CRMP2过表达及沉默对Aβ_(25-35)诱导SH-SY5Y细胞损伤的影响

Effects of CRMP2 overexpression and knockdown on Aβ_(25-35)-induced damage in SH-SY5Y cells

目的探究脑衰反应调节蛋白2(CRMP2)过表达及沉默对Aβ_(25-35)诱导SH-SY5Y细胞损伤的影响。方法将含CRMP2过表达载体、CRMP2空载体、CRMP2沉默载体及CRMP2扰乱载体的慢病毒感染并筛选SH-SY5Y细胞,分别命名为CRMP2过表达组、空载体组、CRMP2沉默组及扰乱载体组。采用荧光检测和Western blot分别验证感染效率和CRMP2过表达及沉默效果。四组细胞均用终浓度为3μmol/L的Aβ_(25-35)孵育处理24 h诱导细胞损伤,采用MTT实验、LDH实验及Hoechst 33258实验分别检测细胞活性、细胞毒性及细胞凋亡。结果荧光检测结果显示,各组慢病毒感染效率均达95%以上。Western blot检测结果显示,与扰乱载体组相比,CRMP2沉默组CRMP2蛋白表达减少(P<0.01)。与空载体组相比,CRMP2过表达组CRMP2蛋白表达增加(P<0.01)。MTT实验、LDH实验及Hoechst 33258实验结果显示,与扰乱载体组相比,CRMP2沉默组细胞活性增加、细胞毒性降低及细胞凋亡减少(均P<0.05);与空载体组相比,CRMP2过表达组细胞活性降低、细胞毒性增加及细胞凋亡增多(均P<0.01)。结论CRMP2过表达对Aβ_(25-35)诱导的SH-SY5Y细胞损伤具有促进作用,CRMP2沉默对Aβ_(25-35)诱导的SH-SY5Y细胞损伤具有抑制作用。

Objective To explore the effects of overexpression and knockdown of collapsin response mediator protein 2(CRMP2)on Aβ_(25-35)-induced damage in SH-SY5Y cells.Methods SH-SY5Y cells were infected with lentivirus containing CRMP2 overexpression vector,CRMP2 empty vector,CRMP2 knockdown vector and CRMP2 scramble vector in CRMP2 overexpression group,empty vector group,CRMP2 knockdown group and scramble vector group,respectively.Fluorescence detection and Western blot were used to validate the efficacy of transfection,CRMP2 overexpression and knockdown,respectively.Cells in the four groups were incubated with Aβ_(25-35) at 3μmol/L for 24 h to induce cell damage.The cell viability,the cytotoxicity and the apoptosis were detected by MTT assay,LDH assay and Hoechst 33258 assay,respectively.Results Fluorescence detection results showed that the infection efficacy of lentivirus in each group was more than 95%.Western blot analysis showed that compared with scramble vector group,the CRMP2 protein expression was significantly decreased in CRMP2 knockdown group(P<0.01).Compared with empty vector group,CRMP2 protein expression was significantly increased in CRMP2 overexpression group(P<0.01).MTT assay,LDH assay and Hoechst 33258 assay results showed that compared with scramble vector group,the cell activity was increased in CRMP2 knockdown group,while the cytotoxicity and the apoptosis were decreased(P<0.05).Compared with empty vector group,the cell activity was decreased in CRMP2 overexpression group,while the cytotoxicity and the apoptosis were increased(P<0.01).Conclusion CRMP2 overexpression can promote Aβ_(25-35)-induced damage in SH-SY5Y cells,while CRMP2 knockdown can inhibit Aβ_(25-35)-induced damage in SH-SY5Y cells.