流式细胞术检测大鼠Th1/Th2细胞的刺激方案研究

Stimulus plan research of flow cytometry in the detection of Th1/Th2 cells of rats

目的探讨流式细胞术检测大鼠Thl／Th2细胞时使用刺激剂的最佳方案。方法用不同浓度佛波酯（PMA）联合不同浓度离子霉素（Ion）作为激活剂，刺激大鼠外周血T淋巴细胞，采用流式细胞仪三色法方案。观察和分析在不同浓度组合的激活剂刺激下检测T淋巴细胞内干扰素-γ／（INF-γ）、白介素-4（IL-4）的分泌效果。结果PMA浓度为25ng／mL时，联合浓度分别为0、2、5、10、15、20μg／mL的Ion刺激大鼠淋巴细胞，胞内IFN-γ／IL-4的表达阳性率分别为（1．19±0．26）％／（2．46±0132）％、（1．23±0．21）％／（2．56±0．86）％、（3．08±0．56）％／（12．01±2．01）％、（4．01±O．48）％／（9．80±1．80）％、（3．98±1．78）％／（11．85±4．83）％、（3．76±1．08）％／（10．78±2．35）％。结论选用工作浓度为5～10μg／mL的Ion联合工作浓度为25ng／mL的PMA刺激培养大鼠淋巴细胞即可起到较好的激活作用，而且不会带来如CD4细胞下调、流式细胞术设门难度增加等干扰因素。在该浓度下，CD3／CD8和CD4两种设门方案均可用于大鼠Thl／Th2细胞检测。

Objective To investigate the preferred plan of flow cytometry （FCM） in testing Thl/Th2 ceils of rats by stimulants. Methods Combination of different concentration of phorbol esters （PMA） with different concentration of ionomycin （I0n） was taken as activator to stimulate the peripheral blood T lymphocytes in rats. The three-color scheme of PMA was adopted to observe and analyze the effect of secretion of interferon-γ（INF-γ） and interleukin-4 （IL-4） under the different concentration combination activator stimulus to detect T cell. Results When the concentration of PMA was 25 ng/mL, the positive rate of intra cellular IFN-γ/IL-4 was（ 1.19±0.26）%/（2.46±0.32）%, （ 1.23±0.21 ）%/（2.56±0.86）%, （3.08±0.56）%/（ 12.01±2.01 ）%, （4.01± 0.48）%/（9.80±1.80）%, （3.98±1.78）%/（11.85±4.83）%, （3.76±1.08）%/（10.78±2.35）% respectively with the joint concentration of Ion of 0 μg/mL, 2 μg/mL, 5μg/mL, 10 μg/mL, 15 μg/mL and 20 μg/mL to stimulate lymphocytes of rats. Conclusion Selec- tion the concentration of 5 -10 μg/mL Ion combined with joint concentration of 25 ng/mL PMA has a good activation effect to stimulate and cultivate lymphocytes of rats. At the same time, it will not bring interference factors such as reduction of CD4 cells or increase the difficulty of PMA. Under the concentration, both plans of the CD3/CD8 and CD4 can be used in detection of Th 1/Th2 cells in rats.