Development of catecholamine producing cells by the transfection of CV40 TS gene and its application to intracranial neural transplantation.

1.将SV40T/TS基因导入胎儿或新生儿中脑(MS)细胞、纹状体(STR)细胞和肾上腺(AD)细胞，在融合的2个细胞上培养或在含有SV40T/TS逆转录病毒颗粒的2个细胞条件培养液中培养。在培养液中加入EGF、成纤维细胞生长因子或纹状体细胞条件培养液时，转基因细胞生长较好。在标准或浓缩培养液中培养4周后，在含有G418的培养液中培养，筛选出8个G418抗性细胞(MS-1、2、3；STR-1、2；AD-1、2、3)。它们在33℃的温度下生长。C并在39岁以下停止生长。C，具有热敏特性。倍增时间约为4周。仅MS-1和AD-1对酪氨酸羟化酶(TH)呈阳性反应。MS-2、3和STR-1、2对神经丝和GFAP均呈免疫阳性反应。在体外生长后，将MS-1和AD-1移植到帕金森病动物模型的纹状体内，将STR-1、2移植到脑缺血动物模型的纹状体内。每个细胞在宿主大脑中存活了两周。然而，4周后，他们变得不健康，最终消失了。因此，它们不能长期存活，从而能够进行功能恢复。移植的细胞为什么不能在宿主大脑中长期存活的原因尚不清楚。各种微环境因素调节着移植细胞的存活。热敏感的特性可能是细胞不能在脑内长期存活的原因之一，因此寻找一种内源性启动子使转基因基因能够长期表达是迫切需要解决的问题。

1. Fetal or neonatal mesencephalic (MS) cells, striatal (STR) cells and adrenal (AD) cells were transfected with SV40T/TS gene by culturing on the confluent 2 cells or culturing in the conditioned medium of 2 cells that have retroviral particles containing SV40T/TS.2. Transfected cells grew better when EGF, FGF or conditioned medium of striatal cells were added in the culture medium.3. After the growing period for 4 weeks in the standard or enriched medium, the transfected cells were cultured in the medium that contains G418 and eight G418-resistant cells were selected (MS-1, 2, 3 ; STR-1, 2 ; AD-1, 2, 3).4. They grew under the temperature of 33。C and stopped growing under 39。C, thus they had thermosensitive character. The doubling time was approximately 4 weeks.5. Only MS-1 and AD-1 were immunoreactive for tyrosine hydroxylase (TH). MS-2, 3 and STR-1, 2 were immunoreactive for both neurofilament and GFAP.6. After growing in vitro, MS-1 and AD-1 were transplanted in the striatum of an animal model of Parkinson's disease, and STR-1, 2 were transplanted in the striatum of an animal model of brain ischemia. Each cells survived in the host brain for two weeks. However, after 4 weeks they became unhealthy and finally disappeared. Thus, they could not survive for a long perid that enables the functional restoration.The reason why the grafted cells can not survive for a long period in the host brain is not clear yet. Various micro-environmental factors modulate the survival of the grafted cells. The characteristic of thermosensitivity might be a reason for that the cells cannot survive for a long period in the brain.Thus, it is an urgent problem to find an endogenous promoter that enables the expression of transfected genes for a long period.