无辅助病毒HSV-1载体介导LacZ基因在培养皮层神经元中的表达

Helper-virus-free herpes simplex virus-1 vector mediated LacZ gene expression in cultured cortical neurons

目的:探讨无辅助单纯疱疹病毒(herpessimplexvirustype 1,HSV 1)载体的包装及其介导LacZ基因在体外培养神经元表达的作用, 解决该载体系统在包装及体外培养神经元应用中的有关问题。方法:将一组含HSV 1基因组的粘粒以限制性内切酶PacⅠ酶切、纯化后,与含LacZ和酪氨酸羟化酶 (tyrosinehydroxylase,TH)及神经微丝(neurofilament,NF)嵌合启动子的HSV 1质粒DNA在脂质体作用下,共转染 2 2细胞;在 34℃条件下培养 72h,收获病毒颗粒。将含病毒颗粒的上清液,加入BHK细胞培养液中,继续培养 24h后,加X gal染液作用 4h,观察表达LacZ基因的蓝染细胞。取培养第 3天的胚胎大鼠大脑皮层神经元,加入含病毒颗粒的培养液,过夜后更换新鲜培养液,分组继续培养 1, 7和 14d,进行X gal染色,光镜下观察。结果:包装形成的病毒颗粒感染BHK细胞, 24h后可见表达LacZ基因的蓝染细胞;感染培养第 3天的皮层神经元,继续培养 1, 7和 14d后均可见蓝染神经元。结论:无辅助病毒包装系统能够使含TH NF嵌合启动子的HSV 1载体形成有效的病毒颗粒,并且在体外培养神经元中持续稳定地表达外源基因,为在神经系统进行基因转移和基因功能研究提供了有效的工具。

Objective: To obtain knowledge about the helper-virus-free packaging system of HSV-1 vector and its use in cultured neurons by investigating helper-virus-free packaging of HSV-1 vector and LacZ gene expression mediated by the vector in rat cultured cortical neurons. Methods: The cosmids with inserts of HSV-1 genome were digested by Pac Ⅰ, purified and cotransfected with HSV plasmid vector DNA (with LacZ gene) into 2-2 cells. After 3 hour incubation at 37 ℃, the media were changed into DMEM with 6%CCS. And the 2-2 cells were incubated for 72 hours at 34 ℃. Then the virus particles were harvested. The medium with the virus particles was added to BHK cells. After 24 hour further culture, X-gal staining was performed. The virus particles were added to 3 day cultured cortical neurons. After 1, 7 and 14 day further cultures, neurons were stained by X-gal and observed under microscope. Results: The BHK cells (stained blue) expressing LacZ gene could be seen after 24 hour further culture. After 1, 7 and 14 day further cultures, neurons stained blue by X-gal staining could be seen all the time in different groups. Conclusion: Helper-virus-free package system can produce effective virus particles from HSV-1 plasmid vector with A tyrosine TH-NF chimeric promoter and mediate stable exogenous gene expression in cultured neurons, thus providing a useful tool for gene transfer and study of gene function in the nervous system.