CAJ | 학술논문

目的：探讨利用流式细胞术检测小鼠腹腔巨噬细胞吞噬荧光微球能力试验方法的灵敏性、稳定性以及易操作性，以期获得一套最优化的方案。方法：取ICR小鼠腹腔巨噬细胞，以原液和1∶1稀释液使用，采用三个微球浓度（5×106/孔、1×107/孔和1.5×107/孔），经1 h、1.5 h和2 h孵育，通过酶消化或细胞刮刀法将贴壁细胞消化处理后，利用流式细胞仪检测含不同数量荧光微球的巨噬细胞数值，计算吞噬率及吞噬指数。为验证实验条件的可靠性，取ICR小鼠每日灌胃金葵素胶囊30 d后，取腹腔巨噬细胞分别用流式细胞术和传统鸡红细胞方法检测吞噬能力。结果：微球浓度越高，吞噬率和吞噬指数越大；细胞浓度为1×105～2×105 ml-1，孵育时间1.5 h，微球浓度为1.5×107/孔时吞噬率（PP）和吞噬指数（PI）最高（89.87％，1.54），孵育至2 h时出现下降（57.71％，1.51）；细胞浓度对吞噬率和吞噬指数的影响与荧光微球浓度呈负相关。贴壁巨噬细胞经胰酶加EDTA消化后PP为44.51％，PI为0.68，单独使用EDTA消化后PP为37.92％，PI为0.57，均低于使用细胞刮刀处理组。流式细胞术法与鸡红细胞法测定金葵素胶囊对小鼠腹腔巨噬细胞吞噬能力影响的结果一致，均为高剂量组吞噬率与对照组相比差异有统计学意义（P＜0.05）。结论：小鼠腹腔巨噬细胞浓度调整为1×105～2×105，孵育1 h，1×107/孔微球浓度可使实验快速而又精确地反映巨噬细胞的吞噬率和吞噬指数，且与传统方法结果有良好的一致性。
목적：탐토이용류식세포술검측소서복강거서세포탄서형광미구능력시험방법적령민성、은정성이급역조작성，이기획득일투최우화적방안。방법：취ICR소서복강거서세포，이원액화1∶1희석액사용，채용삼개미구농도（5×106/공、1×107/공화1.5×107/공），경1 h、1.5 h화2 h부육，통과매소화혹세포괄도법장첩벽세포소화처리후，이용류식세포의검측함불동수량형광미구적거서세포수치，계산탄서솔급탄서지수。위험증실험조건적가고성，취ICR소서매일관위금규소효낭30 d후，취복강거서세포분별용류식세포술화전통계홍세포방법검측탄서능력。결과：미구농도월고，탄서솔화탄서지수월대；세포농도위1×105～2×105 ml-1，부육시간1.5 h，미구농도위1.5×107/공시탄서솔（PP）화탄서지수（PI）최고（89.87％，1.54），부육지2 h시출현하강（57.71％，1.51）；세포농도대탄서솔화탄서지수적영향여형광미구농도정부상관。첩벽거서세포경이매가EDTA소화후PP위44.51％，PI위0.68，단독사용EDTA소화후PP위37.92％，PI위0.57，균저우사용세포괄도처리조。류식세포술법여계홍세포법측정금규소효낭대소서복강거서세포탄서능력영향적결과일치，균위고제량조탄서솔여대조조상비차이유통계학의의（P＜0.05）。결론：소서복강거서세포농도조정위1×105～2×105，부육1 h，1×107/공미구농도가사실험쾌속이우정학지반영거서세포적탄서솔화탄서지수，차여전통방법결과유량호적일치성。
To explore a sensitive , stable and handleable method for evaluating phagocytosis of mouse peritoneal macrophages by flow cytometry , and get a set of optimized solutions.Methods: The peritoneal macrophages obtained from ICR mice were divided into two part.One part was used directly ,and another part was 1∶1 diluted.Three fluorescent microsphere concentrations were used (5×106/well,1×107/well and 1.5×107/well).Incubation time were respective 1 h,1.5 h and 2 h.The adherent cells were digested by enzyme or cell scraper.The percentage of phagocytic cells ( PP) and the phagocytic index ( PI) were determined by flow cy-tometry.To verify and confirm the reliability of experiment conditions , effect of JKS on phagocytosis of mouse macrophages were evaluated with flow cytometric assays and chicken red blood-cell method.Results:The higher concentration of fluorescent microspheres meant PP and PI were higher.When cell concentration was 1×105-2×105 ml-1 ,incubation time was 1.5 h,concentration of fluorescent microspheres was 1.5 ×107/well,the PP and PI were the highest (89.87%,1.54).When incubation time was 2 h,the PP and PI declined(57.71%,1.51).Effect of cell concentration on the PP and PI were negatively correlated with fluorescent microspheres .After adherent macrophages were digested by trypsin+EDTA,the PP and PI were 44.51%,0.68.The PP and PI were 37.92%,0.57 after di-gestion by EDTA.The results were lower than using cell scraper.The PP(1 485 mg/kg group) of JKS were higher than control group that were evaluated with flow cytometric assays and chicken red blood-cell method.The difference was statistically significant ( P<0.05 ).Conclusion: These are the optimized solutions for the experiment such as the concentration of peritoneal macrophaes is (1-2)×105,the incubation time is 1 h and the concentration of fluorescent microspheres is 1×107/well.