原钙黏蛋白7对小鼠皮层神经元细胞凋亡的影响

Influence of protocadherin 7 on cortex neurons apoptosis in mice

目的探讨原钙黏蛋白7(protocadherin 7,Pcdh7)基因在促进小鼠皮层神经元细胞凋亡中的作用及可能机制。方法原代培养的C57BL/6小鼠皮层神经元细胞,随机分为空载组(转染空白质粒)、Pcdh7过表达组(转染Pcdh7过表达质粒)、Ca^2+通道阻滞组(转染Pcdh7过表达质粒及Ca^2+通道抑制剂西尼地平)。转染48h,采用流式细胞仪检测3组神经元细胞内Ca^2+荧光强度,CCK-8法检测3组神经元细胞存活率,荧光探针法检测3组神经元细胞凋亡率。结果Pcdh7过表达组神经元细胞内Ca^2+荧光强度[(30.57±4.34)MFI]高于空载组[(18.60±0.57)MFI]和Ca^2+通道阻滞组[(18.63±1.97)MFI](P<0.05),空载组与Ca^2+通道阻滞组比较差异无统计学意义(P>0.05);Pcdh7过表达组神经元细胞存活率吸光度值(0.64±0.04)低于空载组(1.01±0.03)和Ca^2+通道阻滞组(0.84±0.02)(P<0.05),且Ca^2+通道阻滞组低于空载组(P<0.05);Pcdh7过表达组神经元细胞凋亡率[(34.10±4.00)%]高于空载组[(13.56±2.48)%]和Ca^2+通道阻滞组[(13.98±3.00)%](P<0.05),空载组与Ca^2+通道阻滞组比较差异无统计学意义(P>0.05)。结论过表达Pcdh7基因可诱导小鼠皮层神经元细胞凋亡,其机制可能与促进细胞内Ca^2+内流有关。

Objective To explore the role of protocadherin 7(Pcdh7)gene in promoting cortex neurons apoptosis in C57BL/6mice and its possible mechanism.Methods The primary cultured neurons were randomly transfected with vector(control group),overexpressed Pcdh7(Pcdh7group),and overexpressed Pcdh7combined cilnidipine(Pcdh7+cilnidipine group),respectively.The relative fluorescence intensity of Fluo-3AM was detected with flow cytometer,the activity of primary cultured cortex neurons was measured with CCK-8method,and the apoptosis rate was detected with fluorescent probe method in three groups.Results The relative fluorescence intensity of Fluo-3AM was significantly higher in Pcdh7group((30.57±4.34)MFI)than that in control group((18.60±0.57)MFI)and Pcdh7+cilndipine group((18.63±1.97)MFI)(P<0.05),and showed no significant difference between control group and Pcdh7+cilndipine group(P>0.05).The optical density of cortex neurons was significantly lower in Pcdh7group(0.64±0.04)than that in Pcdh7+cilnidipine group(0.84±0.02)and control group(1.01±0.03)(P<0.05),and lower in Pcdh7+cilnidipine group than that in control group(P<0.05).The apoptosis rate was significantly higher in Pcdh7group((34.10±4.00)%)than that in control group((13.56±2.48)%)and Pcdh7+cilnidipine group((13.98±3.00)%)(P<0.05),and showed no significant difference between control group and Pcdh7+cilnidipine group(P>0.05).Conclusion The overexpressed Pcdh7in cultured cortex neurons could induce the neuronal apoptosis,probably by promoting extracellular Ca^2+entry.