CAJ | 학술논문

目的：该研究通过探讨不同的刺激培养条件和检测方法对T h17细胞频率的影响，建立和优化流式细胞术检测人外周血Th17细胞频率实验方案。方法采用不同浓度的佛波酯（PMA ，终浓度50、100、150、200、250、300 ng/mL）与离子霉素（Ion-omycin）1μg/mL对健康人外周血进行刺激培养，分别于刺激后4、6、8、10和12 h用流式细胞术分析 Th17细胞频率，并检测PM A刺激前后细胞表面与胞膜内CD4平均荧光强度（M FI）和CD4阳性T细胞比例。结果 PM A 150 ng/m＋ Ionomycin 1μg/mL刺激全血6 h后，Th17细胞频率高，刺激的效果较好（P＜0．05）。刺激后检测细胞膜内CD4 MFI低于先标记膜表面CD4后刺激组（ P＜0．05），T h17细胞频率和CD4阳性 T 细胞比例均高于后者，差异无统计学意义（ P＞0．05）。结论采用终浓度为PMA 150 ng/mL＋Ionomycin 1μg/mL刺激培养全血6 h后，测定胞浆内CD4和IL-17水平是检测人Th17细胞频率简便有效的方法。
목적：해연구통과탐토불동적자격배양조건화검측방법대T h17세포빈솔적영향，건립화우화류식세포술검측인외주혈Th17세포빈솔실험방안。방법채용불동농도적불파지（PMA ，종농도50、100、150、200、250、300 ng/mL）여리자매소（Ion-omycin）1μg/mL대건강인외주혈진행자격배양，분별우자격후4、6、8、10화12 h용류식세포술분석 Th17세포빈솔，병검측PM A자격전후세포표면여포막내CD4평균형광강도（M FI）화CD4양성T세포비례。결과 PM A 150 ng/m＋ Ionomycin 1μg/mL자격전혈6 h후，Th17세포빈솔고，자격적효과교호（P＜0．05）。자격후검측세포막내CD4 MFI저우선표기막표면CD4후자격조（ P＜0．05），T h17세포빈솔화CD4양성 T 세포비례균고우후자，차이무통계학의의（ P＞0．05）。결론채용종농도위PMA 150 ng/mL＋Ionomycin 1μg/mL자격배양전혈6 h후，측정포장내CD4화IL-17수평시검측인Th17세포빈솔간편유효적방법。
Objective To investigate the effect of different stimulating culture conditions and detecting methods on the frequency of Th17 cells and to set up and optimize the experimental method for detecting the frequencies of human Th 17 cells in peripheral blood by flow cytometry (FCM ) .Methods The peripheral blood in the healthy people was performed the stimulating culture by phorbol myristate acetate (PMA) at different final concentrations(50 ,100 ,150 ,200 ,250 ,300 ng/mL) and ionomycin 1 μg/mL .The frequencies of Th17 cells were assessed by FCM after different stimulating time points (4 ,6 ,8 ,10 ,12 h) .The mean fluorescence in-tensity(MFI) and percentage of the ratio of CD4 positive cells on the cellular surface and inside cell membrane were measured be-fore and after PMA stimulation .Results The frequency of Th17 cells was highest with the good stimulation effect under the condi-tion of PMA 150 ng/mL and ionomycin1μg/mL stimulation for 6 h (P<0 .05) .MFI of CD4 inside cellular membrane after stimula-tion was lower than that in the cells with stimulation after labeling membrane surface CD 4(P<0 .05) ,and the Th17 cell frequencies and the ratio of CD4 positive cells were higher than those in the latter ,but the differences were not statistically significant (P>0 .05) .Conclusion After stimulating whole blood with the final concentration of PMA 150ng/mL and ionomycin1μg/mL for 6 h , measuring intracytoplasm CD4 and IL-17 levels is the simple and effective method for detecting the frequency of human Th 17 cells .