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逆向胆固醇转运机理的研究对防治动脉粥样硬化具有理论和实践意义,Stein等利用~3H-胆固醇示踪的方法对细胞胆固醇外流进行过较多研究。我们为了同时观察细胞LDL受体表达及胆固醇外流,曾利用薄层层析的方法测量细胞的胆固醇外流。以上两种方法均含有繁琐的用有机溶剂从培养液和细胞中萃取脂质的步骤。为探索一个简便的方法来评价细胞内胆固醇外流的相对值,我们参照Stein等的~3H-胆固醇示踪的方法,采用容水量大的甲苯-Triton闪烁液直接与收集的培养液或细胞Triton萃取液混合后计数,观察了兔缺乏脂蛋白血清对体外培养的兔皮肤成纤维细胞胆固醇外流的影响。 实验前细胞用每毫升含~3H-胆固醇0.5微居里的培养液培养。实验时吸去标记培养液,细胞先后经PBS冲洗两次,含1％牛血清白蛋白的EMEM和不含血清的EMEM各孵育15分钟后,对照组和实验组的细胞分别换不含血清的EMEM和含10％兔缺乏脂蛋白血清的EMEM
Reverse cholesterol transport mechanism of prevention and treatment of atherosclerosis has theoretical and practical significance, Stein and other methods using ~ 3H-cholesterol tracer on cellular cholesterol efflux have done more research. In order to observe both cellular LDL receptor expression and cholesterol efflux at the same time, we used the method of TLC to measure the cellular cholesterol efflux. Both of these methods contain tedious steps of extracting lipids from the culture broth and cells with organic solvents. To explore an easy way to assess the relative value of intracellular cholesterol efflux, we used the ~ 3H-cholesterol tracer method of Stein et al. To extract Triton directly from the collected culture or Triton using a toluene -Triton scintillation cocktail with high water content After the liquid was mixed and counted, the effects of rabbit serum lipoprotein serum on the cholesterol efflux of rabbit dermal fibroblasts cultured in vitro were observed. Before the experiment, the cells were cultured in a medium containing 0.5 microcurrents of ~ 3H-cholesterol per ml. During the experiment, the labeled medium was aspirated and the cells were rinsed twice with PBS. After incubating EMEM containing 1% bovine serum albumin and EMEM without serum for 15 minutes, the cells in the control group and the experimental group were replaced with serum-free EMEM and EMEM with 10% rabbit lacking lipoprotein serum