TYR/BDNF基因慢病毒载体的构建及其在大鼠脑梗死模型中的应用  

Construction of Lentiviral Vector Containing TYR/BDNF Gene and Its Application in Rats with Cerebral Infraction

作　　者：刘玉[1] 刘影[1] 伍志发 鹿静雅 Liu Yu;Liu Ying;Wu Zhifa(Department of Radiology,the First Affiliated Hospital of USTC,Division of Life Sciences and Medicine,University of Science and Technology of China,Auhui 230001,China)

机构地区：[1]中国科学技术大学附属第一医院影像科,合肥230001

出　　处：《华中科技大学学报（医学版）》2022年第3期360-365,374,共7页Acta Medicinae Universitatis Scientiae et Technologiae Huazhong

基　　金：中央高校基本科研业务费专项基金资助项目(No.WK9110000097)。

摘　　要：目的 构建携带酪氨酸酶基因(tyrosinase, TYR)和脑源性神经营养因子(brain-derived neurotrophic factor, BDNF)基因的重组慢病毒载体,建立TYR和BDNF基因过表达的骨髓间充质干细胞株(BMSCs),并利用光学成像技术监测BMSCs在大鼠脑梗死模型脑内分布情况。方法 应用RT-PCR技术扩增TYR及BDNF基因,然后克隆到H7656载体,构建慢病毒表达载体H16189,转染293T细胞,包装并测定其滴度,转染BMSCs后,应用RT-PCR、Western blot和ELISA检测目的基因及蛋白的表达。构建大鼠大脑中动脉梗塞模型,纹状体注射慢病毒感染的BMSCs,小动物光学成像观察BMSCs脑内分布情况。结果 成功构建慢病毒表达载体H16189(PLENTI-CBH-TYR-3XFLAG-P2A-LUC2-TCMV-BDNF-6XHIS-F2A-PURO-WPRE),病毒滴度为2.6×10;TU/mL。慢病毒感染BMSCs后,RT-PCR检测到TYR和BDNF mRNA的表达,Western blot或ELISA法成功检测到TYR和BDNF蛋白表达。光学成像结果显示BMSCs可以在脑内迁徙,且主要聚集在脑梗死区域。结论 成功构建了携带TYR及BDNF基因的慢病毒表达载体,光学成像显示携带TYR及BDNF基因的BMSCs可以将重组质粒运输到缺血梗死区,为下一步进行MRI显像及疗效评价提供了实验依据。Objective To construct the lentiviral vector carrying tyrosinase(TYR)and brain-derived neurotrophic factor(BDNF)gene and the bone mesenchymal stem cells(BMSCs)with over-expression of TYR and BDNF genes, and to detect the distribution of BMSCs in rats with cerebral infraction by optical imaging.Methods The TYR and BDNF genes were amplified by RT-PCR,and then cloned into the vector H7656.The lentiviral vector H16189 was constructed.It was then transfected to 293 T cells and a titer test was conducted.BMSCs were transfected with the lentiviral vector, and the target gene and protein expression levels were detected by RT-PCR,Western blotting and ELISA.After the rat model of middle cerebral artery infarction was established, BMSCs were injected into the striatum, and the distribution of stem cells in the brain was observed by optical imaging.Results The recombinant lentiviral vector H16189(PLENTI-CBH-TYR-3 XFLAG-P2 A-LUC2-TCMV-BDNF-6 XHIS-F2 A-PURO-WPRE)was successfully constructed and its titer was 2.6×10~8TU/mL.The expression of TYR and BDNF mRNA was successfully detected by RT-PCR,and the protein expression of TYR and BDNF was detected by Western blotting or ELISA.Optical imaging result showed that BMSCs could migrate in the brain, and mainly concentrated in the cerebral infarction area.Conclusion In this study, lentiviral vector carrying TYR and BDNF genes was successfully constructed.The optical imaging showed that BMSCs carrying TYR and BDNF genes could transport the recombinant plasmid to the ischemic infarction area, which provided experimental basis for MRI imaging and efficacy evaluation.

关 键 词：骨髓间充质干细胞 酪氨酸酶基因 脑源性神经营养因子 脑梗死 

分 类 号：R743.3[医药卫生—神经病学与精神病学]