Eaton M J, Frydel B R, Lopez T L, Nie X T, Huang J, McKillop J, Sagen J
The Miami Project To Cure Paralysis, University of Miami School of Medicine, Miami, Florida 33136, USA.
J Cell Biochem. 2000 Jul 19;79(1):38-57.
Adrenal chromaffin cells have been successfully used to attenuate chronic pain when transplanted near the spinal cord, but primary cells are neither homogeneous nor practical for routine use in human therapy. Conditional immortalization with the temperature-sensitive allele of the large T antigen (tsTag) and creation of stable chromaffin cell lines would advance our understanding of both the use and limits of cell lines that contain this immortalization gene for such therapies. Cultures of embryonic day 17 rat adrenal and neonatal bovine adrenal cells were immortalized with the temperature-sensitive allele of SV40 tsTag and chromaffin cell lines established. The rat chromaffin line, RAD5.2, and the bovine chromaffin cell line, BADA.20, both expressed immunoreactivities (ir) for all the catecholamine enzymes: tyrosine hydroxylase (TH), the first enzyme in the synthetic pathway for catecholamines, dopa-beta-hydroxylase (DbetaH), and phenylethanolamine-N-methyltransferase (PNMT). At permissive temperature (33 degrees C), these chromaffin cells are proliferative, have a typical rounded chromaffinlike morphology, and contain detectable TH-, DbetaH-, and PNMT-ir. At nonpermissive temperature (39 degrees C), these cells stop proliferating, decrease Tag expression, and change the expression of TH-, DbetaH-, and PNMT-ir in vitro, suggesting increased differentiation at nonpermissive temperature. The chromaffin cell lines also express immunoreactivity for the opioid met-enkephalin (ENK) at permissive and nonpermissive temperatures. The expression of TH-ir in the bovine chromaffin cells is upregulated by the addition of dexamethasone (DEX) or forskolin during differentiation; TH-ir is not affected by the addition of DEX or forskolin in the rat chromaffin cells. The addition of forskolin during differentiation upregulates the expression of DbetaH-ir in the rat chromaffin cells. PNMT-ir is not affected by differentiation or agents in either cell line. However, catecholamine synthesis was not detectable by high-performance liquid chromatography, suggesting incomplete differentiation under current conditions, or influence by continued low levels of Tag expression. Both cell lines have been carried over many passages in vitro for more than 3 years and were repeatedly frozen and thawed. These data describe an initial step in the conditional immortalization of chromaffin cells that can maintain the phenotype of primary chromaffin cells in vitro over long periods. The use of such chromaffin cell lines that are able to deliver neuroactive molecules offers a novel approach to pain management.
肾上腺嗜铬细胞移植到脊髓附近已成功用于减轻慢性疼痛，但原代细胞既不均匀，也不适合在人类治疗中常规使用。利用大T抗原（tsTag）的温度敏感等位基因进行条件永生化并创建稳定的嗜铬细胞系，将有助于我们了解含有这种永生化基因的细胞系在这类治疗中的用途和局限性。用SV40 tsTag的温度敏感等位基因使胚胎第17天大鼠肾上腺细胞和新生牛肾上腺细胞永生化，并建立嗜铬细胞系。大鼠嗜铬细胞系RAD5.2和牛嗜铬细胞系BADA.20均对所有儿茶酚胺合成酶表现出免疫反应性（ir）：酪氨酸羟化酶（TH），儿茶酚胺合成途径中的第一种酶、多巴-β-羟化酶（DβH）和苯乙醇胺-N-甲基转移酶（PNMT）。在允许温度（33℃）下，这些嗜铬细胞具有增殖能力，具有典型的圆形嗜铬样形态，并含有可检测到的TH-ir、DβH-ir和PNMT-ir。在非允许温度（39℃）下，这些细胞停止增殖，Tag表达降低，并在体外改变TH-ir、DβH-ir和PNMT-ir的表达，表明在非允许温度下分化增加。嗜铬细胞系在允许温度和非允许温度下均对阿片样物质甲硫氨酸脑啡肽（ENK）表现出免疫反应性。在分化过程中添加地塞米松（DEX）或福斯高林可上调牛嗜铬细胞中TH-ir的表达；在大鼠嗜铬细胞中，添加DEX或福斯高林对TH-ir无影响。在分化过程中添加福斯高林可上调大鼠嗜铬细胞中DβH-ir的表达。在任一细胞系中，PNMT-ir均不受分化或试剂的影响。然而，通过高效液相色谱法未检测到儿茶酚胺合成产物，这表明在当前条件下分化不完全，或受Tag持续低水平表达的影响。这两种细胞系在体外已传代许多次，历时3年多，并反复冻融。这些数据描述了嗜铬细胞条件永生化的初步步骤，该步骤可在体外长期维持原代嗜铬细胞的表型。使用这种能够递送神经活性分子的嗜铬细胞系为疼痛管理提供了一种新方法。
