高糖对体外培养的Cajal间质细胞P2X7嘌呤能受体表达的影响

High concentration of glucose enhances the expression of P2X7 purine receptor in interstitial cells of Cajal in vitro

目的:观察P2X7嘌呤能受体在体外培养的Cajal间质细胞(ICC)的表达及高糖的影响,探讨P2X7嘌呤能受体在糖尿病胃肠功能紊乱的细胞机制.方法:以小鼠小肠组织为来源,联合使用机械分离法和酶解法分离ICC,培养在50mL/LCO2温箱里.同时应用ICC特异的c-Kit抗体和P2X7受体抗体进行免疫荧光染色,在激光共聚焦显微镜下进行鉴定,以确定P2X7受体是否表达于ICC.实验分组:正常对照组(葡萄糖浓度为5.5mmol/L),高糖组(葡萄糖浓度为33mmol/L).通过倒置显微镜观察细胞形态,RT-PCR对P2X7和c-Kit受体的表达进行比较.结果:激光共聚焦显微镜下P2X7受体在c-Kit抗体阳性的细胞上的免疫荧光染色是阳性的;与正常组比较,加入葡萄糖后的ICC细胞变大,突起变短,与周围细胞的网络连接减少;RT-PCR结果显示P2X7受体表达于ICC,加入高糖后的ICC的c-Kit受体表达减弱,而P2X7受体的表达是增强的.结论:P2X7受体表达于体外培养的ICC上,高糖使ICC的形态发生了改变、减弱了c-Kit的表达、加强了P2X7受体的表达.这可能在糖尿病胃肠功能紊乱的发生中发挥一定的作用.

AIM：To investigate the effects of high concentration of glucose on the expression of P2X7 purine receptor in the interstitial cells of Cajal （ICC） in vitro and to explore the mechanisms underlying gastrointestinal dysmotility in diabetic mellitus.METHODS：ICC were isolated from the intestine of newborn mice by enzymatic dissociation and centrifugation and cultured in an incubator containing 50 mL/L CO2.Cultured ICC were identified by immunofluorescence staining us-ing antibodies directed against c-Kit receptor and P2X7 receptor.ICC were then divided into two groups：control group and experimental group,which were treated with normal and high concentrations of glucose,respectively.After treatment,cell morphology was observed under an inverted light microscope.The expression of P2X7 receptor and c-Kit receptor mRNAs in ICC was detected by reverse transcriptionpolymerase chain reaction （RT-PCR）.RESULTS：Immunofluorescence staining demonstrated that both P2X7 receptor and c-Kit receptor were positive on ICC cells.After treatment with high concentration of glucose,ICC became bigger,and cell processes became shorter.RT-PCR analysis proved the expression of P2X7 receptor in ICC.The expression level of c-Kit receptor mRNA was weaker and that of P2X7 receptor mRNA was stronger in the experimental group than in the control group.CONCLUSION：P2X7 receptor is expressed in ICC.Hyperglycemia may alter cell morphology,decrease the expression of c-Kit receptor,enhance the expression of P2X7 receptor in ICC,and thereby play a role in the pathogenesis of gastrointestinal dysmotility in diabetic mellitus.