不同浓度吗啡对小胶质细胞极化状态的影响

EFFECT OF DIFFERENT MORPHINE CONCENTRATIONS ON THE POLARIZATION OF MICROGLIAL CELLS

目的探讨高、低浓度吗啡对小胶质细胞M1/M2极化状态的影响。方法体外培养大鼠小胶质细胞HAPI,将稀释好的细胞混悬液接种于6孔板中,当细胞融合度约达70%时,采用随机数字表法将细胞分为空白对照组(C组)、10-7mol/L吗啡组(L组)和10-4mol/L吗啡组(H组)。C组细胞不做任何处理,L组和H组细胞分别加入终浓度为10-7mol/L和10-4mol/L的吗啡溶液孵育24 h。采用Western Blotting、细胞免疫荧光法检测各组小胶质细胞M1型活化物标记物CD86、一氧化氮合酶(iNOS)及M2型活化物标记物CD206、重组人精氨酸酶1(Arg-1)的表达水平;采用实时荧光定量PCR方法检测各组小胶质细胞中白细胞介素1β(IL-1β)和脑源性神经营养因子(BDNF)mRNA的表达。结果3组小胶质细胞中CD86、CD206、iNOS、Arg-1蛋白的表达量比较均有显著差异(F=30.660~85.968,P<0.05),其中L组与H组比较,上述蛋白的表达量差异均有显著意义(t=3.992~8.278,P<0.05)。3组小胶质细胞中BDNF、IL-1βmRNA的表达量比较差异均有显著性(F=236.808、38.532,P<0.05),其中L组与H组比较差异有显著性(t=11.843、3.901,P<0.05)。结论不同浓度吗啡诱导小胶质细胞不同极化状态,高浓度吗啡诱导小胶质细胞活化为M1型,低浓度吗啡诱导小胶质细胞活化为M2型,这可能是吗啡耐受和痛觉过敏的机制。

Objective To investigate the effect of high and low concentrations of morphine on M1/M2 polarization of microglial cells.Methods Rat microglial cells(HAPI)were cultured in vitro and the diluted cell suspension was inoculated into a 6-well plate.When cell fusion degree reached 70%,the cells were divided into blank control group(group C),10-7 mol/L morphine group(group L),and 10-4 mol/L morphine group(group H)using a random number table.The cells in group C were not given any treatment,and those in groups L and H were incubated with morphine solution at a final concentration of 10-7 mol/L and 10-4 mol/L,respectively,for 24 hours.Western blotting and immunofluorescence assay were used to measure the expression levels of the M1-type markers CD86 and inducible nitric oxide synthase(iNOS)and the M2-type markers CD206 and Arg-1 in the microglial cells in each group,and quantitative real-time PCR was used to measure the mRNA expression levels of interleukin-1β(IL-1β)and brain-derived neurotrophic factor(BDNF)in microglial cells.Results There were significant differences in the protein expression levels of CD86,CD206,iNOS,and Arg-1 in microglial cells between groups C,L,and H(F=30.660-85.968,P<0.05),as well as between group L and group H(t=3.992-8.278,P<0.05).There were significant differences in the mRNA expression levels of BDNF and IL-1βin microglial cells between groups C,L,and H(F=236.808,38.532,P<0.05),as well as between group L and group H(t=11.843,3.901,P<0.05).Conclusion Different concentrations of morphine can induce diffe-rent polarization states of microglial cells.High concentration of morphine can induce the M1-type activation of microglial cells,while low concentration of morphine can induce the M2-type activation of microglial cells,which may be the mechanisms of morphine tolerance and hyperalgesia.