即将举行的网络研讨会:实现无需离心的流式细胞术样品制备工作流程的标准化
液体中可溶性成分的流式细胞术分析
传统的流式细胞术只能分析细胞及其组分,而不能分析液体中的可溶性组分。然而,如果液体中的可溶性成分结合在一种类似于细胞大小的颗粒上(如乳胶颗粒),则可以通过流式细胞术分析。这是近年来发展起来的流式细胞分析(CBA)技术。CBA的原理是将不同大小的包覆有抗原或抗体的微球与液体中的相应成分反应,形成抗原和抗体的复合物,然后加入第二个荧光素标记的抗体。
结合在微球上的抗原或抗体的分子数量与其荧光强度呈线性关系。因此,可对微球包被抗原或抗体分子所对应的组分在测试液中进行定性或定量分析,如同时测定血清或细胞培养液中的多种细胞因子,同时测定血清中的多种自身抗体。虽然CBA的发展时间很短,可以测试的项目不多,但其发展潜力不容忽视。目前已知的细胞因子检测方法很多,包括靶细胞功能分析、酶联免疫吸附试验(ELISA)、斑点酶免疫试验(Elispots)等,但CBA具有高达2pg/mL的高灵敏度,可以在单个标本中同时检测多种细胞因子。
流式细胞术分子表型分析
分子表型是利用流式细胞术检测细胞中特定的核酸序列或特定的基因异常。流式细胞术结合免疫表型分析为检测特定细胞亚群的特异性核酸序列(如癌基因、病毒核酸等)提供了非常有用的工具,具有广阔的应用前景。将流动分子表型与免疫表型分析相结合的基本技术路线如下:
(1)针对第一细胞和特异性单克隆抗体反应细胞,(2)固定并穿透细胞,(3)通过PCR引物进行特异性核酸序列扩增,(4)应用特异性扩增产物的寡核苷酸探针用荧光原位杂交(FISH)标记,(5)加入耐药细胞的单秒标记抗体。(6)流式细胞术检测及数据分析。例如,流式细胞术免疫分型结合聚合酶链反应和荧光原位杂交(PCR-FISH)检测血液CD4+细胞中hiv特异性DNA或RNA,对检测病程、治疗反应和预后具有重要价值。
端粒长度可由流鱼测定。细胞端粒由2~20Kb串联短片段重复序列(TTAGGG)n和一些结合蛋白组成。端粒长度越长,重复碱基数量越多。荧光素标记核酸-(CCCTAA)3端粒特异性探针处理FISH后,流式细胞术检测荧光强度可反映端粒长度。flow-FISH测量的精度可小于3Kb的端粒长度差,对肿瘤的发生发展、治疗及预后的研究具有一定的价值。
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