模拟微重力下瘦素对肺T淋巴细胞蛋白激酶C信号转导途径的影响

Leptin on protein kinase C of pulmonary T lymphocytes under simulated microgravity

目的探讨在模拟微重力下瘦素对肺T淋巴细胞蛋白激酶C(PKC)信号转导途径的影响。方法分离肺T淋巴细胞,利用旋转式细胞组织培养系统(RCCS)模拟微重力条件。培养T淋巴细胞后,采用PepTag非放射性PKC检测试剂盒测定T淋巴细胞PKC活性;酶联免疫吸附试验(ELISA)测定T淋巴细胞的白细胞介素-2(IL-2)生成;采用流式细胞技术检测T淋巴细胞表面抗原CD25、CD71的表达。结果在普通细胞培养容器组,刀豆蛋白A(ConA)组与瘦素组、ConA+瘦素组与ConA组相比,PKC活性增强,IL-2生成增加,表面表达CD25分子的细胞增多;模拟微重力条件下,与普通细胞培养容器培养的T淋巴细胞相比较,ConA刺激后,PKC活性、IL-2水平,表面表达CD25分子的细胞减少(P<0.01);在模拟微重力条件下,瘦素组与ConA组相比,相同指标无明显变化,而且加入瘦素的ConA组与ConA组比较,仍无明显变化。RCSS组中的ConA+瘦素组,与普通细胞培养容器组中的ConA+瘦素组相比较,上述指标均降低,差异有统计学意义(P<0.01);且在模拟微重力条件下,瘦素组和ConA+瘦素组与同样条件下的ConA+12-肉豆蔻酰-13-乙酸佛波酯(PMA)组相比,上述所有指标均降低(P<0.01)。结论模拟微重力条件下,T淋巴细胞的激活受到抑制;PKC的活性降低,表面表达CD25的细胞数降低,T淋巴细胞分泌的IL-2降低;同样条件下,PMA对T淋巴细胞的上述指标有部分恢复作用。

Objective To investigate the effects of leptin on protein kinase C （PKC） of pulmonary T lympho-cytes under a simulated microgravity. Methods Lung T lymphocytes isolated from rats were cultured by using rotary cell culture system （RCCS） technology which simulated microgravity. Following incubation of T lymphocytes, the cel-lular PKC activity of T lymphocytes was determined by using PepTag′s non-radioactive PKC assay kit. The level of IL-2 was measured by enzyme-linked immunosorbent assay kits （double-antibody）. And flow cytometry was applied to measure the expression of surface antigen CD25 and CD71. Results In the normal gravity environment, the PKC ac-tivity, IL-2 production, the activation of cellular surface CD25 were increased when ConA group compared with leptin group and ConA＋leptin group compared with ConA group （both P〈0.01）. Exposure to microgravity conditions, when compared with normal gravity group, resulted in attenuated PKC activity, IL-2 production and the activation of cellular surface CD25 in ConA group （all P〈0.01）. However, the differences in PKC activity, IL-2 production and the activa-tion of cellular surface CD25 were unremarkable among leptin group, ConA group and ConA＋leptin group （all P〉0.05） when exposed to microgravity conditions. Compared with normal gravity, the RCCS was associated with significantly lower levels of the aforementioned parameter in ConA＋leptin group （all P〈0.01）. When to microgravity conditions, lep-tin group and ConA＋leptin group were associated with lower levels of the aforementioned parameters as compared with ConA＋PMA group （all P〈0.01）. Conclusion Exposure to microgravity conditions accounts for inhibited T lympho-cyte activation, attenuated PKC activity , reduced number of CD25 expressing cells and IL-2 production, which could be restored by PMA.