Boer G J, van Esseveldt K E, Hermens W T, Liu R, Verhaagen J
Graduate School Neurosciences Amsterdam, Netherlands Institute for Brain Research, Amsterdam.
Exp Neurol. 1997 Jun;145(2 Pt 1):536-45. doi: 10.1006/exnr.1997.6489.
Ex vivo gene transfer to fetal suprachiasmatic nucleus (SCN)-containing solid piece neurografts was explored using a first-generation prototype adenoviral vector containing the reporter gene LacZ (Ad-LacZ). Transgene expression was examined at different intervals following grafting in the IIIrd ventricle of rat brain and was compared to that of explant cultures. Large numbers of beta-galactosidase-positive cells were observed 8 days postgrafting. The number of stained cells had decreased considerably at 21 days but transduced cells were still present at 70 days. In vitro culturing of infected SCN tissue revealed high expression up to 21 days, indicating that the in vivo and in vitro fates of Ad-LacZ-infected cells were different. The main reason for this difference appeared to be cell loss by necrosis in the initial phase after transplantation, a phenomenon not related to the infection with Ad-LacZ since it similarly occurred in control grafts. In vivo inflammatory responses, observed after immunostaining for macrophages and T-lymphocytes, were also comparable in control and Ad-LacZ-treated transplants, except that cytotoxic T-cells were observed in the Ad-LacZ-treated transplants and not in controls. The recruitment of these cells was, however, minor and primarily observed at 8 days postgrafting, indicating that a major immunological rejection of the transduced graft did not occur. In both control and Ad-LacZ-infected transplants similar survival and intraimplant neuritic growth of SCN cells were visible. Ex vivo gene transfer of solid piece fetal SCN grafts with adenoviral vectors therefore appeared to be a nontoxic long-term gene-introducing procedure. This would in principle enable the local production of neurotrophic factors within the transplant and has the potential to improve functional SCN neurografting.
利用携带报告基因LacZ的第一代腺病毒载体原型（Ad-LacZ），探索了对含有胎儿视交叉上核（SCN）的实体块神经移植物进行离体基因转移。在将移植物植入大鼠脑第三脑室后的不同时间间隔检查转基因表达，并与外植体培养物进行比较。移植后8天观察到大量β-半乳糖苷酶阳性细胞。在21天时，染色细胞数量显著减少，但在70天时转导细胞仍然存在。对感染的SCN组织进行体外培养显示，其高表达可持续21天，这表明Ad-LacZ感染细胞在体内和体外的命运不同。这种差异的主要原因似乎是移植后初始阶段细胞因坏死而丢失，这一现象与Ad-LacZ感染无关，因为在对照移植物中也同样发生。对巨噬细胞和T淋巴细胞进行免疫染色后观察到的体内炎症反应，在对照移植和Ad-LacZ处理的移植中也相当，只是在Ad-LacZ处理的移植中观察到了细胞毒性T细胞，而在对照中未观察到。然而，这些细胞的募集很少，主要在移植后8天观察到，这表明未发生对转导移植物的主要免疫排斥反应。在对照移植和Ad-LacZ感染的移植中，都可见到SCN细胞有相似的存活和移植物内神经突生长。因此，用腺病毒载体对胎儿SCN实体块移植物进行离体基因转移似乎是一种无毒的长期基因导入程序。这原则上能够使移植体内局部产生神经营养因子，并有可能改善功能性SCN神经移植。
