密度梯度离心法分离脐血干细胞：分离介质的筛选★

摘要

背景：应用脐血分离干细胞的目的是获得以干细胞为主要群体的单个核细胞群，密度梯度离心法是最简单有效的方法之一。密度梯度离心法使用的分离介质以聚蔗糖泛影葡胺最为常用，但哪种浓度获得干细胞最多，目前尚未深入研究。目的：探讨密度梯度离心法分离人脐血干细胞分离介质的最佳浓度，建立临床级干细胞分离应用方案。方法：采用两步法分离脐血流程，先用羟乙基淀粉沉淀脐血红细胞，再使用质量浓度分别为(1.0730±0.0001)，(1.0750±0.0001)，(1.0770±0.0001) g/mL的分离液，分离沉淀脐血红细胞后的上清液，得到单个核细胞，分别计数细胞获得率及细胞存活率。采用流式细胞仪测定单个核细胞表面标志物，将各亚组分绘制成直方图或散点图，分析所得脐血单个核细胞中所含单个核细胞亚组分的比例和绝对数量。结果与结论：应用质量浓度(1.0730±0.0001) g/mL的分离液可得到最大比例间充质干细胞群，是分离间充质干细胞的最佳质量浓度。使用质量浓度(1.0750±0.0001) g/mL的分离液可得到较高比例的造血干细胞群，是分离造血干细胞的最佳质量浓度。使用质量浓度(1.0770±0.0001) g/mL的分离液得到细胞总数最高，但获得的间充质干细胞、造血干细胞比例最低。采用两步法分离干细胞流程，建立严格的实验室条件和标准，可获得密度梯度离心法分离人脐血干细胞的最佳分离方案。%　　BACKGROUND: The purpose for isolating stem cel s from the cord blood is to obtain the mononuclear cel populations with the stem cel s as the major group, and density gradient centrifugation is one of the simplest and most effective ways. Polysucrose diatrizoate is the most commonly used separation medium for density gradient centrifugation, but there is no in-depth research on which method can obtain more stem cel s. OBJECTIVE: To investigate the optimal density of separation medium for isolating human umbilical cord blood stem cel s using density gradient centrifugation method, and to establish the isolation method of stem cel s for clinical application. METHODS: Umbilical cord blood was separated with two-step method. Firstly, hydroxyethyl starch was used to sediment cord blood erythrocyte, and the suspension was obtained, then the separation media with three kinds of concentrations (1.073 0±0.000 1), (1.075 0±0.000 1) and (1.077 0±0.000 1) g/mL was used to isolate the suspension and the mononuclear cel s were obtained. The harvest rate and the survival rate of the cel s were counted. Their surface markers of mononuclear cel s were identified with flow cytometry; histogram or scatter plot of each subset was plotted to analyze their proportions and absolute numbers of sub-cel populations. RESULTS AND CONCLUSION: The separation medium with the concentration of (1.073 0±0.000 1) g/mL could obtain the mesenchymal stem cel populations with the largest proportion, which was the optimal density for isolating the mesenchymal stem cel s. The separation medium with the concentration of (1.075 0± 0.000 1) g/mL could obtain the hemopoietic stem cel populations with the larger proportion, which was the optimal density for isolating the hemopoietic stem cel s. The separation medium with the concentration of (1.077 0±0.000 1) g/mL could obtain the largest number of stem cel s, but the obtained mesenchymal stem cel s and hematopoietic stem cel s had the lowest proportion. Application of two-step method for isolating stem cel s and the establishment of strict laboratory conditions and standards are the optimal programs for isolating the human umbilical cord blood stem cel s using density gradient centrifugation method.