methods Human embryonic kidney cells HEK-293 were culturedin vitro. The mTOR small interfering RNA (mTOR-siRNA) expression plasmid transfection lentivirus was constructed, and non-specific sequence plasmid with no homology to mTOR gene was set as the control. Seventy-two healthy male Sprague-Dawley (SD) rats were randomly divided into normal saline (NS) control group, paraquatmodel group, mTOR unrelated sequence group, and mTOR-siRNA group, with 18 rats in each group. Paraquat poisoning animal model was reproduced by intraperitoneally injecting 20% paraquat solution 15 mg/kg, while the NS control group was intraperitoneally injected the same volumes of NS. Rats in the mTOR unrelated sequence group and mTOR-siRNA group were injected 1×109 TU/mL lentivirus solution 50μL into the airway, respectively, while in the NS control group and paraquat model group were injected the same volumes of NS. At 7, 14 and 28 days after treatment, 6 rats in each group were sacrificed respectively for lung tissue, the pathological changes and fibrosis of lung tissues were observed under light microscope. The levels of hydroxyproline (HYP) in lung tissues were determined by alkaline hydrolysis. The mRNA and protein expressions of mTOR in lung tissues were determined by reverse transcription-polymerase chain reaction (RT-PCR) and Western Blot.Results Under light microscope, there was no obvious pathological changes in the lung tissues in the NS control group, while in the paraquat model group and mTOR unrelated sequence group, lung tissue in rats were damaged, there were a lot of inflammatory cell infiltration, a large number of matrix collagen and fibrous tissues hyperplasia, and gradually increased with time, and it was consistent with paraquat-induced lung tissue fibrosis process. The pathological and fibrotic changes in lung tissue of mTOR-siRNA group were obviously reduced after silencing mTOR gene. The levels of HYP and the expression levels of mTOR mRNA and mTOR protein of lung tissues in the paraquat model group and mTOR unrelated sequence group were continuously increased in time-dependent manner, and they were significantly higher than those in the NS control group at all of the time points, but no significant difference was found between mTOR unrelated sequence group and paraquat model group. In mTOR-siRNA group, silencing mTOR gene could inhibit paraquat poisoning induced HYP increase in lung tissue, and the expressions increase in mTOR mRNAand mTOR protein, the values were close to the levels of NS control group, and the significant difference was found as compared with paraquat model group at 7 days or 14 days, and the change was maintained to 28 days [7 days: HYP (μg/mg) was 1.13±0.06 vs. 1.25±0.07; 14 days: HYP (μg/mg) was 1.19±0.09 vs. 1.29±0.12, mTOR mRNA (2-ΔΔCt) was 0.99±0.11 vs. 1.94±0.12, mTOR protein (gray value) was 0.39±0.08 vs. 0.75±0.09; 28 days: HYP (μg/mg) was 1.28±0.06 vs. 1.40±0.05, mTOR mRNA (2-ΔΔCt) was 1.15±0.13 vs. 2.85±0.15, mTOR protein (gray value) was 0.45±0.10 vs. 0.86±0.12, allP < 0.05].Conclusion Lentivirus-mediated mTOR-siRNA could effectively inhibit the expressions of mTOR in lung tissues of paraquat-poisoned rats, and reduce the damage and fibrosis of lung tissues caused by paraquat.