Wu-Fienberg Yuewei, Moore Amy M, Marquardt Laura M, Newton Piyaraj, Johnson Philip J, Mackinnon Susan E, Sakiyama-Elbert Shelly E, Wood Matthew D
Division of Plastic and Reconstructive Surgery, Department of Surgery, Washington University School of Medicine, Campus Box 8238, 660 South Euclid Avenue, St. Louis, Missouri, 63110.
Biotechnol Bioeng. 2014 Sep;111(9):1886-94. doi: 10.1002/bit.25247. Epub 2014 Apr 24.
Glial cell-line-derived neurotrophic factor (GDNF) is a potent neurotrophic factor known to enhance motor nerve regeneration following its delivery. However, recent studies have determined that extended GDNF delivery to regenerating axons can entrap motor axons at the site of GDNF delivery. This entrapment leads to reduced motor axons available to reinnervate muscle. To address this issue, we designed a cell-based GDNF expression system that can temporally regulate protein expression using an inducible gene excision mechanism to prevent entrapment at the site of expression. To design this system for regulation of GDNF expression, we transduced two lentiviral vectors, one containing a constitutively active GDNF transgene flanked by two loxP sites, and the other containing a tetracycline-inducible cre transgene along with its constitutively active transactivator, into Schwann cells (SCs). These SCs over-express GDNF, but expression can be suppressed through the administration of tetracycline family antibiotics, such as doxycycline. The engineered SCs produced significantly more GDNF as compared to untransduced controls, as measured by enzyme-linked immunosorbent assay (ELISA). Following doxycycline treatment, these SCs produced significantly lower levels of GDNF and induced less neurite extension as compared to untreated SCs. Engineered SCs treated with doxycycline showed a marked increase in Cre recombinase expression, as visualized by immunohistochemistry (IHC), providing evidence of a mechanism for the observed changes in GDNF expression levels and biological activity. This cell-based GDNF expression system could have potential for future in vivo studies to provide a temporally controlled GDNF source to promote axon growth.
胶质细胞源性神经营养因子（GDNF）是一种强效神经营养因子，已知其在递送后可促进运动神经再生。然而，最近的研究表明，向再生轴突持续递送GDNF会使运动轴突被困在GDNF递送部位。这种被困导致可用于重新支配肌肉的运动轴突减少。为了解决这个问题，我们设计了一种基于细胞的GDNF表达系统，该系统可以利用诱导型基因切除机制在时间上调节蛋白质表达，以防止在表达部位被困。为了设计这个调节GDNF表达的系统，我们将两种慢病毒载体转导到雪旺细胞（SCs）中，一种载体含有一个由两个loxP位点侧翼的组成型活性GDNF转基因，另一种载体含有一个四环素诱导型cre转基因及其组成型活性反式激活因子。这些SCs过度表达GDNF，但通过施用四环素类抗生素（如强力霉素）可以抑制表达。通过酶联免疫吸附测定（ELISA）测量，与未转导的对照相比，工程化SCs产生的GDNF明显更多。在用强力霉素处理后，与未处理的SCs相比，这些SCs产生的GDNF水平显著降低，并且诱导的神经突延伸更少。用强力霉素处理的工程化SCs通过免疫组织化学（IHC）观察到Cre重组酶表达显著增加，这为观察到的GDNF表达水平和生物学活性变化提供了一种机制的证据。这种基于细胞的GDNF表达系统可能对未来的体内研究具有潜力，以提供一个时间可控的GDNF来源来促进轴突生长。
