Castillo B, del Cerro M, Breakefield X O, Frim D M, Barnstable C J, Dean D O, Bohn M C
Department of Neurobiology and Anatomy, University of Rochester Medical Center, NY 14642.
Brain Res. 1994 May 30;647(1):30-6. doi: 10.1016/0006-8993(94)91395-1.
Genetically engineered cells carrying genes for neurotrophic factors have potential application for treatment of neurodegenerative diseases and injuries to the nervous system. Brain-derived neurotrophic factor (BDNF) promotes the survival of specific neurons, including retinal ganglion cells (RGC). To determine whether genetically engineered astrocytes might be used for delivering bioactive BDNF, we infected primary type 1 rat astrocytes with a retrovirus harboring a human prepro-BDNF cDNA and assayed the medium conditioned by these astrocytes for effects on survival of rat RGCs in vitro. High levels of BDNF mRNA were expressed by infected astrocytes, but not by control astrocytes as determined by RNase protection assay using a BDNF specific probe. To test for secretion of bioactive BDNF from the transgenic astrocytes, embryonic day 17 rat retinas were dissociated and grown in medium conditioned (CM) for 24 h by astrocytes infected with a replication deficient retrovirus carrying BDNF, NGF, or alkaline phosphatase (AP) cDNA. After 3 days, the number of Thy-1 immunoreactive RGCs was counted. BDNF astrocyte CM significantly enhanced RGC survival by 15-fold compared to the AP control. NGF astrocyte CM had no significant effect. The rate of BDNF secretion was estimated at 83-166 pg/10(5) cells/h. This study demonstrates that astrocytes can be genetically engineered to synthesize and secrete bioactive BDNF. These techniques may be applicable to rescuing neurons from degenerative processes and also for enhancing their survival following transplantation.
携带神经营养因子基因的基因工程细胞在治疗神经退行性疾病和神经系统损伤方面具有潜在应用价值。脑源性神经营养因子（BDNF）可促进特定神经元的存活，包括视网膜神经节细胞（RGC）。为了确定基因工程改造的星形胶质细胞是否可用于递送具有生物活性的BDNF，我们用携带人前体BDNF cDNA的逆转录病毒感染原代1型大鼠星形胶质细胞，并检测这些星形胶质细胞条件培养基对体外大鼠RGC存活的影响。通过使用BDNF特异性探针的核糖核酸酶保护试验确定，感染的星形胶质细胞表达高水平的BDNF mRNA，而对照星形胶质细胞则不表达。为了测试转基因星形胶质细胞分泌具有生物活性的BDNF，将胚胎第17天的大鼠视网膜解离，并在携带BDNF、NGF或碱性磷酸酶（AP）cDNA的复制缺陷型逆转录病毒感染的星形胶质细胞条件培养基（CM）中培养24小时。3天后，对Thy-1免疫反应性RGC的数量进行计数。与AP对照相比，BDNF星形胶质细胞CM显著提高RGC存活率15倍。NGF星形胶质细胞CM没有显著影响。估计BDNF的分泌速率为83 - 166 pg/10(5)细胞/小时。这项研究表明，星形胶质细胞可以通过基因工程改造来合成和分泌具有生物活性的BDNF。这些技术可能适用于从退行性过程中拯救神经元，也适用于提高其移植后的存活率。
