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

1.通过在汇合的2细胞上培养或在具有含SV 40 T/TS的逆转录病毒颗粒的2细胞的条件培养基中培养，用SV 40 T/TS基因转染胎儿或新生儿中脑（MS）细胞、纹状体（STR）细胞和肾上腺（AD）细胞。培养基中加入EGF、FGF或纹状体细胞条件培养液，转染细胞生长良好.在标准或富集培养基中生长4周后，将转染的细胞在含有G418的培养基中培养，并选择8个G418抗性细胞（MS-1，2，3 ; STR-1，2 ; AD-1，2，3）。它们在33 ℃下生长。C，并在39岁以下停止生长。C，具有温敏性。倍增时间约为4周。只有MS-1和AD-1的酪氨酸羟化酶（TH）的免疫反应。MS-2、3和STR-1、2均为神经丝和GFAP免疫反应阳性。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中，并将STR-1、2移植到脑缺血动物模型的纹状体中。每个细胞在宿主脑中存活两周。然而，4周后，他们变得不健康，最后消失了。移植细胞不能在宿主脑内长期存活的原因尚不清楚。各种微环境因素调节移植细胞的存活。细胞的温敏特性可能是导致其不能在脑内长期存活的原因之一，因此寻找一种能够使转染基因长期表达的内源性启动子是一个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.