本文關(guān)鍵詞： 骨髓間充質(zhì)干細(xì)胞 二氧化硅 急性肺損傷 巨噬細(xì)胞 白細(xì)胞介素-1β　出處：《山西醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:研究BMSC移植對(duì)矽塵所致急性肺損傷的療效及作用機(jī)制。方法:采用隨機(jī)分配法將60只小鼠分為對(duì)照組、SiO_2組和BMSC移植組3個(gè)組。氣管暴露法建立實(shí)驗(yàn)動(dòng)物模型,對(duì)照組經(jīng)氣管注射0.90%氯化鈉20μL;SiO_2組和BMSC移植組經(jīng)氣管注射20μL濃度為250 g/L的SiO_2混懸液,即5 mg/只,6 h后,上述兩組小鼠分別經(jīng)尾靜脈給予100μL的0.90%氯化鈉或細(xì)胞密度為5×109個(gè)/L的BMSC。常規(guī)喂養(yǎng)3天后處死小鼠,觀察3組小鼠肺組織損傷情況,Wright-Giemsa染色BALF中細(xì)胞并計(jì)數(shù)。ELISA檢測(cè)小鼠血清、BALF中細(xì)胞因子表達(dá)水平,qPCR檢測(cè)小鼠肺組織中細(xì)胞因子mRNA的表達(dá)情況;FCM檢測(cè)小鼠BALF中巨噬細(xì)胞百分比,免疫組化檢測(cè)肺組織中巨噬細(xì)胞數(shù)目,WB檢測(cè)肺組織中IL-1β、Caspase-1蛋白表達(dá)水平。Transwell共培養(yǎng)BMSC和巨噬細(xì)胞,ELISA檢測(cè)巨噬細(xì)胞培養(yǎng)上清中IL-1β表達(dá)水平,WB檢測(cè)巨噬細(xì)胞中IL-1β、Caspase-1蛋白表達(dá)水平。結(jié)果:肉眼觀察可見對(duì)照組和BMSC移植組小鼠肺臟表面光滑,SiO_2組小鼠肺臟出現(xiàn)明顯的腫脹、充血。病理切片顯示對(duì)照組和BMSC移植組小鼠肺組織結(jié)構(gòu)無破壞,肺泡腔大小均勻、完整,肺泡結(jié)構(gòu)完整;SiO_2組小鼠肺組織有大量炎性細(xì)胞浸潤(rùn)、聚集,肺泡腔有炎性滲出物,肺泡塌陷。SiO_2組小鼠肺系數(shù)高于對(duì)照組(P0.05),BMSC移植組較SiO_2組降低(P0.05)。SiO_2組小鼠BALF中總細(xì)胞數(shù)目和巨噬細(xì)胞數(shù)目分別高于對(duì)照組(P0.05),BMSC移植組小鼠上述兩個(gè)指標(biāo)分別低于SiO_2組(P0.05)。SiO_2組小鼠血清、BALF中IL-1β、IL-6水平分別高于對(duì)照組,BMSC移植組上述兩個(gè)指標(biāo)較SiO_2組降低(P0.05)。Si O2組小鼠肺組織中IL-1β、IL-6 m RNA相對(duì)表達(dá)水平分別高于對(duì)照組(P0.05),BMSC移植組上述兩個(gè)指標(biāo)較SiO_2組降低(P0.05)。SiO_2組小鼠BALF中巨噬細(xì)胞百分比較對(duì)照組升高,BMSC移植組巨噬細(xì)胞百分比較SiO_2組降低。免疫組化結(jié)果顯示SiO_2組小鼠肺組織中有較多巨噬細(xì)胞聚集,BMSC移植組巨噬細(xì)胞較少。SiO_2組小鼠BALF中CCL-3表達(dá)水平高于對(duì)照組(P0.05),BMSC移植組較SiO_2組降低(P0.05)。SiO_2組小鼠肺組織中IL-1β、Caspase-1表達(dá)較對(duì)照組升高,BMSC移植組上訴兩個(gè)指標(biāo)較SiO_2組降低;而Pro-IL-1β、Pro-Caspase-1表達(dá)水平在3組間比較無差異。體外實(shí)驗(yàn)發(fā)現(xiàn)不同濃度SiO_2作用巨噬細(xì)胞后IL-1β表達(dá)水平升高,并呈濃度依賴;分別與BMSC共培養(yǎng)后IL-1β表達(dá)水平降低。巨噬細(xì)胞中IL-1β、Caspase-1表達(dá)情況與體內(nèi)實(shí)驗(yàn)結(jié)果一致。結(jié)論:1、BMSC可以減輕矽塵所致的急性肺損傷。2、其作用機(jī)制可能是BMSC通過作用于巨噬細(xì)胞NLRP3炎性小體通路,減少IL-1β的產(chǎn)生,從而減輕炎癥反應(yīng)。
[Abstract]:Objective: to study the effect and mechanism of BMSC transplantation on acute lung injury induced by silica dust. Methods: 60 mice were randomly divided into control group. The experimental animal model was established by trachea exposure in SiO_2 group and BMSC transplantation group, and the control group was injected with 20 渭 L sodium chloride 0.90% 渭 L through trachea. In SiO_2 group and BMSC transplantation group, 20 渭 L SiO_2 suspension (250g / L) was injected through trachea for 6 h. The mice in the two groups were given 100 渭 L sodium chloride (0.90% 渭 L) via tail vein or BMSCs with cell density of 5 脳 10 9 / L. The mice were sacrificed after 3 days of routine feeding. The lung tissue injury in the three groups was observed and the expression of cytokines in the serum of BALF was detected by Wright-Giemsa staining. The expression of cytokine mRNA in lung tissue of mice was detected by qPCR. The percentage of macrophages in BALF and the number of macrophages in lung tissue were detected by FCM and WB respectively. Expression