摘要
目的构建含有人脑源性神经营养因子(human brain-derived neurotrophic factor,hBDNF)的逆转录病毒载体pLXSN(hBDNF-pLXSN),鉴定hBDNF生物活性。方法提取自愿捐献5月龄胎儿脑组织基因组mRNA,应用RT-PCR技术获得hBDNF基因序列,体外构建hBDNF-pLXSN感染的成纤维细胞NIH/3T3。对其进行病毒滴度检测,采用免疫组织化学染色鉴定NIH/3T3细胞中hBDNF的表达。通过对PC12细胞和大鼠背根神经节的培养,分析hBDNF生物学活性。结果测序结果表明成功构建了hBDNF-pLXSN。被感染的NIH/3T3细胞免疫组织化学染色显示胞浆中有hBDNF表达。hBDNF-pLXSN具有促进PC12细胞增殖的作用,并可以使培养的背根神经节长出神经突。结论hBDNF-pLXSN病毒可以感染NIH/3T3细胞并使其表达和分泌具有生物学活性的hBDNF,可以作为面瘫基因治疗研究的工具。
Objective To construct human brain-derived neurotrophic factor retroviral vector-pLXSN（hBDNF-pLXSN）, and to evaluate the bioactivity of hBDNF.Methods The genome mRNA was extracted from embryonic brain tissue of a 5-month-old infant, the hBDNF gene sequence was obtained with RT-PCR technology, and hBDNF-pLXSN constructed in vitro was used to infect the broblasts（NIH/3T3）.The expression of hBDNF was ident ed by the immunohistochemistry method, and the NIH/3T3 and BDNF biological activities were determined by culture of the PC12 cells and dorsal root ganglia.Results The hBDNF-pLXSN was constructed successfully by sequencing analyses.The infected NIH/3T3 showed positive expression of hBDNF.The infected NIH/3T3 could product hBDNF.Bioactivity of the products could support the PC12 cell survival and neurite growth in the primary cultures of dorsal root ganglia neurons of mice.Conclusion hBDNF-pLXSN virus has the ability to infect NIH/3T3 and make it expressed and secreted hBDNF with the biological activity.It can be used to treat facial paralysis as a gene therapy.
出处
《中国修复重建外科杂志》
CAS
CSCD
北大核心
2009年第5期588-591,共4页
Chinese Journal of Reparative and Reconstructive Surgery
