Cyclophilin A通过与Caveolin-1协同作用抑制RAW264.7细胞胆固醇蓄积
发布时间：2018-07-30 08:00
【摘要】：目的:构建鼠源性Cyclophilin A和Caveolin-1基因重组质粒,瞬时转染RAW264.7细胞,建立稳定表达细胞株,然后通过ox-LDL诱导细胞荷脂,观察胆固醇转运复合物中关键蛋白Cyclophilin A对Caveolin-1介导RAW264.7细胞胆固醇含量的影响。 方法:从鼠源性RAW264.7细胞中提取总RNA,设计特异性引物,通过RT-PCR法扩增Cyclophilin A和Caveolin-1基因编码区序列(CDS)片段,将其插入到质粒中,经双酶切和测序鉴定,确定重组质粒是否构建成功;将构建成功的重组质粒瞬时转染RAW264.7细胞,然后采用100mg/L ox-LDL与RAW264.7细胞共同孵育48h,建立RAW264.7细胞荷脂模型,酶荧光法观察细胞内胆固醇酯比率变化,油红O染色观察细胞内脂滴的形成情况,免疫共沉淀检测转染细胞Cyclophilin A和Caveolin-1的相互作用,免疫荧光法定位检测转染细胞Cyclophilin A和Caveolin-1蛋白的表达改变。 结果:以重组载体为模板扩增得到的片段大小与已知Cyclophilin A和Caveolin-1基因大小相同,酶切也得到目的基因片段,测序结果也证实Cyclophilin A和Caveolin-1基因片段插入序列和方向正确;瞬时转染的RAW264.7细胞,与正常组和空转组比较,Cyclophilin A、Caveolin-1的基因和蛋白表达均增强,有显著性差异(P0.05),尤其以转染48h明显;将100mg/L ox-LDL与瞬时转染Caveolin-1组和共转染Cyclophilin A和Caveolin-1组的RAW264.7细胞共同孵育48h后,酶荧光定量检测分析两组细胞内胆固醇酯占总胆固醇含量比率较未处理组和空转组均减少,尤以后者减少更为明显;油红O染色显示细胞内脂滴较未处理组和空转组减轻,尤以共转染Cyclophilin A和Caveolin-1组减少更为明显;免疫共沉淀结果也说明Caveolin-1转染和Cyclophilin A和Caveolin-1共转染的RAW264.7细胞,Cyclophilin A和Caveolin-1的相互作用增强;间接免疫荧光定位检测发现转染Caveolin-1和共转染Cyclophilin A和Caveolin-1的RAW264.7细胞中Cyclophilin A和Caveolin-1在胞膜、胞浆中的表达均增强,以后者为甚。 结论: 1、将含Cyclophilin A和Caveolin-1质粒共同导入RAW264.7细胞可增加Cyclophilin A和Caveolin-1的结合。 2、将含Caveolin-1质粒导入RAW264.7细胞可抑制ox-LDL诱导的胆固醇蓄积。 3、将含Cyclophilin A和Caveolin-1质粒共同导入RAW264.7细胞可增强抑制ox-LDL诱导的胆固醇蓄积作用。
[Abstract]:Objective: to construct murine Cyclophilin A and Caveolin-1 gene recombinant plasmids, transfect them into RAW264.7 cells, establish stable expression cell lines, and then induce lipids by ox-LDL. To observe the effect of Cyclophilin A, a key protein in cholesterol transport complex, on cholesterol content in RAW264.7 cells mediated by Caveolin-1. Methods: total RNAs were extracted from murine RAW264.7 cells and specific primers were designed. The (CDS) fragments of Cyclophilin A and Caveolin-1 gene coding regions were amplified by RT-PCR method and inserted into plasmids. The recombinant plasmids were identified by double enzyme digestion and sequencing to determine whether the recombinant plasmids were successfully constructed. After transient transfection of the constructed recombinant plasmid into RAW264.7 cells, 100mg/L ox-LDL and RAW264.7 cells were incubated for 48h to establish the RAW264.7 cell model. The changes of the ratio of cholesterol ester in the cells were observed by enzyme fluorescence method. The formation of lipid droplets was observed by oil red O staining, the interaction between Cyclophilin A and Caveolin-1 was detected by immunoprecipitation, and the expression of Cyclophilin A and Caveolin-1 protein was detected by immunofluorescence. Results: the fragment size of the recombinant vector was the same as that of known Cyclophilin A and Caveolin-1 genes, and the target gene fragment was obtained by restriction endonuclease digestion. The sequencing results also confirmed that the insertion sequence and direction of Cyclophilin A and Caveolin-1 gene fragments were correct. Compared with the control group and the empty group, the gene and protein expression of RAW264.7 cells transfected with transient transfection was significantly higher than that of the control group and the empty group (P0.05), especially at 48 hours after transfection. After 100mg/L ox-LDL was incubated with RAW264.7 cells of transient transfection Caveolin-1 group and co-transfected Cyclophilin A and Caveolin-1 group for 48 h, the ratio of cholesterol ester to total cholesterol in the two groups was lower than that in untreated group and empty group. The oil red O staining showed that the lipid droplets in the cells were less than those in the untreated group and the empty group, especially in the co-transfected Cyclophilin A and Caveolin-1 groups. The results of co-immunoprecipitation also indicated that the interaction between Caveolin-1 and Cyclophilin A in RAW264.7 cells transfected with Caveolin-1 and cotransfected with Cyclophilin A and Caveolin-1 was enhanced, and that Cyclophilin A and Caveolin-1 were found in the cell membrane of Caveolin-1 transfected with Caveolin-1 and cotransfected with Cyclophilin A and Caveolin-1 by indirect immunofluorescence localization. The expression in cytoplasm was increased, especially in the latter. Conclusion: 1. The combination of RAW264.7 cells with Cyclophilin A and Caveolin-1 plasmids can increase the binding of Cyclophilin A and Caveolin-1. 