阻遏矽肺纤维化的实验研究

The experimental study of suppressing silicosis fibrosis

目的比较骨髓间充质样干细胞（BM＋MSCs）与pcDNA3．1肝细胞生长因子（HGF）转染的BM—MSCs对SiO，所致大鼠肺泡炎和早期肺纤维化的影响并探讨其机制。方法收集培养雄性Wistar幼鼠的原代BM-MSCs，并进行4，6-联脒-2-苯基吲哚（DAPI）标记。将50只雄性Wistar大鼠气管滴注40mg／mlSiO2，混悬液1ml造模后，随机分为模型组、BM—MSCs组和pcDNA3．1-HGF＋BM—MSCs组。造模次日，模型组（10只）：尾静脉注入PBS1ml；BM—MSCs组（20只）：尾静脉注入10^6／mlBM-MSCs1ml；pcD-NA3．1-HGF＋BM-MSCs组（20只）：尾静脉注入10^6个／mlpcDNA3．1-HGF转染的BM-MSCslm1。14和28d后，分别处死大鼠，取肺组织冰冻切片，荧光显微镜下检测体内DAPI标记细胞；HE和Masson染色观察肺炮炎和纤维化情况；免疫印迹（Westernblot）法测各组大鼠肺组织的I-IGF表达量；ELISA法检测肺组织中肿瘤坏死因子（TNF）．or．的含量，样本碱水解法检测肺组织中羟脯氨酸（HYP）的含量。结果14、28d时，pcDNA3．1-HGF＋BM-MSCs组肺组织肺泡炎评分（14d：1．16±0.3，28d：0.8＋0．20）均低于同时期BM-SCs组（14d：1．68＋0．17，28d：1．58±031）和模型组（14d：2．36＋_0．17，28d：2．80_＋0．14），BM—MSCs组肺泡炎评分均低于同时期模型组，差异均有统计学意义（P〈0．05）。14、28d时pcDNA3．1-HGF＋BM～MSCs组肺纤维化评分（14d：0．10＋_0．11，28d：1．16＋_0．13）均明显低于同时期BM—MSCs（14d：0．54＋0．15，28d：1．36±0．13）和模型组（14d：0．64＋0．09，28d：1．84＋0．17），差异均有统计学意义（P〈O．05）。14、28d时pcDNA3．1一HGF＋BM—MSCs组肺组织匀浆中TNF—d含量[14d：（280．48＋23．11）pg／mg，28d：（249．78_＋22．33）pg／mg]均明显低于BM—MSCs组[14d：（342．58_＋35．34）pg／mg，28d：（319．5l±17．84）pg／mg]~模型组『14d：（442．29±36．76）pg／mg，28d：（348．53＋_33．95）pghngl，BM—MSCs组肺组织匀浆中TNF—d含量低于模型组，差异均有统计学意义（P〈O．05）。14、28d时peDNA3．1-HGF＋BM—MSCs组肺组织匀浆中HYP表达量f14d：（O．46＋0．04）~g／mg，28d：（0．65_＋0．05）Ixg／mg】均明显低于同时期BM—MSCs组[14d：（0．63＋0．04）~g／mg，28d：（1．04_＋0．07）jxg／mg】和模型组【14d：（0．72_＋0．06）}xg／mg，28d：（1．39＋0．06）jxg／mgl，差异均有统计学意义（P〈0．05）；28d时，BM—MSCs组肺组织匀浆中HYP表达量低于模型组，差异有统计学意义（P〈O．05）。结论应用pcDNA3．1-HGF转染后的BM—MSCs比单独应用BM—MSCs能更有效地对抗SiO：诱导的大鼠肺泡炎和早期肺纤维化，作用机制可能与其减轻肺组织的炎症有关。

Objective To compare the difference of effects on SiO2-induced alveolitis and early fibrosis between bone marrow-derived mesenchymal-like stem cells （BM-MSCs） and BM-MSCs transfected by peD- NA3. I-HGF and to explore the mechanism of this effects. Methods The Primary BM-MSCs from Wistar ma｜e young rats were cultured and labeled by 4, 6-diamidino-2-phenylindole （DAPI）. Fifty Wistar rats were randomly divided into 3 groups：model group （10 rats）,which was administered with SiO2 by the traehe,the next day,in- jected PBS via the tail vein; BM-MSCs group （20 rats） ,which was administered with SiO2 by tbe trache,the nextday,injected with 1 ml suspension of BM-MSCs via the tail vein; pcDNA3.I-HGF plus BM-MSC group （20 rats）, which was administered with SiO2 by the trache, the next day,injected with 1 ml suspension of BM-MSCs transfected by pcDNA3.1-HGF via the tail vein. On the 14th and 28th days after treatment, half of the animals were sacrificed, respectively, and the lungs were harvested for frozen section to observe the cell marked by DAP1.HE staining under a fluorescent microscope, and to observe the pulmonary alveolitis and fibrosis by HE and Masson staining under a light microscope. Western blot assay was used to detect the expression of HGF in rat lungs. The expression levels of tumor necrosis factor-ct （TNF-c0 in pulmonary tissues were analyzed quanti- tatively by ELISA. The contents of HYP in pulmonary tissues were analyzed quantitatively by sample hydrolysis method. Results On the 14th and 28th days after treatment, the scores of pulmonary alveolitis and early fibro- sis in pcDNA3.1-HGF plus BM-MSCs group were 2.36＋_0.17, 2.8＋_0.14 and 0.1 ＋_0.11, 1.16＋_0.13, which were significantly lower than those （ 1.68＋_0.17, 1.58＋_0.31 and 0.54＋_0.15, 1.36＋_0.13） in BM-MSCs group, also which were significantly lower those （2.36＋0.17, 2.80＋0.14 and 0.64＋0.09, 1.84＋0.17） in model group （P〈0.05）; On the 14d＇ and 28d＇ days after treatment, the TNF-ot contents of pulmonary tissues in pcDNA3.1-HGF plus BM- MSCs group were 280.4＋-23.11 and 249.78＋_22.33 pg/mg, which were significantly lower than those （341.58＋_ 35.34, 442.29-＋36.76 pg/mg and 319.51＋17.84, 348.53＋33.95 pg/mg） in BM-MSCs and model groups （P〈 0.05）; On the 14~ and 28~ days after treatment, the HYP contents of puhnonary tissues in pcDNA3.1-HGF piLLs BM-MSCs group were 0.46＋_0.04 and 0.65＋_0.05 ~g/mg, which were significantly lower than those （0.63_＋0.04, 1.04＋_O.07tzg/mg and 0.72＋_0.60, 1.39＋_0.601xg/mg） in BM-MSCs and model groups （P〈0.05）. Conclusion The effects of BM-MSCs transfected by pcDNA3.1-HGF on suppressing pulmonary alveolitis and early fibrosis induced by SiO2 were better than those of BM-MSCs. The mechanism may be associated with the reduced pul-