TROPHIC ALTERATIONS IN DOPAMINE NEURON TRANSPLANTATION

早期临床试验的结果表明，胎儿黑质移植 可能提供了一种令人兴奋的新的治疗策略 帕金森氏病(PD)。然而，与储存相关的组织损失 移植前流产的胎儿组织，知识不足 关于患者年龄和/或疾病阶段对移植物存活率的影响， 而缺乏持续的移植组织来源是 在影响原本直截了当的外科手术的因素中 程序。我们实验室最近的研究表明，多巴胺(DA) “预适应”多巴胺能神经元可提高神经元存活率 储存过程中的营养分子。我们还证明了营养活性 在大鼠的纹状体中，随着年龄的增长而减少，并成正比增加 到DA去神经的程度。这些数据表明，患者的年龄和 帕金森病期可能对营养有显著影响 移植组织被移植到的环境。在特定目标中1 因此，我们将系统地评估补充的效果 含有营养分子(碱性成纤维细胞生长因子、脑源性神经营养因子、胶质细胞源性神经营养因子、表皮生长因子)、抗氧化剂的储存介质 (U83836E和U74389G)和组织提取物(纹状体、中脑、 胎盘血清)。在此条件下，大鼠中脑可保存5天 然后在这些条件下与纹状体细胞共培养。DA神经元 在这些培养物中的存活率将通过评估K+诱发的DA来评估 释放和酪氨酸羟化酶免疫反应(TIR)细胞计数。 将进一步评估最佳存储条件，方法是植入 “增强”细胞进入单侧损毁(6OHDA)大鼠。具体目标2 将重点关注移植受者年龄和DA程度的影响 去神经对移植物存活率有影响。增强的细胞将被移植到 2月龄、9月龄、17月龄和22月龄大鼠的纹状体和Thir细胞计数 植入部位将在不同组之间进行比较。使用脑室内 6OHDA模型，17月龄大鼠双侧失神经35，50，75， 90%，移植了增强的细胞。THIR细胞计数将再次 在不同的群体之间进行比较。尽管优化了胎儿组织存储 条件并确定DA失神经的年龄和程度 最大化DA神经元的存活率可能会立即产生积极的影响 对目前正在进行的移植计划的影响，这些目标不 解决有关获取连续的DA神经元来源的问题 丰富的组织。在这方面，我们最近表明，一种自然的， 多能祖细胞系可从胎儿和成人中获得 中脑。新鲜中脑细胞转化后的共培养 其中许多细胞进入DA神经元表型，而已知的营养 但因素并不是这样。这些文化中的许多都高度丰富了 细胞。因此，具体目标3将重点放在未来对 多能祖细胞作为连续传代细胞；2-优化和 鉴定和鉴定中脑的粗制成分 负责将其转化为DA表型；以及3-嫁接 60OHDA损伤大鼠的祖细胞。成功地实施了 该提案的具体目标涉及以下几个实际方面 可以很容易地移植到临床环境中。

Results form early clinical trials suggest that fetal nigral transplants may offer an exciting new therapeutic strategy for the treatment of Parkinson's disease (PD). However, tissue loss associated with the storage of aborted fetal tissue prior to transplantation, inadequate knowledge about the impact of patient age and/or stage of disease on graft survival, and the lack of a continuous source of tissue for transplantation are among the factors that compromises an otherwise straight forward surgical procedure. Recent work in our laboratory suggests that dopamine (DA) neuron survival can be increased by "preconditioning" DA neurons with trophic molecules during storage. We have also shown that trophic activity in the rat striatum is reduced by aging and increased in direct proportion to the degree of DA denervation. These data suggest that patient age and Parkinsonian stage may have a significant impact on the trophic environment into which grafted tissue is transplanted. In Specific Aim 1 we will therefore systematically evaluate the effect of supplementing storage media with trophic molecules (bFGF, BDNF, GDNF, EGF), antioxidants (U83836E and U74389G), and tissue extracts (striatal, mesencephalic, placental serum). Rat mesencephalon will be stored for up to 5 days under these conditions and then co-cultured with striatal cells. DA neuron survival in these cultures will be evaluated by assessing K+-evoked DA release and tyrosine hydroxylase immunoreactive (THir) cell counts. Optimal storage conditions will be further evaluated by implanting the "enhanced" cells into unilaterally lesioned (6OHDA) rats. Specific Aim 2 will focus on the effects the transplant recipients age and degree of DA denervation has on graft survival. Enhanced cells will be implanted into the striata of 2,9, 17, and 22 month old rats and THir cell counts in the implant site will be compared across groups. Using the intraventricular 6OHDA model, 17 month old rats will be bilaterally denervated 35,50,75, and 90% and transplanted with enhanced cells. THir cell counts will again be compared across groups. Although optimizing fetal tissue storage conditions and identifying the age and degree of DA denervation that maximizes DA neuron survival is likely to have an immediate, positive impact on transplant programs currently under way, these Aims do not address the issues surrounding access to a continuous source of DA neuron enriched tissue. In this regard, we have recently shown that a natural, pluripotent, progenitor cell line can be derived from fetal and adult mesencephalon. Co-culture with freshly harvested mesencephalon converted many of these cells into the DA neuron phenotype whereas known trophic factors did not. Many of these cultures were highly enriched with THir cells. Specific Aim 3 will therefore focus on the future use of the pluripotent progenitor cell as a continuous passage; 2-optimizing and identifying and identifying the crude components of the mesencephalon responsible for its conversion to the DA phenotype; and 3- grafting the progenitor cells in 6OHDA lesioned rats. The successful implementation of the Specific Aims of this proposal addresses several practical aspects of transplantation that could be readily transferred to the clinical setting.