Smith G M, Hale J, Pasnikowski E M, Lindsay R M, Wong V, Rudge J S
Department of Anesthesiology and Pain Management, University of Texas Southwestern Medical School, Dallas 75235, USA.
Exp Neurol. 1996 May;139(1):156-66. doi: 10.1006/exnr.1996.0090.
Neurotrophic factors have been shown to ameliorate neuronal death in several in vitro and in vivo models of neurodegenerative disease. However, delivery of polypeptide growth factors to compromised neurons in the CNS is problematic as the blood-brain barrier prevents systemic delivery, and chronic in-dwelling cannulae are required for intraparenchymal delivery. To circumvent these problems and specifically target neurotrophic factors to the environment surrounding degenerating neurons in the CNS, we have generated replication-defective adenovirus (Ad) vectors that contain a secretable form of ciliary neurotrophic factor (sCNTF) or neurotrophin-3 (NT-3). In this study, we demonstrate that sCNTF/Ad and NT-3/Ad can efficiently infect primary astrocytes, resulting in gene transcription and the production of functional protein. Using Northern blot analysis, dose-dependent expression of sCNTF or NT-3 mRNA was detected 7 days after infection. The levels of mRNA expressed in transgenic astrocytes was dependent on virus titer and increased with increasing virus concentration. sCNTF or NT-3 protein was also detected in astrocyte supernatants by immunoblot analysis and 2-site ELISA. ELISA indicated that astrocytes infected with sCNTF/Ad or NT-3/Ad secreted neurotrophic factors at a rate of approximately 120 pg/10(6) cells/h and 350 pg/10(6) cells/h, respectively. To test for secretion of bioactive sCNTF or NT-3 protein, E8 chick ciliary ganglion or nodose ganglion neurons were grown in medium conditioned by control astrocytes or astrocytes treated with sCNTF/Ad or NT-3/Ad, showing a robust and dose-dependent increase in neuronal survival when compared to control supernatant. In addition, motor neurons plated onto astrocyte monolayers pretreated with sCNTF/Ad showed a two- to fourfold increase in ChAT activity when compared to those grown on astrocytes pretreated with Lac-Z/Ad. This study demonstrates that, using replication-defective adenovirus, primary astrocytes can be efficiently engineered to secrete bioactive sCNTF or NT-3, resulting in enhanced survival of responsive peripheral and central neuronal populations.
神经营养因子已被证明能在多种神经退行性疾病的体外和体内模型中改善神经元死亡。然而，将多肽生长因子递送至中枢神经系统中受损的神经元存在问题，因为血脑屏障会阻碍全身递送，而实质内递送则需要长期留置套管。为了规避这些问题，并将神经营养因子特异性地靶向中枢神经系统中退化神经元周围的环境，我们构建了复制缺陷型腺病毒（Ad）载体，其包含可分泌形式的睫状神经营养因子（sCNTF）或神经营养素-3（NT-3）。在本研究中，我们证明sCNTF/Ad和NT-3/Ad能够有效感染原代星形胶质细胞，从而导致基因转录并产生功能性蛋白。通过Northern印迹分析，在感染后7天检测到sCNTF或NT-3 mRNA的剂量依赖性表达。转基因星形胶质细胞中表达的mRNA水平取决于病毒滴度，并随病毒浓度的增加而升高。通过免疫印迹分析和双位点ELISA也在星形胶质细胞上清液中检测到了sCNTF或NT-3蛋白。ELISA表明，感染sCNTF/Ad或NT-3/Ad的星形胶质细胞分别以约120 pg/10(6)细胞/小时和350 pg/10(6)细胞/小时的速率分泌神经营养因子。为了测试生物活性sCNTF或NT-3蛋白的分泌情况，将E8鸡睫状神经节或结状神经节神经元培养在由对照星形胶质细胞或经sCNTF/Ad或NT-3/Ad处理的星形胶质细胞条件化的培养基中，与对照上清液相比，神经元存活率显示出强劲且剂量依赖性的增加。此外，与在经Lac-Z/Ad预处理的星形胶质细胞上生长的运动神经元相比，接种到经sCNTF/Ad预处理的星形胶质细胞单层上的运动神经元的ChAT活性增加了两到四倍。本研究表明，使用复制缺陷型腺病毒，可以有效地改造原代星形胶质细胞以分泌生物活性sCNTF或NT-3，从而提高反应性外周和中枢神经元群体 的存活率。
