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目的:观察低浓度一氧化碳暴露对营养相对匮乏情况下大鼠肝前体细胞系生长的影响并探索其作用机制。方法:分别在营养充足(10%胎牛血清,10%FBS)和营养相对匮乏(4%胎牛血清,4%FBS)条件下对大鼠肝脏前体细胞系WB-F344进行体外培养,观察细胞的生长情况;同时,在不同两组4%FBS培养基中分别加入无活性的一氧化碳释放分子iCORM-2和有活性的一氧化碳释放分子CORM-2,两者的浓度均为100μM,观察比较低浓度一氧化碳暴露是否有利于WB-F344的生长和增殖;利用吖啶橙染色观察不同组别培养细胞酸性自噬小体的形成情况,并进行Western-Blot,检测自噬相关蛋白LC3-Ⅰ和LC3-Ⅱ的转化情况,探讨其可能的作用分子机制。结果:在含有10%FBS的培养基中,WB-F344细胞生长良好,呈指数生长,并在第5天达到生长高峰;与其相比,4%FBS培养基对WB-F344的生长有明显抑制,细胞生长相对缓慢,并且相同时间点的细胞增殖数量明显不足。在进行浓度为100μM的一氧化碳分子暴露后,WB-F344细胞生长情况有明显改善,细胞增殖的峰值有显著增加。吖啶橙染色显示,4%FBS培养基条件下培养的WB-F344细胞,细胞内出现橘红色荧光,在暴露于低浓度的一氧化碳分子后,橘红色荧光的强度有明显增加。进一步的Western-Blot检测发现,营养相对匮乏的培养基(4%FBS)能使WB-F344细胞在生长过程产生LC3-Ⅰ向LC3-Ⅱ的转化,在暴露于低浓度的一氧化碳分子后,LC3-Ⅱ的量进一步增加,LC3-Ⅱ/LC3-Ⅰ的比值显著增大(P<0.05);进一步的研究显示在此过程中伴随着p-AKT和mTOR蛋白的表达降低,同时,HO-1蛋白的表达增加。结论:低浓度一氧化碳暴露能促进WB-F344细胞产生自噬,有利于大鼠肝前体细胞系的生长和增殖,同时这和AKT/mTOR信号途径的抑制及HO-1的表达增加有关。
Objective: To observe the effect of low concentration carbon monoxide exposure on the growth of rat hepatic progenitor cell line with relative lack of nutrition and to explore its mechanism. Methods: The rat hepatic progenitor cell line WB-F344 was cultured in vitro under the conditions of adequate nutrition (10% fetal bovine serum, 10% FBS) and nutrient deficient (4% fetal bovine serum, 4% FBS) At the same time, inactive carbon monoxide releasing molecule iCORM-2 and active carbon monoxide releasing molecule CORM-2 were respectively added to 4% FBS medium in two different groups, the concentration of both was 100 μM, and the observation was relatively low The concentration of carbon monoxide exposure was beneficial to the growth and proliferation of WB-F344. The formation of acidic autophagy in different groups of cultured cells was observed by acridine orange staining. Western-Blot was used to detect the expression of autophagy-related proteins LC3-I and LC3 -II transformation, to explore its possible role in molecular mechanisms. Results: WB-F344 cells grew well and exponentially in medium containing 10% FBS and peaked on day 5; 4% FBS medium significantly inhibited the growth of WB-F344 , The cell growth is relatively slow, and the number of cell proliferation at the same time point is obviously insufficient. After exposure to carbon monoxide at a concentration of 100 μM, the growth of WB-F344 cells was significantly improved and the peak of cell proliferation was significantly increased. Acridine orange staining showed that orange-red fluorescence appeared in WB-F344 cells cultured in 4% FBS medium, and the intensity of orange-red fluorescence increased significantly after exposure to low concentration of carbon monoxide molecules. Further Western blot analysis showed that LC3-I was converted to LC3-II by WB-F344 cells in a relatively nutrient-deficient medium (4% FBS). After exposure to low concentrations of carbon monoxide, LC3 (P <0.05). Further studies showed that the expression of p-AKT and mTOR protein was decreased in this process, meanwhile, the expression of HO-1 Increased protein expression. CONCLUSION: Exposure to low concentrations of carbon monoxide can promote the autophagy in WB-F344 cells, which is beneficial to the growth and proliferation of rat hepatic progenitor cell lines. At the same time, it is related to the inhibition of AKT / mTOR signal pathway and the increase of HO-1 expression.