level of Caspase-1 protein. Transwell co-cultured BMSC and macrophages were used to detect the expression of IL-1 尾 in the supernatant of macrophage culture by enzyme-linked immunosorbent assay (Elisa). Results: the lung surface of mice in control group and BMSC transplantation group was smooth. The lung of SiO_2 group showed obvious swelling and hyperemia. The pathological sections showed that the lung tissue structure of the control group and BMSC transplantation group was not damaged, the alveolar cavity was uniform and intact, and the alveolar structure was intact. In SiO_2 group, there were a lot of inflammatory cells in lung tissue, aggregation, inflammatory exudate in alveolar cavity, lung coefficient in group 2 was higher than that in control group (P0.05). Compared with SiO_2 group, the total number of BALF cells and the number of macrophages in BALF of BMSC transplantation group were significantly lower than those of control group (P 0.05). The above two indexes in BMSC transplantation group were lower than those in SiO_2 group (P 0.05). Sio group 2 mice were higher than control group in IL-1 尾 -IL-6 level in serum. Compared with SiO_2 group, the above two indexes in BMSC group decreased the IL-1 尾 in lung tissue of P0.05U 路Sio _ 2 group. The relative expression level of IL-6 m RNA was higher than that of control group (P 0.05). Compared with SiO_2 group, the percentage of macrophages in BALF of BMSC transplantation group was lower than that of SiO_2 group, and the percentage of macrophage in BALF of BALF group was higher than that of control group. The percentage of macrophages in the BMSC group was lower than that in the SiO_2 group. The immunohistochemical results showed that there were more macrophages in the lung tissue of the SiO_2 group. The expression of CCL-3 in BALF of mice in BMSC transplantation group was higher than that in control group (P 0.05). Compared with SiO_2 group, the expression of IL-1 尾 -caspase-1 in lung tissue of BMSC transplantation group was lower than that of SiO_2 group. The two indexes of appeal in BMSC transplantation group were lower than those in SiO_2 group. And Pro-IL-1 尾. There was no difference in the expression of Pro-Caspase-1 between the three groups. In vitro, it was found that the expression of IL-1 尾 in macrophages with different concentrations of SiO_2 increased in a dose-dependent manner. The expression level of IL-1 尾 was decreased after co-culture with BMSC, and the expression of IL-1 尾 -caspase-1 in macrophages was consistent with the experimental results in vivo. Conclusion: 1. BMSC can attenuate acute lung injury induced by silica. The mechanism may be that BMSC reduces the production of IL-1 尾 by acting on the inflammatory corpuscle pathway of NLRP3 in macrophages. This reduces the inflammatory response.
【學(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R135.2

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