低浓度CO暴露促进大鼠肝前体细胞增殖的体外研究

Low-concentration CO Exposure Promote the Proliferation of Rat Hepatic Progenitor Cell in Vitro

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 explore the role and molecular mechanism of hepatic progenitor cell （HPC） exposed to low-concentra- tion exogenous CO molecular against relative nutrition deficiency. Methods： HPCs （WB-F344） were cultured in vitro in 10%FBS and 4% FBS medium respectively. Meanwhile, inactive CORM-2 （iCORM-2） and CORM-2 were administered respectively to different groups of WB-F344 cultured in 4%FBS medium. 100 μM was chosen as the same CO exposure concentration both in iCORM-2 and CORM-2 groups. Cell viability and proliferation were observed in all groups. Acridine orange （AO） staining was used to detect the formation of Acidic autophagosomes in WB-F344 cells. The transformation between LC3-I and LC3-II was detected with western-blot. Results： WB-F344 cells exhibited an exponential growth in 10%FBS medium and achieved growth peak in the fifth day. On the contrary, WB-F344 cells showed an inhibitory viability and very slow growth rate in 4% FBS medium. The proliferative cell number decreased at the same time point compared to the control group. While exposed to 100 μM CORM-2, WB-F344 cells＇ viability and growth situation improved significantly and had an increased growth peak. AO staining displayed obvious orange fluorescent in WB-F344 cells cultured in 4%FBS medium and the intensity of orange fluorescent enhanced after CO molecular exposure. Western-blot assay demonstrated that rel- ative nutrition deficiency （4%FBS） promoted LC3-I to transform into LC3-II in the growth of WB-F344 cells. CO molecular exposure in- creased the production of LC3-II and the ratio between LC3-II and LC3-I （P〈0.05）. Further western-blot detection revealed that the expression of p-AKT and roTOR declined simultaneously in the CO exposure process in concurrence with the increased expression of HO-1. Conclusion： Low-concentration CO exposure could benefit the autophagy formation in W-B-F344 cell against severe environment and promote its viability and proliferation. The molecular mechanism referred to AKT/mTOR signal pathway inhibition and HO-1 increase.