酸环境对大鼠髓核细胞自噬及功能的影响

Acid environment effected on autophagy and function of nucleus pulposus cells in rat

中文关键词:  髓核细胞  酸环境  卵巢癌G蛋白偶联受体1  自噬  细胞功能  大鼠

英文关键词:Nucleus pulposus cells  Acidic environment  Ovarian cancer G-protein-coupled receptor 1  Auophagy  Cellular function  Rat

中文摘要:

  【摘要】 目的：观察酸环境下髓核细胞（nucleus pulposus cells，NPCs）中卵巢癌G蛋白偶联受体1（ovarian cancer G-protein-coupled receptor 1，OGR1）和自噬相关蛋白LC3的表达水平，探讨自噬激活机制，初步分析其对细胞功能的影响。方法：取SD大鼠正常髓核组织，分离培养NPCs，甲苯胺蓝、阿利新染色和Ⅱ型胶原免疫荧光检测鉴定NPCs。扩增培养NPCs，取第2代NPCs，在pH值为6.2、6.4、6.8、7.0、7.2和7.4的培养基中培养24h，免疫荧光观察NPCs中OGR1的表达情况，Western blotting检查细胞中OGR1和LC3-Ⅱ蛋白表达水平；在pH值为6.4的培养基中培养0、3h、6h、12h、24h、48h后，采用Western blotting检测NPCs中LC3-Ⅰ和LC3-Ⅱ蛋白表达水平。构建3个不同靶点的OGR1干扰序列，检测最合适滴度慢病毒，用最适滴度慢病毒转染NPCs，使用Western blotting和Real-time PCR检测不同干扰序列的沉默效果，选取最适干扰系列构建OGR1-shRNA慢病毒载体。取第2代NPCs，分为4组：正常组（DMEM培养基）、pH值6.4培养基组（空白组）、pH值6.4培养基空载体组（空载体组）、pH值6.4培养基OGR1-shRNA慢病毒转染组（转染组），培养24h后用Western blotting检测LC3-Ⅱ和p62蛋白表达水平； Alcian染色观察NPCs中蛋白多糖的表达情况。结果：（1）分离培养的细胞高表达蛋白多糖和Ⅱ型胶原，符合NPCs表型。（2）OGR1蛋白表达水平与培养基pH值相关，pH值<7.0时，OGR1和LC3-Ⅱ蛋白表达水平显著性升高（P<0.05），pH值6.4时表达量最高。（3）在pH值6.4的培养基中培养时，细胞中LC3-Ⅰ和LC3-Ⅱ的表达量与培养时间有关，24h时达高峰，48h仍较高。（4）3个干扰序列对OGR1均有沉默作用，与对照组比较有显著性差异（P<0.05），OGR1-shRNA1沉默效果最佳。（5）转染组细胞在pH值6.4的培养基中培养24h后，LC3-Ⅱ表达水平显著性低于空载体组（P<0.05）；p62表达水平显著性高于空载体组（P<0.05）；蛋白多糖的表达低于空载体组（P<0.05）。结论：酸环境可促进大鼠NPCs中OGR1蛋白表达和自噬水平升高，OGR1介导了细胞自噬的激活，并影响NPCs的生物学功能。

英文摘要:

  【Abstract】 Objectives: To explore the autophagy activation and cell function of nucleus pulposus cells(NPCs) effected by acid environment, through observing the expression levels of ovarian cancer G-protein-coupled receptor 1(OGR1) and autophagy-related protein LC3 in rat. Methods: NPCs in normal nucleus pulposus tissues of SD rats were isolated and cultured, which were then identified by toluidine blue, alixin staining and Ⅱ collagen immunofluorescence. The cells were expanded, and the second generation NPCs were cultured in medium with pH 6.2, 6.4, 6.8, 7.0, 7.2 and 7.4 for 24h. Immunofluorescence was used to observe the expression of OGR1 in NPCs. Western blotting was used to examine OGR1. After incubation at pH 6.4 for 0h, 3h, 6h, 12h, 24h, 48h, the expression levels of LC3-Ⅰ and LC3-Ⅱ in NPCs were detected by Western blotting. The OGR1 interference sequences of three different targets were constructed, and the most suitable titer lentivirus was detected. NPCs cells were transfected with the optimal titer lentivirus, and the silencing effects of different interference sequences were detected by Western blotting and Real-time PCR. The interference series were used to construct the OGR1-shRNA lentiviral vector. The second generation NPCs were divided into 4 groups: normal group(DMEM medium), pH 6.4 medium group(blank group), pH 6.4 medium empty vector group(empty vector group), pH 6.4 medium OGR1 -shRNA lentiviral transfection group(transfection group), and the expressions of LC3-Ⅱ and p62 protein were detected by Western blotting after 24 hours of culture. Alcian staining was used to observe the expression of proteoglycan in NPCs. Results: (1)The isolated cultured cells highly expressed proteoglycan and type Ⅱ collagen, which were consistent with the NPCs phenotype. (2)The expression level of OGR1 protein was correlated with the pH value of the medium. When the pH value lower than 7.0, the expression levels of OGR1 and LC3-Ⅱ protein significantly increased(P<0.05), and the expression level was the highest at pH 6.4. (3)When cultured in a medium with a pH of 6.4, the expression levels of LC3-Ⅰ and LC3-Ⅱ in the cells were related to the culture time, which peaked at 24h and remained high at 48h. (4)Three interfering sequences had silent effect on OGR1, which was significantly different from the control group(P<0.05), and OGR1-shRNA1 had the best silencing effect. (5)After the cells were cultured for 24 hours in the medium with pH 6.4 in the transfection group, the expression level of LC3-II was significantly lower than that in the empty group(P<0.05); the p62 expression in the transfection group was significantly higher than that in the empty vector group(P<0.05). The expression of proteoglycan in the transfection group was lower than that in the empty group(P<0.05). Conclusions: The acid environment can promote OGR1 protein expression and autophagy of NPCs in rats. OGR1 mediates the activation of autophagy and affects the function of NP cells.