CAJ | 학술논문

目的评价磷酸二酯酶4B(PDE4B)在脂多糖诱导大鼠小胶质细胞炎性因子释放中的作用.方法采用随机数字表法,将原代大鼠小胶质细胞分为5组(n=24):对照组、脂多糖组、单纯载体组、错配小干扰RNA组(siRNA)组和PDE4B-siRNA组.对照组和脂多糖组不转染;单纯载体组给予脂质载体1 μl;错配siRNA组和PDE4B-siRNA组分别加入错配siRNA 2μl、PDE4B-siRNA 2 μl与脂质载体1μl(加入100μl无血清培养基中,siRNA终浓度为20 nmol/L),各组转染或培养48 h后,采用Western blot法和RT-PCR法分别测定PDE4B蛋白与mRNA的表达,除对照组外其余各组加入含有脂多糖100 ng/ml的无血清培养基,培养24 h后采用ELISA法测定TNF-α及IL-1β释放水平,采用Western blot 法检测小胶质细胞细胞外调节蛋白激酶(ERK)、磷酸化ERK(p-ERK)的蛋白表达.结果与对照组相比,其余4组TNF-α和IL-1β释放水平升高,小胶质细胞p-ERK表达上调,PDFB-siRNA组小胶质细胞PDE4B蛋白及其mRNA表达下调(P＜0.05),脂多糖组、单纯载体组和错配siRNA组差异无统计学意义(P＞0.05);与脂多糖组相比,PDE4B-siRNA组TNF-α和IL-1β释放水平降低,p-ERK表达下调(P＜0.05),单纯载体组和错配siRNA组差异无统计学意义(P＞0.05).5组小胶质细胞ERK表达水平差异无统计学意义(P＞0.05).结论 PDE4B可促进脂多糖诱导的小胶质细胞炎性因子释放,机制与激活ERK有关.
목적 평개린산이지매4B(PDE4B)재지다당유도대서소효질세포염성인자석방중적작용.방법 채용수궤수자표법,장원대대서소효질세포분위5조(n=24):대조조、지다당조、단순재체조、착배소간우RNA조(siRNA)조화PDE4B-siRNA조.대조조화지다당조불전염;단순재체조급여지질재체1 μl;착배siRNA조화PDE4B-siRNA조분별가입착배siRNA 2μl、PDE4B-siRNA 2 μl여지질재체1μl(가입100μl무혈청배양기중,siRNA종농도위20 nmol/L),각조전염혹배양48 h후,채용Western blot법화RT-PCR법분별측정PDE4B단백여mRNA적표체,제대조조외기여각조가입함유지다당100 ng/ml적무혈청배양기,배양24 h후채용ELISA법측정TNF-α급IL-1β석방수평,채용Western blot 법검측소효질세포세포외조절단백격매(ERK)、린산화ERK(p-ERK)적단백표체.결과 여대조조상비,기여4조TNF-α화IL-1β석방수평승고,소효질세포p-ERK표체상조,PDFB-siRNA조소효질세포PDE4B단백급기mRNA표체하조(P＜0.05),지다당조、단순재체조화착배siRNA조차이무통계학의의(P＞0.05);여지다당조상비,PDE4B-siRNA조TNF-α화IL-1β석방수평강저,p-ERK표체하조(P＜0.05),단순재체조화착배siRNA조차이무통계학의의(P＞0.05).5조소효질세포ERK표체수평차이무통계학의의(P＞0.05).결론 PDE4B가촉진지다당유도적소효질세포염성인자석방,궤제여격활ERK유관.
Objective To evaluate the role of phosphodiesterase 4B (PDE4B) in lipopolysaccharide (LPS)-induced release of inflammatory factors in rat microglias.Methods The pri mary cultured microglial cells were randomly divided into 5 groups (n =24 each) using a random number table:control group (group C),LPS group,vehicle group,mismatch small interfering RNA (siRNA) group and PDE4B-siRNA group.The cells were incubated for 48 h in C and LPS groups.The cells were transfected with lipofectaminTM RNAiMAX 1 μl for 48 h in vehicle group.In mismatch siRNA and PDE4B-siRNA groups,mismatch siRNA 2 μl and PDE4B-siRNA 2 μl were added to 100μl serum-free culture medium (final concentration of siRNA 20 nmol/L),respectively,lipofectaminTM RNAiMAX 1 μl was added simultaneously and the cells were then transfected for 48 h.The expression of PDE4B protein and mRNA was determined by Western blot and real-time PCR,respectively.The cells were cultured for 24 h in serum-free culture medium containing LPS 100 ng/ml in LPS,vehicle,mismatch siRNA and PDF4B-siRNA groups.Then the release of TNF-α and IL-1β was measured by ELISA and the expression of extracellular signalregulated protein kinase (ERK) and phosphorylated ERK (p-ERK) was detected by Western blot.Results Compared with C group,the expression of PDE4B protein and mRNA was significantly down-regulated in group PDE4BsiRNA (P ＜ 0.05),no significant changes were found in LPS,vehicle and mismatch siRNA groups (P ＞ 0.05),and the release of TNF-α and IU1β was increased and the expression of p-ERK was up-regulated in the other four groups (P ＜ 0.05).Compared with LPS group,the release of TNF-α and IL-1β was decreased and the expression of p-ERK was down-regulated in PDE4B-siRNA group,and no significant changes were found in vehicle and mismatch siRNA groups (P ＞ 0.05).There was no significant difference in ERK expression between the five groups (P ＞ 0.05).Conclusion PDF4B can promote LPS-induced release of inflammatory factors in rat microglias and activation of ERK is involved in the mechanism.