【摘要】：目的:構(gòu)建鼠源性Cyclophilin A和Caveolin-1基因重組質(zhì)粒,瞬時(shí)轉(zhuǎn)染RAW264.7細(xì)胞,建立穩(wěn)定表達(dá)細(xì)胞株,然后通過ox-LDL誘導(dǎo)細(xì)胞荷脂,觀察膽固醇轉(zhuǎn)運(yùn)復(fù)合物中關(guān)鍵蛋白Cyclophilin A對(duì)Caveolin-1介導(dǎo)RAW264.7細(xì)胞膽固醇含量的影響。 方法:從鼠源性RAW264.7細(xì)胞中提取總RNA,設(shè)計(jì)特異性引物,通過RT-PCR法擴(kuò)增Cyclophilin A和Caveolin-1基因編碼區(qū)序列(CDS)片段,將其插入到質(zhì)粒中,經(jīng)雙酶切和測(cè)序鑒定,確定重組質(zhì)粒是否構(gòu)建成功;將構(gòu)建成功的重組質(zhì)粒瞬時(shí)轉(zhuǎn)染RAW264.7細(xì)胞,然后采用100mg/L ox-LDL與RAW264.7細(xì)胞共同孵育48h,建立RAW264.7細(xì)胞荷脂模型,酶熒光法觀察細(xì)胞內(nèi)膽固醇酯比率變化,油紅O染色觀察細(xì)胞內(nèi)脂滴的形成情況,免疫共沉淀檢測(cè)轉(zhuǎn)染細(xì)胞Cyclophilin A和Caveolin-1的相互作用,免疫熒光法定位檢測(cè)轉(zhuǎn)染細(xì)胞Cyclophilin A和Caveolin-1蛋白的表達(dá)改變。 結(jié)果:以重組載體為模板擴(kuò)增得到的片段大小與已知Cyclophilin A和Caveolin-1基因大小相同,酶切也得到目的基因片段,測(cè)序結(jié)果也證實(shí)Cyclophilin A和Caveolin-1基因片段插入序列和方向正確;瞬時(shí)轉(zhuǎn)染的RAW264.7細(xì)胞,與正常組和空轉(zhuǎn)組比較,Cyclophilin A、Caveolin-1的基因和蛋白表達(dá)均增強(qiáng),有顯著性差異(P0.05),尤其以轉(zhuǎn)染48h明顯;將100mg/L ox-LDL與瞬時(shí)轉(zhuǎn)染Caveolin-1組和共轉(zhuǎn)染Cyclophilin A和Caveolin-1組的RAW264.7細(xì)胞共同孵育48h后,酶熒光定量檢測(cè)分析兩組細(xì)胞內(nèi)膽固醇酯占總膽固醇含量比率較未處理組和空轉(zhuǎn)組均減少,尤以后者減少更為明顯;油紅O染色顯示細(xì)胞內(nèi)脂滴較未處理組和空轉(zhuǎn)組減輕,尤以共轉(zhuǎn)染Cyclophilin A和Caveolin-1組減少更為明顯;免疫共沉淀結(jié)果也說明Caveolin-1轉(zhuǎn)染和Cyclophilin A和Caveolin-1共轉(zhuǎn)染的RAW264.7細(xì)胞,Cyclophilin A和Caveolin-1的相互作用增強(qiáng);間接免疫熒光定位檢測(cè)發(fā)現(xiàn)轉(zhuǎn)染Caveolin-1和共轉(zhuǎn)染Cyclophilin A和Caveolin-1的RAW264.7細(xì)胞中Cyclophilin A和Caveolin-1在胞膜、胞漿中的表達(dá)均增強(qiáng),以后者為甚。 結(jié)論: 1、將含Cyclophilin A和Caveolin-1質(zhì)粒共同導(dǎo)入RAW264.7細(xì)胞可增加Cyclophilin A和Caveolin-1的結(jié)合。 2、將含Caveolin-1質(zhì)粒導(dǎo)入RAW264.7細(xì)胞可抑制ox-LDL誘導(dǎo)的膽固醇蓄積。 3、將含Cyclophilin A和Caveolin-1質(zhì)粒共同導(dǎo)入RAW264.7細(xì)胞可增強(qiáng)抑制ox-LDL誘導(dǎo)的膽固醇蓄積作用。
[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.
【學(xué)位授予單位】：南華大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R329

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