摘要
目的建立稳定表达脑源性神经营养因子(BDNF)基因的神经干细胞并观察其对于体外培养的脊髓背根神经节(DRG)细胞存活及突起生长的影响。方法以PCR技术从人脑cDNA文库中获取人BDNF基因片段。采用分子克隆技术构建带有绿色荧光蛋白报告基因的多顺反子的MSCVBDNFIRES2EGFP重组鼠干细胞病毒表达载体,病毒经PT67细胞包装后感染神经干细胞株C17.2。通过免疫组化,RTPCR,ELISA检测BDNF的表达,在双室共培养系统中共培养3,10d后观察经筛选的稳定表达株对原代培养DRG细胞的生物学作用。结果RTPCR证实BDNF基因在C17.2细胞中转录,ELISA分析显示转基因C17.2培养上清中在转染24h后即可检测到BDNF表达,并在3个月后仍表达(14.6±0.8)ng·d-1·10-1细胞;免疫细胞化学显示,与对照组相比较,BDNF基因修饰的神经干细胞组,DRG细胞突起生长较长交织呈网状的,节细胞存活较多。结论干细胞病毒介导转染的BDNF基因能够在神经干细胞C17.2中稳定表达,并且BDNF基因修饰的神经干细胞对体外培养的DRG细胞的存活及突起、延伸具有显著促进作用。
Objective To gain stable genetic modification of neural stem cells （NSC） that constitutively secrete brain-derived neurotropic factor （ BDNF ） and to explore the biological role on the survival and neurite outgrowth of cultured dorsal root ganglion （DRG） neurons. Methods BDNF gene fragment from human brain cDNA libraries was obtained by using PCR. With molecular cloning technique, the recombinant stem cell viral vector with report gene was constructed, which is that MSCV-BDNF-IRES2-EGFP vector could encode Polycistronic mRNA. Viral particle was packaged by PT67 cell line and infected neural stem cells （mouse clone：C17. 2）. After selection with cloning cylinder, the expression of BDNF was assessed by immunohistochemistry enzyme linked immunosorbent assay （ELISA） and reversetranscriptase polymerase chain reaction （RT-PCR）. The effects of stable gene-modified neural stem cells on embryonic mouse DRG neurons were evaluated in a dual-chambered cocultivation system at 3th.10th day. Results RT-PCR analysis demonstrated expression of mRNA for human-BDNF. ELISA confirmed the presence of secreted BDNF 24 h after transfection and showed that the level of BDNF production by NSC-BDNF transfectedC17.2 was at a rate of （14.6±0.8）ng·d^-1·10^-1 cells even after 3 months. With immunohistochemical analysis, compared with the control, the longer neurite outgrowth of cultured DRG cells and the more survival neurons were observed in NSC-BDNF transfected cells group. Conclusions BDNF gene could be stably expressed in C17.2 cell line by MSCV , and the BDNF gene-modified NSC markedly enhances the survival and neurite outgrowth of cultured DRG neurons.
出处
《中华外科杂志》
CAS
CSCD
北大核心
2005年第24期1609-1612,共4页
Chinese Journal of Surgery
基金
国家自然科学基金资助项目(30271322)
