Low Hann, Hoang Anh, Sviridov Dmitri
Baker IDI Heart and Diabetes Institute.
J Vis Exp. 2012 Mar 6(61):e3810. doi: 10.3791/3810.
Cholesterol content of cells must be maintained within the very tight limits, too much or too little cholesterol in a cell results in disruption of cellular membranes, apoptosis and necrosis. Cells can source cholesterol from intracellular synthesis and from plasma lipoproteins, both sources are sufficient to fully satisfy cells' requirements for cholesterol. The processes of cholesterol synthesis and uptake are tightly regulated and deficiencies of cholesterol are rare. Excessive cholesterol is more common problem. With the exception of hepatocytes and to some degree adrenocortical cells, cells are unable to degrade cholesterol. Cells have two options to reduce their cholesterol content: to convert cholesterol into cholesteryl esters, an option with limited capacity as overloading cells with cholesteryl esters is also toxic, and cholesterol efflux, an option with potentially unlimited capacity. Cholesterol efflux is a specific process that is regulated by a number of intracellular transporters, such as ATP binding cassette transporter proteins A1 (ABCA1) and G1 (ABCG1) and scavenger receptor type B1. The natural acceptor of cholesterol in plasma is high density lipoprotein (HDL) and apolipoprotein A-I. The cholesterol efflux assay is designed to quantitate the rate of cholesterol efflux from cultured cells. It measures the capacity of cells to maintain cholesterol efflux and/or the capacity of plasma acceptors to accept cholesterol released from cells. The assay consists of the following steps. Step 1: labelling cellular cholesterol by adding labelled cholesterol to serum-containing medium and incubating with cells for 24-48 h. This step may be combined with loading of cells with cholesterol. Step 2: incubation of cells in serum-free medium to equilibrate labelled cholesterol among all intracellular cholesterol pools. This stage may be combined with activation of cellular cholesterol transporters. Step 3: incubation of cells with extracellular acceptor and quantitation of movement of labelled cholesterol from cells to the acceptor. If cholesterol precursors were used to label newly synthesized cholesterol, a fourth step, purification of cholesterol, may be required. The assay delivers the following information: (i) how a particular treatment (a mutation, a knock-down, an overexpression or a treatment) affects the capacity of cell to efflux cholesterol and (ii) how the capacity of plasma acceptors to accept cholesterol is affected by a disease or a treatment. This method is often used in context of cardiovascular research, metabolic and neurodegenerative disorders, infectious and reproductive diseases.
细胞内的胆固醇含量必须维持在非常严格的限度内，细胞内胆固醇过多或过少都会导致细胞膜破坏、细胞凋亡和坏死。细胞可以从细胞内合成以及血浆脂蛋白中获取胆固醇，这两种来源都足以充分满足细胞对胆固醇的需求。胆固醇合成和摄取过程受到严格调控，胆固醇缺乏的情况很少见。胆固醇过多是更常见的问题。除肝细胞以及在一定程度上肾上腺皮质细胞外，细胞无法降解胆固醇。细胞有两种降低胆固醇含量的选择：将胆固醇转化为胆固醇酯，但由于细胞内胆固醇酯过载也有毒性，所以这种选择的能力有限；以及胆固醇流出，这是一种能力可能无限的选择。胆固醇流出是一个特定过程，受多种细胞内转运蛋白调控，如ATP结合盒转运蛋白A1（ABCA1）和G1（ABCG1）以及B1型清道夫受体。血浆中胆固醇的天然受体是高密度脂蛋白（HDL）和载脂蛋白A-I。胆固醇流出测定旨在定量培养细胞中胆固醇流出的速率。它测量细胞维持胆固醇流出的能力和/或血浆受体接受细胞释放胆固醇的能力。该测定包括以下步骤。步骤1：通过向含血清培养基中添加标记胆固醇并与细胞孵育24 - 48小时来标记细胞胆固醇。这一步骤可与细胞加载胆固醇相结合。步骤2：将细胞置于无血清培养基中孵育，以使标记胆固醇在所有细胞内胆固醇池中达到平衡。此阶段可与细胞胆固醇转运蛋白的激活相结合。步骤3：将细胞与细胞外受体孵育，并定量标记胆固醇从细胞向受体的移动。如果使用胆固醇前体来标记新合成的胆固醇，则可能需要第四步，即胆固醇的纯化。该测定可提供以下信息：（i）特定处理（突变、敲低、过表达或某种处理）如何影响细胞流出胆固醇的能力；（ii）疾病或处理如何影响血浆受体接受胆固醇的能力。这种方法常用于心血管研究、代谢和神经退行性疾病、感染性和生殖性疾病等领域。
