μ阿片受体在大鼠结肠平滑肌细胞中的表达

Expression ofμopioid receptors in rat colonic smooth muscle cells

目的通过培养大鼠结肠原代平滑肌细胞,探索μ阿片受体(MOR)在结肠平滑肌细胞中的表达特性。方法分离、培养和鉴定结肠平滑肌细胞;运用免疫荧光组织化学双重标记法观察MOR、内吗啡肽2(EM2)、钙调蛋白(CaM)在结肠平滑肌细胞的分布特性;施加乙酰胆碱(ACh)(1×10^(-3)mol/L)或EM2(2μmol/L)后,Western blot技术检测CaM蛋白表达变化;单独或依次施加ACh(1×10^(-3)mol/L)和EM2(2μmol/L)后,钙离子成像法观察平滑肌细胞内钙离子浓度的变化。结果鉴定结肠平滑肌细胞:α-肌动蛋白(α-SMA)标记的阳性细胞占细胞总数的95%以上;免疫荧光组织化学双重标记显示,在结肠平滑肌细胞有MOR和EM2的分布,且所有MOR和EM2阳性细胞均与α-SMA有共存;Western blot检测发现,施加ACh 10 min后,CaM表达升高(P<0.05);施加EM210 min后,CaM表达降低(P<0.05);钙离子成像结果显示,单独施加ACh,平滑肌细胞内钙离子浓度明显升高(P<0.05);单独施加EM2,结肠平滑肌细胞内钙离子浓度下调(P<0.05),依次施加ACh和EM2后,EM2显著抑制了ACh诱发的钙离子浓度升高(P<0.05)。结论MOR和EM2在结肠平滑肌细胞有表达,且EM2可通过MOR抑制CaM的表达与降低钙离子浓度。

Objective To investigate the expression characteristics of mu opioid receptor(MOR)in rat colon smooth muscle cells by cultured rat primary colonic smooth muscle cells.Methods colonic smooth muscle cells were isolated,cultured and identified;immunofluorescence double labeling method was used to observe the distribution characteristics of MOR,Endomorphin-2(EM2),and calmodulin(CaM)in colonic smooth muscle cells;Western Blot method was used to detect the expression of MOR and EM2 in smooth muscle cells of the colon.After the intervention measures Acetylcholine(ACh)(1×10^(-3)mol/L)and EM2(2μmol/L)were applied,the changes of CaM protein expression were observed;The calciumion imaging method was used to detect the changes of calciumion concentration in smooth muscle cells.Results The colonic smooth muscle cells were cultured and identified.The positive cells labeled withα-smooth muscle actin(α-SMA)accounted for more than 95%of the total number of cells.Immunofluorescence double labeling showed that there were MOR and EM2 distributions in colonic smooth muscle cells,and all MOR and EM2 positive cells coexisted withα-SMA.Western Blot results showed that there were MOR and EM2 expressions in colonic smooth muscle cells.CaM in the ACh group significantly increased at 10 minutes(P<0.05),CaM in the EM2 group significantly decreased(P<0.05);The calciumion imaging results showed that alone applied ACh,the calciumion concentration in smooth muscle cells significantly increased(P<0.05);Alone applied EM2,the calciumion concentration in colonic smooth muscle cells was down-regulated(P<0.05);Applied ACh and EM2 sequentially,EM2 significantly reduced the increase of the calciumion concentration in smooth muscle cells induced by ACh(P<0.05).Conclusion MOR and EM2 are expressed in colonic smooth muscle cells,and EM2 may inhibit the expression of CaM and reduce the concentration of calcium ions through MOR.