结核多肽特异性细胞内因子多色流式分析

目的建立能同时检测CD3、CD4、CD8表型及胞内因子IFN-γ、IL-4、TNF-α的多色胞内细胞因子染色流式细胞术;评价六条结核特异性多肽的性能。方法将结核患者组及健康对照组的PBMC,用结核特异性多肽或PMA刺激培养后,通过胞内细胞因子流式检测术,收集荧光标记阳性的淋巴细胞等;从而确定各亚群IFN-γ、IL-4、TNF-α阳性细胞的数量及百分率,得出两组间及各多肽间的性能差异。结果 1.各多肽均能刺激活化结核患者PB-MC产生IFN-γ等细胞因子,但百分率较低;IFN-γ及IL-4多数小于1%,而TNF-α则在大约10%左右。与对照组比较,IFN-γ及TNF-α的均值分别高1-12倍或1-3倍,IL-4＋/CD4＋T淋巴细胞活化率的倍数较小,而IL-4＋/CD8＋的倍数则较大;2.不同多肽刺激产生细胞内因子的比较：除多肽H256与H210活性能刺激CD4＋亚群分泌更多IFN-γ外,其余各多肽刺激产生因子的性能无统计学差异;3.对于IFN-γ及IL-4因子,各多肽均无CD4＋或CD8＋亚群差异,但各多肽均可刺激CD8＋细胞亚群产生更多TNF-α;4.双阳性（IFN-γ及TNF-α阳性）多功能T细胞分析,经多克隆刺激剂（PMA）活化后,IFN-γ＋/CD4＋及IFN-γ＋/CD8＋T细胞分别有83.0%及79.9%（均值）为双阳性细胞,而经多肽刺激后则为68.1%及65.7%（均值）。结论多色流式胞内因子染色能很好地检测特异性多功能细胞;所用的6条多肽均能活化结核特异性淋巴细胞产生细胞因子,但细胞因子的活化率较低。

Detection of peptide activated TB-specific T cell Cytokines by Multiparameter Flow Cytometry

Objective This study measured frequencies of activated T lymphocytes by MTB-specific peptides from patients with active tuberculosis,and comparing their differences among peptides and different lymphocyte subpopula- tions. Methods The PBMC of TB patients or the healthy control were stimulated by TB specific pepties or PMA,via intracellular cytokine staining, the IFN-γ and/or TNF-α and/or IL-4 cytokine positive CD4＋ or CDS＋T cells were counted. Results i） After peptide stimulation it was found that TB patients had higher frequencies of cytokine positive T cells compared to the heahhy control,the means of IFN-~/and TNF-a raised 1 to 12 fold or 1 to 3 fold respectively. IL- 4 had low fold in CD4＋ subpopulation but higher in CD8＋. The cytokine response frequencies were low, near 1% for IFN-~＇ and ]L-4,TNF-a may come to 10~//00. ii） All six peptides have more ability to active TNF-a positive CD8 ＋ sub- population than CD4＋ suhpopulation,there is not significant differences in cytokines IFN--/and IL-4 between CD4＋ and CD8＋ subpopulation T cells. After PMA stimulation, the mean of multifunctional IFN-γ＋/TNF-α＋ cells were 83.0% and 79.9% of IFN-γ＋CD4＋ or IFN-γ＋CD8＋ cells respectively. And they were 68.1% and 65.7% respectively,af- ter peptide stimulation. Conclusion The technique of intracellular cytokine staining using multi-color immunofluores- cent labeling and flow cytometry could fully served to identify peptide specific T ceils. These six MTB-specifie peptides have the potential to identify individuals with MTB infection as a specific antigen.