蛋氨酸脑啡肽对人外周血淋巴细胞的阿片受体表达和增殖作用研究

The effect of methionine encephalin on the opioid receptors expression and proliferation of human peripheral lymphocytes

目的探讨蛋氨酸脑啡肽(methionine enkephalin,MENK)对未活化人外周血淋巴细胞阿片受体表达和细胞增殖的影响。方法无菌分离5名健康志愿者人外周血淋巴细胞,将取得细胞平均随机分为对照组和MENK组,MENK组在1×10^-12mol/L MENK下培养48 h,对照组在RPMI1640培养基中行相同时间培养。实时定量PCR检测阿片受体μ和阿片生长受体(opioid growth factor receptor,OGFr)mRNA表达变化,Western blot检测组间OGFr蛋白表达变化。使用MENK以1×10^-3、1×10^-6、1×10^-8、1×10^-10、1×10^-12和1×10^-14mol/L作用于未活化人外周血淋巴细胞24、48、72、96 h,CCK-8检测不同MENK浓度下淋巴细胞增殖情况。结果1×10^-12mol/L MENK刺激未活化人外周血淋巴细胞可显著上调阿片受体μ[(1.257±0.078)比(1.000±0.000),t=3.289,P<0.05]和OGFr[(2.787±0.494)比(1.000±0.000),t=3.618,P<0.05]mRNA表达,并增加OGFr蛋白表达[(2.174±0.281)比(1.000±0.000),t=4.183,P<0.05]。48 h时1×10^-8 mol/L[(1.220±0.090)比(1.000±0.000),t=2.455,P<0.05]、1×10^-10 mol/L[(1.220±0.089)比(1.000±0.000),t=2.480,P<0.05]、1×10^-12 mol/L[(1.174±0.060)比(1.000±0.000),t=2.904,P<0.05]MENK组均可显著刺激未活化人外周血淋巴细胞增殖;72 h时1×10^-3 mol/L[(0.839±0.039)比(1.000±0.000),t=4.152,P<0.05)]、1×10^-12 mol/L[(0.878±0.023)比(1.000±0.000),t=5.247,P<0.05]、1×10^-14 mol/L[(0.919±0.027)比(1.000±0.000),t=2.997,P<0.05]MENK组对未活化人外周血淋巴细胞增殖有显著抑制作用。结论单独低浓度MENK刺激可对未活化人外周血淋巴细胞上阿片受体μ和OGFr表达产生调节作用;不同浓度MENK在不同时间下对未活化人外周血淋巴细胞增殖产生调节作用不同。

Objective To explore the effect of methionine enkephalin (MENK) on the expression of opioid receptors and proliferation of human peripheral blood lymphocytes.Methods Lymphocytes from 5 healthy volunteers peripheral lymphocytes were sterilizedly isolated and divided into control group and MENK group.The MENK group was cultured at 1×10^-12mol/L MENK for 48 h, while the control group was cultured in RPMI1640 medium for the same time.The effect of MENK on μ opioid receptor and opioid growth factor receptor mRNA expression were measured by Real-time PCR, and the protein expression changes were measured by Western blot.The effect of different concentrations of MENK(1×10^-3、1×10^-6、1×10^-8、1×10^-10、1×10^-12 and 1×10^-14mol/L)on the proliferation of lymphocytes were measured by CCK-8 assay.Resultsm RNA expression of μ opioid receptor[(1.257±0.078)vs(1.000±0.000), t=3.289, P<0.05]and opioid growth factor receptor[(2.787±0.494)vs(1.000±0.000), t=3.618, P<0.05) of the human peripheral blood lymphocytes were increased significantly with 1×10^-12 mol/L MENK.The protein expression of growth factor receptor were increased significantly with 1×10^-12mol/L MENK[(2.174±0.281)vs(1.000±0.000), t=4.183, P<0.05]. After incubated 48 h, the proliferation of human peripheral blood lymphocytes were increased significantly with 1×10^-8 mol/L[(1.220 ± 0.090)vs(1.000±0.000), t=2.455, P<0.05], 1×10^-10 mol/L[(1.220 ± 0.089)vs(1.000±0.000), t=2.480, P<0.05], 1×10^-12mol/L[(1.174 ± 0.060)vs(1.000±0.000), t=2.904, P<0.05]MENK.1×10^-3 mol/L[(0.839 ± 0.039)vs(1.000±0.000), t=4.152, P<0.05)]、1×10^-12 mol/L[(0.878 ± 0.023)vs(1.000±0.000), t=5.247, P<0.05]、1×10^-14 mol/L[(0.919 ± 0.027 )vs(1.000±0.000), t=2.997, P<0.05)MENK significantly reduced the number of human peripheral blood lymphocytes after 72 h incubation.Conclusion The opioid growth factor receptor and μ opioid receptor expression on human peripheral blood lymphocytes were regulated by MENK.MENK at different concentrations have different effect on the human peripheral blood lymphocytes proliferation.