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目的观察铀矿尘对小鼠肺成纤维细胞合成胶原蛋白的影响并探讨其机制。方法①取小鼠巨噬细胞RAW264.7细胞分为溶剂对照组和染尘组,分别以终浓度为0、120 mg/L的铀矿尘混悬液处理0、2、4、8、16、24和32 h后,收集细胞上清液,采用酶联免疫吸附实验(ELISA)法检测转化生长因子-β1(TGF-β1)水平,选择最佳染尘时间,分离制备该时间点的2组细胞上清液作为条件培养液(CM)进行后续实验。②取小鼠肺成纤维细胞L929细胞分为对照组和铀矿尘组,分别予溶剂对照组和染尘组的CM处理0、6、12、18、24、30、36 h后,采用免疫组化法检测细胞内α-平滑肌肌动蛋白(α-SMA)的表达,ELISA法检测细胞上清液中Ⅰ型胶原蛋白(COLⅠ)、III型胶原蛋白(COLⅢ)和羟脯氨酸(HYP)的水平。结果①染尘组RAW264.7细胞上清液中TGF-β1水平从染尘后4 h时间点起开始较溶剂对照组出现有统计学意义的升高(P<0.05);在染尘后4~24 h呈随染尘时间的增加而升高的时间-效应关系(P<0.05),至染尘后24 h达到峰值。取染尘后24 h时间点的2组细胞上清液作为CM进行后续实验。②相较同时间点对照组,铀矿尘组在经相应CM处理后18~36 h,L929细胞中α-SMA表达水平均上调(P<0.05),在CM处理后12~36 h,L929细胞上清液中COLⅠ、COLⅢ和HYP的水平均升高(P<0.05),且α-SMA表达水平和COLⅢ、HYP的水平均不同程度地呈现随CM作用时间的增加而升高的时间-效应关系(P<0.05)。结论铀矿尘刺激巨噬细胞后可导致细胞分泌TGF-β1增加,继而作用于成纤维细胞,上调α-SMA表达,增加COLⅠ、COLⅢ和HPY合成,可能是铀矿尘致肺纤维化的机制之一。
Objective To observe the effects of uranium dust on the synthesis of collagen in mouse lung fibroblasts and its mechanism. Methods ① Mouse macrophages RAW264.7 cells were divided into solvent control group and dyed group, respectively, with 0,120 mg / L uranium dust suspension at the final concentration of 0,2,4,8,16 , 24 and 32 h later, the supernatant was collected and the level of transforming growth factor-β1 (TGF-β1) was detected by enzyme-linked immunosorbent assay (ELISA) Group cell supernatant as conditioned medium (CM) for follow-up experiments. (2) The L929 cells from mouse lung fibroblasts were divided into control group and uranium mine dust group, and then treated with CM and CMV for 0, 6, 12, 18, 24, 30 and 36 h respectively, The expression of α-smooth muscle actin (α-SMA) in the cells was detected by histochemistry and the expressions of COL Ⅰ, COL Ⅲ and HYP )s level. Results ① The level of TGF-β1 in the RAW264.7 cell supernatant in the dust-exposed group was significantly higher than that in the solvent-treated group at 4 hours after dusting (P <0.05) ~ 24 h showed the time-effect relationship (P <0.05) increased with the increase of dusting time, reaching its peak at 24 h after dusting. Two groups of cell supernatants at 24 h after dying were used as CM for subsequent experiments. ② Compared with the control group at the same time point, the expression of α-SMA in L929 cells was up-regulated (P <0.05) at 18-36 h after treatment with corresponding CM in the uranium mine dust group. At 12-36 h after CM treatment, L929 The levels of COLⅠ, COL Ⅲ and HYP in supernatants of the cells increased (P <0.05), and the levels of α-SMA and COL Ⅲ, HYP in the supernatant of the cells increased to different degrees with the increase of CM action time- Effect (P <0.05). CONCLUSIONS: Uranium dust-stimulated macrophages can induce the secretion of TGF-β1, then act on fibroblasts, increase the expression of α-SMA and increase the COLⅠ, COLⅢ and HPY synthesis, which may be the mechanism of dust-induced pulmonary fibrosis in uranium mines one.