逆转录病毒介导的人NT-3在嗅神经鞘细胞中的表达及活性检测

本文构建了pRev-TRF-NT-3的逆转录病毒表达载体,通过Fcopack293细胞将其与pRev-Tet-On分别进行包装,制备了重组缺陷型的hNT-3和Tet-on逆转录病毒,用这两种病毒感染原代培养大鼠嗅神经鞘细胞(olfactory ensheathing cells,OECs),并经强力霉素(Dox)诱导,获得Dox浓度依赖性hNT-3表达的OECs,最后经过Western-Blot检测hNT-3的表达以及通过DRG与NT-3转染后OECs联合培养来对表达的hNT-3活性进行鉴定,结果:(1)hNT-3-pcDNA的多克隆位点,用EcoR Ⅰ和Hind Ⅲ酶切,PCR扩增,再用Sall和Hind Ⅲ切下全长编码的hNT-3 cDNA,定向连接到pRev-TRF上,pRev-TRF全长为6.5kb,NT-3全长为0.78kb,pRev-TRE-NT-3重组体经过鉴定,确认插入子的方向和完整性,结果与预期相符。(2)hNT-3修饰的OECs培养上清经Western-blot检测,与hNT-3抗体结合的蛋白条带分子量约为28kd,hNT-3修饰过的OECs上清表达量明显高于未转染组OECs的表达量,且hNT-3表达量与Dox浓度有依赖性。(3)与hNT-3修饰的OECs联合培养背根神经节(dorsal root ganglion,DRG),有许多DRG细胞向外迁移,这些细胞折光性强,突起较长而纤细,并形成复杂的网络。而未修饰的OECs组,只有少量的细胞迁移生长,细胞迁移和突起生长都很局限,空白对照组的DRG没有向外迁移的细胞和向外延伸的突起;且对照组的突起生长长度明显比NT-3修饰的实验组短(P<0.01)。这些结果表明,Tet-On调控NT-3表达在OECs转染成功,为进一步体内移植修复损伤提供了理想的材料。

EXPRESSION AND BIOLOGICAL ACTIVITY OF HUMAN NEUROTROPHIN-3 IN OLFACTORY ENSHEATHING CELLS MEDIATED BY RETROVIRAL VECTOR

We successfully constructed a retrovirus expression vector pRev-TRE-NT3. The supernatant with highest retroviral liters was obtained by transfection of two retovirus vector, pRev-TRE-NT3 and pRev-Tet-On, into an ecotropic Ecopack-293 cells. Primarily cultured rat olfactory ensheathing cells (OECs) were co-injected with retrovirus following pRev-TRE-NT-3 and pRev- Tet-On system of produced retrovirus. by adding different concentrations of Doxycline, genetically modified OECs were induced to secrete human N'F-3. The secreted NT-3 in OEC culture supernatant was detected with western-blot, and its biological activity was confirmed by the growth bioassay of dorsal root ganglion (DRG) cells. The results are as follows: (1)0. 78kb hNT-3 cDNA was harvested and successfully cloned into pRev-TRE vector by PCR and restriction enzyme digestion methods. The constructed plasmid was identified with electrophoresis, and the direction of hNT-3 cD-NA inserting pRev-TRE vector was integrated correctly; (2) NT-3-modified OEC culture supernatant contained 28kd N'F-3 which can bind NT-3 antibody in western-blot ting assay, and NT-3 expression was concentration-dependent on Doxycline. NT-3 was not detected in control groups; and (3) Numerous DRG cells migrated from DRG tissue mass in NT-3-modified OECs experimental groups, and these cells had long and thin processes with strong dioptre. These processes formed complicated networks. As for unmodified OEC control groups, a few DRG cells migrated from the tissue mass, and the processes of these cells were rather short. In blank groups, no cells migrated and grew. And their processes in control groups are significantly shorter than NT-3-modified OECs experimental groups. Our results indicate that Tet-On-regulated NT-3 expression in OECs was efficient, offering novel material of in vivo transplantation for the repair of CNS injury.