【作者】 陈敏；

【导师】 王家富； 李祥安；

【作者基本信息】 泰山医学院 ， 病理学与病理生理学， 2008， 硕士

【摘要】 目的:观察研究SR-BI上C323位点突变后,对介导细胞选择性摄取胆固醇酯及游离胆固醇释放作用的影响,以进一步明确SR-BI上C323位点在SR-BI功能发挥中的作用。方法:1.免疫印记(Western Blot)检测细胞株SR-BI表达情况。2.1.通过Caveolae Prep.,从蛋白水平检测变异细胞株SR-BI的分布情况。2.2.用免疫荧光标记细胞株SR-BI,通过共聚焦显微镜观察变异细胞株SR-BI分布情况。2.3.通过生物素技术,标记变异细胞株细胞表面SR-BI,明确变异细胞株细胞表面SR-BI分布状况。3.1.改进iodine monochloride method法[1],用125I标记HDL,改进Gwynne and Mahaffee文献中所述方法[3],用3H标记125I-HDL。3.2.使用同位素示踪技术,检测SR-BI介导细胞选择性摄取胆固醇酯情况,使用γ-计数器读取125I放射活性值,使用β-计数器读取3H的放射活性值。3.3. BCA法测定同位素样品中的细胞蛋白浓度。4.参考Nancy Webb文献中所述方法[3] ,使用3H标记胆固醇,同位素示踪细胞内游离胆固醇释放情况,使用β-计数器读取3H的放射活性值。5.酶切法切除SR-BI上糖基,应用免疫印记法检测酶切后SR-BI分子量的大小。结果:1.免疫印记结果:CHO-SR-BIC323G细胞株与阳性组细胞CHO-SR-BI一样,表达SR-BI,分子量为75KD;变异细胞株还表达分子量为100KD的SR-BI,其表达量少于分子量为75KD的SR-BI。2.1.Caveolae Prep.结果:在细胞内,CHO-SR-BIC323G细胞株中的SR-BI与caveolin-1蛋白分布位置相同:均在代表细胞膜成分的PNS和代表Caveolae成分的CM里表达。2.2.免疫荧光标记后的细胞在共聚焦显微镜下显示:CHO-SR-BIC323G细胞株的SR-BI与CHO-SR-BI细胞株SR-BI分布位置相同,均与caveolin-1蛋白分布位置一样,位于细胞膜上的Caveolae内。2.3.生物素实验:CHO-SR-BIC323G细胞株与阳性对照CHO-SR-BI细胞株一样,在细胞表面有SR-BI的表达。3.1.表示细胞中SR-BI与HDL结合量的125I放射活性值: CHO-SR-BIC323G细胞株与CHO-SR-BI细胞株两两比较,CHO-SR-BI细胞株的125I放射活性值明显高于CHO-SR-BIC323G细胞株,有统计学意义,P<0.05;与CHO-vector细胞株两两比较,CHO-SR-BIC323G细胞株125I放射活性值稍高于CHO-vector细胞株,有统计学意义,P<0.05;3.2.表示SR-BI介导细胞选择性摄取胆固醇酯的3H放射活性值: CHO-SR-BIC323G细胞株与CHO-SR-BI细胞株两两比较,CHO-SR-BI细胞株明显高于CHO-SR-BIC323G细胞株,有统计学意义,P<0.05;与CHO-vector两两比较,CHO-SR-BIC323G与CHO-vector相同,无统计学意义,P=0.184。4.SR-BI介导游离胆固醇释出的平均百分比:在各个时间点上,CHO-SR-BI细胞株明显高于CHO-SR-BIC323G细胞株,两两比较有统计学意义,P<0.05,CHO-SR-BIC323G细胞株与CHO-vector两两比较,二者相同,无统计学意义,P=0.119。5.CHO-SR-BIC323G细胞株中SR-BI去糖基后,其分子量与阳性对照CHO-SR-BI细胞株SR-BI去糖基后分子量相同,为56KD。结论:1. C323位点是SR-BI上的关键位点,在SR-BI介导细胞选择性摄取胆固醇酯及游离胆固醇释放过程中,是不可缺少的。2.C323位点的突变未改变SR-BI在细胞内外的分布位置。3.SR-BI上C323位点突变后,其介导细胞选择性摄取胆固醇酯及游离胆固醇释放的能力显著下降。更多还原

【Abstract】 Purpose:To determine the change of the ability of SR-BI mediating cholesteryl esters (CEs) uptake from HDL and free cholesterol efflux from cell after C323 point mutated. And to further determine the role of C323 in SR-BI’s functions.Methods:1.Western Blot is used to investigate the expression of SR-BI in cells.2.By Caveolae Prep., we determine the SR-BI localization, and double confirme it via confocal immunofluorescence images and biotinlation.3.Use iodine monochloride method to iodinate HDL and Gwynne and Mahaffee’s method to trace the HDL-associated cholesteryl ester with nonhydrolyzable [1, 2(n)-3H] cholesteryl oleoyl ether. To determine the ability of SR-BI mediated cholesteryl ester uptake, selective uptake assay is carried on using double labeled HDL to trace the amount of HDL binding to cell and cholesteryl ester taken up into cells.4.SR-BI’s ability of mediating free cholesterol efflux is determined by 3H cholesterol with cholesterol efflux experiment.5. Deglycosytlation is used to determine the reasons of the expression of SR-BI with molecular weight of 100KD in CHO-SR-BIC323G mutant cell line.Results:1.Western Blot indicates that CHO-SR-BIC323G cell mutant still stably express SR-BI except one difference—SR-BI in CHO-SR-BI C323G mutant express two kinds of SR-BI protein with molecular weight of 75KD and 100KD.2.Cavelae prep. indicates that SR-BI in CHO-SR-BI C323G mutant locate in the plasma membrane in caveolae, same as in positive control cell line—CHO-SR-BI , confocal images and biotin results double confirme it. 3.In cholesterol uptake assay, the results show that SR-BI of CHO-SR-BIC323G cell mutant has lost the function of binding to HDL and mediating cholesteryl ester uptake from it.4.The same phenomenon appeared in cholesterol efflux assay as in cholesterol uptake assay.5.The deglycosytlation experiment results show that the expression of SR-BI with mocecular weight of 100KD is caused by glycosylation.Conclusions:1. C323 of SR-BI is critical and required in its mediating cholesteroyl ester uptake and free cholesterol efflux.2. C323 mutating has no effect on SR-BI localization in mutant cell line.3. SR-BI of CHO-SR-BIC323G has lost the function of mediating cholesteryl ester uptake from HDL and free cholesterol efflux from cell.更多还原