Department of Neurosurgery, University of Kentucky College of Medicine, Lexington, KY 40536-0305, USA.
Cell Transplant. 2009;18(10):1183-96. doi: 10.3727/096368909X12483162196881. Epub 2009 Jun 22.
Previously it was established that infusion of glial cell line-derived neurotrophic factor (GDNF) protein into grafts of embryonic dopamine cells has a neurotrophic effect on the grafted cells. In this study we used a nonviral technique to transfer the gene encoding for GDNF to striatal cells. Plasmid DNA encoding for GDNF was compacted into DNA nanoparticles (DNPs) by 10 kDa polyethylene glycol (PEG)-substituted lysine 30-mers (CK(30)PEG10k) and then injected into the denervated striatum of rats with unilateral 6-hydroxydopamine lesions. Sham controls were injected with saline. One week later, experimental animals received either a ventral mesencephalic (VM) tissue chunk graft or a cell suspension VM graft implanted into the denervated striatum. Grafts were allowed to integrate for 4-6 weeks and during this period we monitored spontaneous and drug-induced motor activity. Using stereological cell counting we observed a 16-fold increase in the number of surviving TH(+) cells within tissue chunk grafts placed into the striatum pretreated with pGDNF DNPs (14,923 +/- 4,326) when compared to grafts placed into striatum pretreated with saline (955 +/- 343). Similarly, we observed a sevenfold increase in the number of TH(+) cells within cell suspension grafts placed into the striatum treated with pGDNF DNPs when compared to cell suspension grafts placed into the saline dosed striatum. Behaviorally, we observed significant improvement in rotational scores and in spontaneous forepaw usage of the affected forelimb in grafted animals receiving prior treatment with compacted pGDNF DNPs when compared to grafted animals receiving saline control pretreatment. Data analysis for protein, morphological, and behavioral measures suggests that compacted pGDNF DNPs injected into the striatum can result in transfected cells overexpressing GDNF protein at levels that provide neurotrophic support for grafted embryonic dopamine neurons.
先前的研究已经证实，胶质细胞源性神经营养因子（GDNF）蛋白的输注对胚胎多巴胺细胞移植的细胞具有神经营养作用。在这项研究中，我们使用非病毒技术将编码 GDNF 的基因转移到纹状体细胞中。编码 GDNF 的质粒 DNA 通过 10 kDa 聚乙二醇（PEG）取代的赖氨酸 30 聚体（CK（30）PEG10k）被压缩成 DNA 纳米颗粒（DNPs），然后注入单侧 6-羟多巴胺损伤的大鼠去神经纹状体。假手术对照注射生理盐水。一周后，实验组动物接受腹侧中脑（VM）组织块移植物或植入去神经纹状体的 VM 细胞悬浮液移植物。移植物允许整合 4-6 周，在此期间我们监测自发和药物诱导的运动活动。使用立体学细胞计数，我们观察到在预先用 pGDNF DNPs 预处理的纹状体中放置组织块移植物的存活 TH（+）细胞数量增加了 16 倍（14923+/-4326），与预先用盐水预处理的纹状体中放置的移植物相比（955+/-343）。同样，我们观察到在预先用 pGDNF DNPs 处理的纹状体中放置细胞悬浮液移植物的 TH（+）细胞数量增加了 7 倍，与在盐水处理的纹状体中放置的细胞悬浮液移植物相比。行为上，与接受盐水对照预处理的移植物动物相比，接受预先用 compacted pGDNF DNPs 处理的移植物动物的旋转评分和受影响前肢的自发前爪使用明显改善。对蛋白质、形态和行为测量的数据进行分析表明，注入纹状体的 compacted pGDNF DNPs 可导致转染细胞过度表达 GDNF 蛋白，为移植的胚胎多巴胺神经元提供神经营养支持。
