CAJ | 학술논문

目的探讨内质网三磷酸肌醇受体在fractalkine诱发BV-2小胶质细胞p38丝裂原活化蛋白激酶(p38MAPK)信号通路激活中的作用.方法将BV-2小胶质细胞以1×105个/ml浓度接种于3.5 cm培养皿(5 ml/皿)、50 ml培养瓶(8 ml/瓶)、24孔培养板(1 ml/孔)或6孔培养板(2 ml/孔),采用随机数字表法,将其随机分为5组(n=25)∶正常对照组(C组)、fractalkine组(F组)、CX3C趋化因子受体1抗体anti-CX3CR1+ fractalkine组(CF组)、内质网三磷酸肌醇受体拮抗剂2-APB+ fractalkine组(AF组)及p38MAPK抑制剂SB203580+ fractalkine组(SF组).除C组外,其他4组加入10 nmol/L fractalkine,CF组、AF组及SF组于加入fractalkine前1h分别加入15 μmol/L anti-CX3CR1、50 μmol/L 2-APB及10 μmol/L SB203580.测定fractalkine孵育10 min期间细胞内Ca2+浓度([Ca2+]i)的最大值作为[Ca2+]i,于fractalkine孵育即刻、30、60、120、240 min时测定p38MAPK磷酸化水平,于fractalkine孵育24h时测定细胞培养液白细胞介素-1β(IL-1β)和肿瘤坏死因子-α(TNF-α)浓度.结果与C组比较,F组、CF组、AF组和SF组[Ca2+]i、p38MAPK磷酸化水平、IL-1β和TNF-α浓度升高(P＜0.05)；与F组比较,CF组和AF组[Ca2+]i降低,CF组、AF组及SF组p38MAPK磷酸化水平、IL-1β和TNF-α浓度降低(P＜0.05).结论内质网三磷酸肌醇受体参与了fractalkine诱发BV-2小胶质细胞p38MAPK信号通路激活.
목적 탐토내질망삼린산기순수체재fractalkine유발BV-2소효질세포p38사렬원활화단백격매(p38MAPK)신호통로격활중적작용.방법 장BV-2소효질세포이1×105개/ml농도접충우3.5 cm배양명(5 ml/명)、50 ml배양병(8 ml/병)、24공배양판(1 ml/공)혹6공배양판(2 ml/공),채용수궤수자표법,장기수궤분위5조(n=25)∶정상대조조(C조)、fractalkine조(F조)、CX3C추화인자수체1항체anti-CX3CR1+ fractalkine조(CF조)、내질망삼린산기순수체길항제2-APB+ fractalkine조(AF조)급p38MAPK억제제SB203580+ fractalkine조(SF조).제C조외,기타4조가입10 nmol/L fractalkine,CF조、AF조급SF조우가입fractalkine전1h분별가입15 μmol/L anti-CX3CR1、50 μmol/L 2-APB급10 μmol/L SB203580.측정fractalkine부육10 min기간세포내Ca2+농도([Ca2+]i)적최대치작위[Ca2+]i,우fractalkine부육즉각、30、60、120、240 min시측정p38MAPK린산화수평,우fractalkine부육24h시측정세포배양액백세포개소-1β(IL-1β)화종류배사인자-α(TNF-α)농도.결과 여C조비교,F조、CF조、AF조화SF조[Ca2+]i、p38MAPK린산화수평、IL-1β화TNF-α농도승고(P＜0.05)；여F조비교,CF조화AF조[Ca2+]i강저,CF조、AF조급SF조p38MAPK린산화수평、IL-1β화TNF-α농도강저(P＜0.05).결론 내질망삼린산기순수체삼여료fractalkine유발BV-2소효질세포p38MAPK신호통로격활.
Objective To evaluate the role of inositol triphosphate receptor (IP3 R) in the fractalkine-induced activation of p38MAPK signaling pathway in BV-2 microglial cells.Methods BV-2 microglial cells were seeded in 3.5 cm diameter dishes (5 ml/dish),50 ml culture flasks (8 ml/flask) or 24-well plates (1 ml/hole) with a density of 1 × 105/ml and randomly divided into 5 groups (n =25 each) ∶ control group (group C),fractalkinegroup (group F),CX3C chemokine receptor 1 (CX3CR1) antibody anti-CX3CR1 + fractalkine group (group CF),IP3R antagonist 2-APB + fractalkine group (group AF) and p38 mitogen-activated protease (p38MAPK) inhibitor SB203580 + fractalkine group (group SF).Fractalkine 10 nmol/L was added to the culture medium in groups F,CF,AF and SF.The anti-CX3CR1 15 μmol/L,2-APB 50 μmol/L and SB203580 10 μmol/L were added to the culture medium in groups CF,AF and SF,respectively,1 h before addition of fractalkine.The cells were then cultured for 24 h.The intracellular Ca2+ concentration ([Ca2+]i) was measured during the 10 min incubation with fractalkine.The phosphorylation of p38MAPK was measured at 0,30,60,120 and 240 min of incubation with fractalkine.The concentrations of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in theculture medium were determined at 24 h of incubation with fractalkine.Results Compared with group C,[Ca2+]i,and the phosphorylation of p38MAPK and concentrations of IL-1β and TNF-α were significantly increased in groups F,CF,AF and SF (P ＜ 0.05).[Ca2+]i was significant lower in groups AF and CF and phosphorylation of p38MAPK and concentrations of IL-1β and TNF-α were significantly lower in groups CF,AF and SF than in group F (P ＜ 0.05).Conclusion IP3 R is involve in the fractalkine-induced activation of p38MAPK signaling pathway in BV-2 microglial cells.