【摘要】 目的构建弓形虫棒状体蛋白ROP18重组腺病毒载体,研究其对神经干细胞C17.2凋亡的影响。方法将弓形虫ROP18从PEGFP-G2-ROP18重组质粒上亚克隆至腺病毒载体pHBAd-MCMV-GFP,重组腺病毒载体pHBAd-MCMVGFP-ROP18经PCR、测序鉴定正确后,与骨架质粒pHBAd-BHG共转染HEK293细胞进行包装,收获、扩增重组腺病毒并对其滴度进行测定。以MOI=70的重组腺病毒感染神经干细胞C17.2,转染不同时间荧光显微镜观察基因的表达情况。转染48h用CCK-8法检测C17.2的增殖情况;转染24h采用Annexin V-APC/PI双染流式细胞术检测细胞凋亡情况;Western blot法检测半胱天冬氨酸蛋白酶3(caspase-3)的蛋白水平。结果重组ROP18过表达腺病毒在C17.2神经干细胞内能有效表达。转染48h,与空载体对照组相比,C17.2细胞增殖活性明显降低(P<0.05);转染24h,流式细胞术检测结果显示,ROP18基因转染组C17.2细胞凋亡率明显增加(P<0.001);Western blot检测显示caspase-3活性片段的表达明显增加(P<0.05)。结论成功构建了弓形虫ROP18重组腺病毒并在神经干细胞C17.2中表达,ROP18重组腺病毒能诱导C17.2神经干细胞凋亡。更多还原

【Abstract】 To study the effect of Toxoplasma rhoptry kinase 18(ROP18)on the apoptosis of C17.2neural stem cells,Toxoplasma gondii ROP18 was subcloned from PEGFP-G2-ROP18 into pHBAd-MCMV-GFP.The recombinant adenovirus vector pHBAd-MCMV-GFP-ROP18 was comfirmed by PCR and sequencing,and the identified recombinant adenovirus vector was transfected into 293 cells for packaging with skeleton plasmid.After amplification,ROP18 recombinant adenovirus(MOI=70)was transfected into C17.2neural stem cell.The proliferation of C17.2cells at 48 horus Post-treatment was detected using CCK-8kit and the apoptosis of C17.2cells at 24 hours Post-treatment was detected by flow cytometer.Western blot was used to determine the expression levels of caspase-3.ROP18 recombinant adenovirus was expressed effectively in C17.2neural stem.Compared with the control group,proliferation of C17.2cells was significantly decreased(P<0.05)in the ROP18 recombinant adenovirus group at 48 hours while the apoptosis of C17.2cells was significantly increased in the ROP18 recombinant adenovirus(P<0.001)at 24 hours Post treatment and the caspases-3expression levels was significantly increased(P<0.05).ROP18 recombinant adenovirus was successfully constructed and was highly expressed in C17.2neural stem cells.ROP18 recombinant adenovirus induced the apoptosis of C17.2neural stem cells.更多还原