多色流式细胞术检测HIV/AIDS病人中的Th细胞亚群

Use of multi-color flow cytometry to detect helper T cell subsets in HIV/AIDS patients

目的建立多色流式方法,检测艾滋病病毒(HIV)感染者/艾滋病(AIDS)病人(简称HIV/AIDS病人)中的辅助性T细胞亚群。方法用3种细胞表面抗体CD3、CD4、CD8,6种细胞内因子抗体γ干扰素(IFN-γ)、粒细胞巨噬细胞集落刺激因子(GM-CSF)、白细胞介素17(IL-17)、肿瘤坏死因子α(TNF-α)、IL-2、L-10,建立多色流式染色方案。根据多色流式荧光搭配原则,制定最佳配色方案。用1例HIV/AIDS病人外周血标本分别进行最适检测电压调节、荧光补偿设定、减一阴性对照(FMO对照)。确定条件后,对3例HIV/AIDS病人外周血标本进行检测分析。结果根据阴性细胞确定检测电压,用补偿微球(CompBeads)单色染色与细胞单色染色确定适宜补偿,减一阴性对照确定阴性边界,建立了九色流式方法检测HIV/AIDS病人外周血中辅助性T淋巴细胞亚群的方案,在单细胞水平同时检测多个因子。结论建立的多色流式方案可检测HIV/AIDS病人外周血中的辅助性T细胞亚群,能在单细胞水平同时检测多个因子,所需样品少,操作简便、相对快速并能准确定量。

Objective To detect helper T cell subsets in HIV/AIDS patients by using multi-color flow cytometry. Methods We used 3 cell-surface antibodies （CD3, CD4, CD8） and 6 intracellular cytokine antibodies （IFN-7, GM-CSF, IL-17, TNF-α, IL-2, L-10） to establish a multi-color flow cytometry staining protocol. In accordance with the principles of multi-color flow fluorescence, we developed the best color scheme. Peripheral blood samples were collected from a HIV/AIDS patient for the optimum PMT test, fluorescence compensation setting and fluores- cence-minus-one control （FMO）. After that, we detected and analyzed the peripheral blood of three HIV/AIDS cases. Results We used negative cells to determine the optimum PMT, single-stained CompBeads and PBMCs to set the appropriate fluorescence compensation and FMO control to define the negative border. Finally, we established a nine-color flow fluorescence to detect various helper T cell subsets in HIV/AIDS patients by detecting multiple factors simultaneously at a single cell level. Conclusion We can use a multi-color flow cytometry to detect helper T cell subsets and multiple factors simultaneously at the single cell level in peripheral blood of HIV/AIDS patients.