瞬时受体电位M8离子通道对冷刺激诱导气道上皮细胞炎症反应的影响
순시수체전위M8리자통도대랭자격유도기도상피세포염증반응적영향
Effects of transient receptor potential melastatin 8 cation channels on inflammatory reaction induced by cold temperatures in human airway epithelial cells
  • 万方数据
    中华结核和呼吸杂志 중화결핵화호흡잡지
    Chinese Journal of Tuberculosis and Respiratory Diseases
    2011年 10期 757-761 ,共5页

    李敏超%Juliy M.Perelman%Victor P.Kolosov%周向东

    리민초%Juliy M.Perelman%Victor P.Kolosov%주향동

    瞬时受体电位通道%蛋白激酶C%白细胞介素6%白细胞介素8%肿瘤坏死因子-α 瞬時受體電位通道%蛋白激酶C%白細胞介素6%白細胞介素8%腫瘤壞死因子-α
    순시수체전위통도%단백격매C%백세포개소6%백세포개소8%종류배사인자-α
    Transient receptor potential channels%Protein kinase C%Interleukin-6%Interleukin-8%Tumor necrosis factor-alpha
    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、TRPM8 shRNA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Ca2+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Ca2+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Ca2+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Ca2+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.
    목적 탐토순시수체전위M8리자통도(TRPM8)재랭자격유도기도상피세포산생염성인자과정중발휘적작용급상관신호전도궤제.방법 용랭공기(18℃)자격인기도상피16HBE세포,이TRPM8통도특이성길항제BCTC、TRPM8 shRNA급단백격매C(PKC)특이성억제제개린산단백C위간예수단,장세포분위대조조(37℃배양)、랭자격조、랭자격+BCTC조、랭자격+전염TRPM8 shRNA조、랭자격+전염대조shRNA조、랭자격+개린산단백C조.Western blot법검측TRPM8 shRNA전염대16HBE세포합성TRPM8단백적간우효솔；개리자성상기술측량전5조세포내매10s간격적상대Ca2+농도동태변화；ELISA법검측각조세포분비적백세포개소(IL)-6、IL-8、종류배사인자(TNF)-α단백함량；실시형광정량PCR검측각조세포중IL-6、IL-8、TNF-α mRNA표체수평.결과 랭자격조세포내상대Ca2+농도최고치위2.36±0.24,현저고우대조조적1.01±0.02(t=12.52,P＜0.01),랭자격+BCTC조、랭자격+전염TRPM8 shRNA조세포내상대Ca2+농도강위1.47±0.17화1.26±0.12,현저저우랭자격조(t치분별위6.69、9.12,균P＜0.01)；랭자격조적IL-6、IL-8、TNF-α적mRNA화단백함량분별위0.66±0.16、0.77±0.15、0.73±0.09、(92±13) ng/L、( 125±22) ng/L、( 88±12) ng/L,교대조조[0.37±0.08、0.32±0.07、0.48±0.10、(52±8)ng/L、(50 ±9) ng/L、(61±8)ng/L]현저승고(t치분별위3.20、5.36、3.36、5.24、6.26、3.74,균P＜0.05),랭자격+ BCTC조[분별위0.42±0.09、0.52±0.13、0.52 ±0.12、(72±8)ng/L、(92±14) ng/L、(68±11)ng/L]、랭자격+전염TRPM8 shRNA조[분별위0.41 ±0.10、0.49±0.08、0.50±0.08、(60±12)ng/L、(89±14)ng/L、(68±11)ng/L]、랭자격+개린산단백C조[분별위0.40±0.07、0.44±0.09、0.47±0.08、(69±9)ng/L、(86±15) ng/L、(61±10) ng/L]교랭자격조현저강저(균P＜0.05)；랭자격+전염대조shRNA조[분별위0.61±0.10、0.69±0.11、0.64±0.13、(89±13) ng/L、( 118±20)ng/L、(79±13)ng/L]여랭자격조비교차이무통계학의의(t치분별위0.48、0.79、1.12、0.35、0.43、1.00,균P＞0.05).결론 랭공기가통과격활기도상피세포상적TRPM8리자통도이유도Ca2+내류진이격활하유PKC신호통로,진이도치대표성염성인자적표체급생성증다.
    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.Cells were divided into 6 groups:a control group ( incubated at 37 ℃ ),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 Ca2 + 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 ±0.24) 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 Ca2+ 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.
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