2. Introducing Caveolin-1 plasmids into RAW264.7 cells can inhibit the accumulation of cholesterol induced by ox-LDL. 3. The co-introduction of Cyclophilin A and Caveolin-1 plasmids into RAW264.7 cells enhanced the inhibition of cholesterol accumulation induced by ox-LDL.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329
本文编号：2154352
[Abstract]:Objective: to construct murine Cyclophilin A and Caveolin-1 gene recombinant plasmids, transfect them into RAW264.7 cells, establish stable expression cell lines, and then induce lipids by ox-LDL. To observe the effect of Cyclophilin A, a key protein in cholesterol transport complex, on cholesterol content in RAW264.7 cells mediated by Caveolin-1. Methods: total RNAs were extracted from murine RAW264.7 cells and specific primers were designed. The (CDS) fragments of Cyclophilin A and Caveolin-1 gene coding regions were amplified by RT-PCR method and inserted into plasmids. The recombinant plasmids were identified by double enzyme digestion and sequencing to determine whether the recombinant plasmids were successfully constructed. After transient transfection of the constructed recombinant plasmid into RAW264.7 cells, 100mg/L ox-LDL and RAW264.7 cells were incubated for 48h to establish the RAW264.7 cell model. The changes of the ratio of cholesterol ester in the cells were observed by enzyme fluorescence method. The formation of lipid droplets was observed by oil red O staining, the interaction between Cyclophilin A and Caveolin-1 was detected by immunoprecipitation, and the expression of Cyclophilin A and Caveolin-1 protein was detected by immunofluorescence. Results: the fragment size of the recombinant vector was the same as that of known Cyclophilin A and Caveolin-1 genes, and the target gene fragment was obtained by restriction endonuclease digestion. The sequencing results also confirmed that the insertion sequence and direction of Cyclophilin A and Caveolin-1 gene fragments were correct. Compared with the control group and the empty group, the gene and protein expression of RAW264.7 cells transfected with transient transfection was significantly higher than that of the control group and the empty group (P0.05), especially at 48 hours after transfection. After 100mg/L ox-LDL was incubated with RAW264.7 cells of transient transfection Caveolin-1 group and co-transfected Cyclophilin A and Caveolin-1 group for 48 h, the ratio of cholesterol ester to total cholesterol in the two groups was lower than that in untreated group and empty group. The oil red O staining showed that the lipid droplets in the cells were less than those in the untreated group and the empty group, especially in the co-transfected Cyclophilin A and Caveolin-1 groups. The results of co-immunoprecipitation also indicated that the interaction between Caveolin-1 and Cyclophilin A in RAW264.7 cells transfected with Caveolin-1 and cotransfected with Cyclophilin A and Caveolin-1 was enhanced, and that Cyclophilin A and Caveolin-1 were found in the cell membrane of Caveolin-1 transfected with Caveolin-1 and cotransfected with Cyclophilin A and Caveolin-1 by indirect immunofluorescence localization. The expression in cytoplasm was increased, especially in the latter. Conclusion: 1. The combination of RAW264.7 cells with Cyclophilin A and Caveolin-1 plasmids can increase the binding of Cyclophilin A and Caveolin-1. 2. Introducing Caveolin-1 plasmids into RAW264.7 cells can inhibit the accumulation of cholesterol induced by ox-LDL. 3. The co-introduction of Cyclophilin A and Caveolin-1 plasmids into RAW264.7 cells enhanced the inhibition of cholesterol accumulation induced by ox-LDL.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329
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