AKT和PKCθ与吗啡抑制辅助性T淋巴细胞分化的关系  

Relationship between AKT and PKCθ and morphine-induced inhibition of differentiation of T helper cells

作　　者：毛毛[1] 孙杰[1] 钱燕宁[1] 

机构地区：[1]南京医科大学第一附属医院麻醉科,210029

出　　处：《中华麻醉学杂志》2014年第2期193-195,共3页Chinese Journal of Anesthesiology

摘　　要：目的 评价蛋白激酶B（AKT）和蛋白激酶Cθ（PKCθ）与吗啡抑制辅助性T淋巴细胞（Th细胞）分化的关系.方法 取20名健康成年志愿者静脉血样各20 ml,采用密度梯度离心法提取外周血单个核细胞（PB MC）,采用免疫磁珠分离法提取纯CD4＋T淋巴细胞.采用随机数字表法,将CD4＋T淋巴细胞分为4组（n=3）：对照组（C组）不给予任何处理;PI组加入佛波酯（PMA）25 ng/ml＋钙离子载体（Ion）1 μg/ml;吗啡组（M组）加入PMA 25 ng/ml＋Ion 1 μg/ml＋吗啡50 ng/ml;纳洛酮组（N组）加入PMA 25 ng/ml＋Ion 1 μg/ml＋吗啡50 ng/ml＋纳洛酮50 ng/ml.细胞置于37℃5％CO2细胞培养箱中孵育4h.采用流式细胞分析法测定IFN-γ和IL-4的表达,以IFN-γ表达反映Th1细胞亚群比例,以IL-4表达反映Th2细胞亚群比例,计算Th1/Th2比值.采用Western blot法检测AKT、磷酸化AKT（p-AKT）、PKCθ和磷酸化PKCθ（p-PKCθ）的表达,计算p-AKT/AKT比值和p-PKCθ/PKCθ比值,分别反映AKT和PKCθ的活性.结果 与C组比较,PI组、M组和N组Th1、Th2细胞亚群比例、Th1/Th2比值升高,PI组和N组AKT和PKCθ的活性升高,M组PKCθ活性升高（P＜0.05）.与PI组比较,M组Th1细胞亚群比例、Th 1/Th2比值和AKT活性降低,N组Th1/Th2比值降低（P＜0.05）,M组和N组PKCθ活性差异无统计学意义（P＞0.05）.与M组比较,N组Th1细胞亚群比例、Th1/Th2比值和AKT活性升高（P＜0.05）,PKCθ活性差异无统计学意义（P＞0.05）.结论 吗啡通过激活阿片受体抑制Th细胞分化的机制与抑制AKT的激活有关,与PKCθ无关.Objective To evaluate the relationship between protein kinase B （AKT） and protein kinase （PKCθ） and morphine-induced inhibition of differentiation of T helper （Th） cells.Methods Peripheral venous blood samples were taken from 20 healthy volunteers..Peripheral blood mononuclear cells （PBMCs） were extracted by density gradient centrifugation method.CD4＋ T lymphocytes extracted were purified by magnetic bead separation.CD4＋ T lymphocytes were randomly assigned into 5 groups （n =3 each） using a random number table.CD4 ＋ T lymphocytes were incubated routinely in group C.CD4 ＋ T lymphocytes were incubated in the presence of PMA 25 ng/ml ＋ ionomycin 1 μg/ml （group PI）,PMA 25 ng/ml ＋ ionomycin 1 μμg/ml ＋ morphine 50 μμg/ml （group M）,or PMA 25 ng/ml ＋ ionomycin 1 μμg/ml ＋ morphine 50 μg/ml ＋ naloxone 50 μμg/ml （group N）.The cells were incubated for 4 h in the incubator containing 5% CO2 at 37 ℃.The expression of interferon （IFN）-γ and interleukin-4 （IL-4） was detected by flow cytometry.The expression of IFN-γ and IL-4 was used to reflect the percentage of Th1 and Th2 cells,respectively.The ratio of Th1/Th2 was calculated.The expression of AKT,phosphorylated AKT （p-AKT）,PKCθ and phosphorylated PKCθ （p-PKCθ） was detected by Western blot,and the ratio of p-AKT/AKT and p-PKCθ/PKCθ was calculated to reflect the activities of AKT and PKCθ,respectively.Results Compared with group C,the percentage of Th1 and Th2 cells and ratio of Th1/Th2 were significantly increased in PI,M and N groups,the activities of AKT and PKCθ were increased in PI and N groups,and the activity of PKCθ was increased in group M （P 〈 0.05）.Compared with group PI,the percentage of Th1 cells,ratio of Th1/Th2 and activity of AKT were significantly decreased in group M,the ratio of Th1/Th2 was decreased in group N （P 〈0.05）,and no significant change in the activity of PKCθ was found in M and N groups （P 〉 0.05）.Compared with group M,the percentage of Th1

关 键 词：吗啡 T淋巴细胞 辅助诱导 蛋白质丝氨酸苏氨酸激酶 蛋白激酶C 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]