Generation of DOPA-Producing Astrocytes by Retroviral Transduction of the Human Tyrosine Hydroxylase Gene:In VitroCharacterization andin VivoEffects in the Rat Parkinson Model
Generation of DOPA-Producing Astrocytes by Retroviral Transduction of the Human Tyrosine Hydroxylase Gene:In VitroCharacterization andin VivoEffects in the Rat Parkinson Model
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DOI：
10.1006/exnr.1996.0079
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发表时间：
1996-05
影响因子：
5.3
通讯作者：
C. Lundberg;P. Horellou;J. Mallet;A. Björklund
中科院分区：
文献类型：
--
作者：
C. Lundberg;P. Horellou;J. Mallet;A. Björklund
通过逆转录病毒介导的人酪氨酸羟化酶（TH）基因转移，已经产生了分泌高水平L-3，4-二羟基苯丙氨酸（DOPA）的星形胶质细胞。从出生前大鼠脑中获得的未成熟星形胶质细胞与TH病毒产生的psi-2细胞共培养，所述细胞已经用有丝分裂抑制剂丝裂霉素-C预处理。在共培养的第一周期间，多巴产量逐渐增加，在7-9天后达到平台。在这个时间点，几乎所有的细胞都是GFAP阳性的，超过80%的细胞表达TH。在转导的星形胶质细胞的多巴生产在很大程度上是独立的外源性辅因子，多巴释放到培养基中不受影响，无论是KCl或河豚毒素或从培养基中除去Ca 2+，表明新合成的多巴组成释放的细胞。TH-转导的星形胶质细胞移植到单侧6-羟基多巴胺损伤大鼠的纹状体，在移植后2周，阿扑吗啡诱导的转向减少了约50%。显微镜分析显示，转导的星形胶质细胞在移植后存活良好，并且一些移植的细胞已经迁移出来，部分沿着血管进入周围的纹状体。TH表达的细胞中观察到的成熟GFAP阳性星形胶质细胞的外观，以及在更不成熟的外观细胞。然而，只有百分之几的所有移植细胞保持显着表达的转基因，TH免疫组织化学测定。结果表明，原代星形胶质细胞可能是非常有用的基因载体，在中枢神经系统的离体基因治疗。随着基因转导程序的进一步改进，TH转基因在体内的表达更有效、更持久，基因工程化的产生多巴的星形胶质细胞作为一种工具有很大的希望，以探索帕金森病的离体基因治疗的潜力。
Astrocytes secreting high levels of L-3,4-dihydroxyphenylalanine (DOPA) have been generated by retrovirus-mediated transfer of the human tyrosine hydroxylase (TH) gene. Immature astrocytes obtained from prenatal rat brain were cocultured with TH virus producing psi-2 cells that had been pretreated with the mitosis inhibitor mitomycin-C. During the first week of coculture DOPA production gradually increased to reach a plateau after 7-9 days. At this time point virtually all cells were GFAP positive and over 80% of them expressed TH. DOPA production in the transduced astrocytes was largely independent of exogenous cofactor, and DOPA release into the medium was not influenced by addition of either KCl or tetrodotoxin or by removal of Ca2+ from the culture medium, indicating that the newly synthesized DOPA was constitutively released from the cells. Transplantation of the TH-transduced astrocytes to the striatum in unilaterally 6-hydroxydopamine lesioned rats reduced apomorphine-induced turning by about 50% at 2 weeks postgrafting. Microscopic analysis revealed that the transduced astrocytes survived very well after transplantation and that some of the grafted cells had migrated out, partly along blood vessels, into the surrounding striatum. TH expression was observed in cells with both the appearance of mature GFAP-positive astrocytes, as well as in more immature-looking cells. However, only a few percent of all transplanted cells maintained significant expression of the transgene, as determined by TH immuno-histochemistry. The results show that primary astrocytes may be highly useful as gene carriers for ex vivo gene therapy in the CNS. With future improvement in the gene transduction procedure for more efficient, sustained expression of the TH transgene in vivo, genetically engineered DOPA-producing astrocytes hold great promise as a tool to explore the potential of ex vivo gene therapy in Parkinson's disease.