流式细胞术检测调节性T细胞方法的建立

目的建立流式细胞术（FCM）检测外周血CD4＋CD25＋调节性T细胞（Tregs）的方法，并观察紫杉醇联合卡铂治疗对晚期肺癌和乳腺癌患者外周血CD4＋CD25＋Tregs数量的影响。 方法19例晚期肺癌和10例乳腺癌患者均给予紫杉醇联合卡铂方案化疗，于化疗前1d和化疗后第7天采集患者外周血，分别加入鼠抗人CD4-FITC（异硫氰酸荧光素）/CD8-PE（藻红蛋白）/CD3-PerCP（多甲藻叶绿素蛋白）、CD25-FITC/CD127-PE/CD4-PerCP、CD3-FITC/CD（16＋56）-PE/CD45-PerCP单抗，并以分别加入同型鼠抗人IgG1-FITC、IgG1-PE、IgG1-PerCP抗体作为阴性对照。采用流式细胞术（FCM）检测化疗前后外周血CD3＋、CD4＋、CD8＋T细胞和CD4＋CD25＋Tregs、NK细胞所占比例并进行数据分析。实验重复3次。 结果与化疗前比较，晚期肺癌和乳腺癌患者化疗后外周血CD4＋CD25＋Tregs的比例均明显降低（6.82%±3.11%vs.5.48%±2.13%，P=0.045；6.38%±1.84%vs.3.88%±1.69%，P=0.007）；晚期肺癌患者化疗后外周血CD4＋T细胞的比例升高（48.84%±16.44%vs.56.35%±14.50%，P=0.006），CD8＋T细胞的比例降低（51.18%±16.44%vs.43.65%±14.50%，P=0.006），CD4＋/CD8＋T细胞比值升高（1.12±0.60vs.1.57±0.88，P=0.008），而CD3＋T细胞和NK细胞的比例均无明显变化；乳腺癌患者化疗后外周血CD3＋、CD4＋、CD8＋T细胞、NK细胞的比例和CD4＋/CD8＋T细胞比值均无明显变化。 结论成功建立了FCM检测CD4＋CD25＋Tregs的方法，联合应用CD4、CD25、CD127检测CD4＋CD25＋Tregs简便可行、重复性好，检测结果可靠、准确，比较适用于临床检验。紫杉醇联合卡铂能够降低晚期肺癌和乳腺癌患者外周血CD4＋CD25＋Tregs的数量。

Development and use of flow cytometry for detection of regulatory T cells

Objective To establish a method to detect CD4+CD25+ regulatory T cells (Tregs) in peripheral blood using flow cytometry (FCM) and evaluate the effect of paclitaxel plus carboplatin on the CD4+CD25+ Tregs in the peripheral blood of patients with advanced lung cancer or breast cancer. Methods A total of 19 patients with advanced lung cancer and 10 patients with breast cancer were treated with paclitaxel combined with carboplatin. Peripheral blood samples were colleted 1 d before the chemotherapy and 7 d after the chemotherapy. Mouse-anti-human CD4-FITC/CD8-PE/CD3-PerCP, CD25-FITC/CD127-PE/CD4-PerCP, or CD3-FITC/CD(16+56)-PE/CD45- PerCP were added into the blood samples respectively. The blood sample added with IgG1-FITC, IgG1-PE and IgG1-PerCP was set as a negative control. The proportion of CD3+, CD4+ and CD8+ T cells, CD4+CD25+ Tregs, and NK cells were evaluated using FCM. Each experiment was repeated 3 times. Results The proportions of CD4+CD25+ Tregs in the patients with advanced lung cancer and breast cancer after the chemotherapy were significantly lower than those before the chemotherapy [(5.48 ± 2.13)% and (3.88 ± 1.69)% vs. (6.82 ± 3.11)% and (6.38 ± 1.84)%; P = 0.045 and P = 0.007, respectively]. In the patients with advance lung cancer, the proportion of CD4+ T cells in peripheral blood increased significantly (48.84% ± 16.44% vs. 56.35% ± 14.50 %, P = 0.006), while that of CD8+ T cells decreased (51.18% ± 16.44% vs. 43.65% ± 14.50%, P = 0.006) and the ratio of CD4+ to CD8+ T cells increased (1.12 ± 0.60 vs. 1.57 ± 0.88, P = 0.008) significantly. No significant difference was detected in the proportions of CD3+ T cells and NK cells between the blood samples collected before and after the chemotherapy. In the patients with breast cancer, the proportion of NK, CD3+, CD4+, CD8+ T cells in peripheral blood did not changed markedly after the chemotherapy. Conclusions We have established a method to determine CD4+CD25+ Tregs using FCM. The method is convenient, repeatable, reliable and precise when CD4, CD25, and CD127 are employed for the detection of Tregs. Paclitaxel plus carboplatin can lower the level of CD4+CD25+ Tregs in the peripheral blood of the patients with advanced lung cancer or breast cancer.