摘要
目的探讨瞬时受体电位M8离子通道（TRPM8）在冷刺激诱导气道上皮细胞产生炎性因子过程中发挥的作用及相关信号转导机制。方法用冷空气（18℃）刺激人气道上皮16HBE细胞，以TRPM8通道特异性拮抗剂BCTC、TRPM8shRNA及蛋白激酶C（PKC）特异性抑制剂钙磷酸蛋白C为干预手段，将细胞分为对照组（37℃培养）、冷刺激组、冷刺激＋BCTC组、冷刺激＋转染TRPM8 shRNA组、冷刺激＋转染对照shRNA组、冷刺激＋钙磷酸蛋白C组。Western blot法检测TRPM8 shRNA转染对16HBE细胞合成TRPM8蛋白的干扰效率；钙离子成像技术测量前5组细胞内每10s间隔的相对Ca^2＋浓度动态变化；ELISA法检测各组细胞分泌的白细胞介素（IL）-6、IL-8、肿瘤坏死因子（TNF）-α蛋白含量；实时荧光定量PCR检测各组细胞中IL-6、IL．8、TNF-α mRNA表达水平。结果冷刺激组细胞内相对Ca^2＋浓度最高值为2．36±0．24，显著高于对照组的1．01±0．02（t=12．52，P〈0．01），冷刺激＋BCTC组、冷刺激＋转染TRPM8 shRNA组细胞内相对Ca^2＋浓度降为1．47±0．17和1．26±0．12，显著低于冷刺激组（t值分别为6．69、9．12，均P〈0．01）；冷刺激组的IL-6、IL-8、TNF-α的mRNA和蛋白含量分别为0．66±0．16、0．77±0．15、0．73±0．09、（92±13）ng／L、（125±22）ng／L、（88±12）ng／L，较对照组[0．37±0．08、0．32±0．07、0．48±0．10、（52±8）ng／L、（50±9）ng／L、（61±8）ng/L]显著升高（t值分别为3．20、5．36、3．36、5．24、6．26、3．74，均P〈0．05），冷刺激＋BCTC组[分别为0．42±0．09、0．52±0．13、0．52±0．12、（72±8）ng／L、（92±14）ng／L、（68±11）ng／L]、冷刺激＋转染TRPM8 shRNA组[分别为0．41±0．10、0．49±0．08、0．50±0．08、（60±12）ng／L、（89±14）ng／L、（68±11）ng/L]、冷刺激＋钙磷酸蛋白C组[分别为0．40±0．07、0．44±0．09、0．47±0．08、（69±9）ng／L、（86±15）ng／L、（61±10）ng／L]较冷刺激组显著降低（均P〈0．05）；冷刺激＋转染对照shRNA组[分别为0．61±0．10、0．69±0．11、0．64±0．13、（89±13）ng/L、（118±20）ng／L、（79±13）ng／L]与冷刺激组比较差异无统计学意义（t值分别为0．48、0．79、1．12、0．35、0．43、1．00，均P〉0．05）。结论冷空气可通过激活气道上皮细胞上的TRPM8离子通道而诱导Ca^2＋内流进而激活下游PKC信号通路，进而导致代表性炎性因子的表达及生成增多。
Objective To explore the role of transient receptor potential melastatin 8 cation channels（TRPM8） in cold-induced production of inflammatory factors in airway epithelial cells and related signal transduction mechanism. Methods The 16HBE human airway epithelial cells were stimulated with cold temperature （18 ℃）. In intervention experiments, cells were pretreated with TRPM8 channel antagonist BCTC, protein kinase C （PKC） specific inhibitor calphostin C and transfected with TRPM8 shRNA or control shRNA respectively, and thereafter cold stimulation was applied. Ceils were divided into 6 groups ： a control group （ incubated at 37 22 ） , a cold stimulation group, a cold stimulation ＋ BCTC group, a cold stimulation ＋ TRPM8 shRNA group, a cold stimulation ＋ control shRNA group, a cold stimulation ＋ calphostin C group. Western blot was performed to show the extent of knockdown in TRPM8 protein expression in the TRPM8 shRNA transfected cells. Dynamics of relative concentration of intracellular Ca^2＋ in the former 5 groups were measured by calcium imaging techniques. Images were taken at one frame per 10 seconds. The levels of interleukin （IL）-6, IL-8, tumor necrosis factor （TNF）-α mRNA and protein were detected by real-time PCR and ELISA respectively. Results The highest relative concentration of intracellular calcium in cold stimulation group （2. 36 ＋ O. 24） was higher than that of control group （ 1.01 ±0. 02） （t = 12. 52, P 〈 0. 01 ）. BCTC and TRPM8 shRNA reduced intracellular calcium （ 1.05 ±0. 09, 1.08 -0. 09）, compared with single cold stimulation group（t =6. 69 and 9. 12 , all P 〈0. 01 ）. IL-6,IL-8, TNF-α mRNA and protein in cold stimulation group[0. 66 ±0. 16,0. 77 ±0. 15,0. 73 ±0. 09 and（92 ±13） ng/L, （125 ±22 ）ng/L, （88 ±12）ng/L ] were significantly higher than those in control group [ 0. 37 ± 0.08,0.32 ±0.07,0.48 ±0. 10 and（52 ±8） ng/L, （50 ±9） ng/L, （61 ±8）ng/L] （t =3.20 -6.26, all P 〈 0.05 ）. IL-6 mRNA, IL-8 mRNA, TNF-α mRNA and protein in cold stimulation ＋ BCTC group [0. 42 ±0. 09,0. 52 ±0. 13,0. 52 ±0. 12 and（72 ±8） ng/L, （92 ±14） ng/L, （68 ±11） ng/L], cold stimulation ＋ TRPM8 shRNA group [ 0. 41 ±0. 10,0. 49 ±0. 08,0. 50 ±0. 08 and （60 ±12） ng/L, （ 89 ＋ 14） ng/L, （ 68 ＋ 11 ） ng/L ] and cold stimulation ＋ calphostin C group [ 0. 40 ±0. 07,0. 44 ±0. 09,0.47 ±0. 08 and （69 ±9） ng/L, （ 86 ±15 ） ng/L, （ 61 ±10） ng/L ] were significantly lower than those in cold stimulation group（t =2.47 -4. 21, all P 〈0. 05）. IL-6 mRNA,IL-8 mRNA,TNF-α mRNA and protein in cold stimulation ＋ control shRNA group [0. 61 ±0. 10,0. 69 ±0. 11,0. 64 ±0. 13 and（89 ±13） ng/L, （118 ±20）ng/L, （79 ＋ 13 ）ng/L] showed no significant change, compared with cold stimulation group （t = 0. 35 - 1.12, all P 〉 0. 05 ）. Conclusion Cold temperature may induce Ca^2＋ influx and up-regulate IL-6, IL-8, and TNF-α expression in 16HBE cells by activating the TRPM8 ion channels, and this is via a signaling pathway involving PKC.
出处
《中华结核和呼吸杂志》
CAS
CSCD
北大核心
2011年第10期757-761,共5页
Chinese Journal of Tuberculosis and Respiratory Diseases
基金
国家自然科学基金（81070031）
国家自然科学基金中俄国际合作项目（81011120108）
中俄政府间合作项目（2009：13-01）
