藻酸钠-多聚赖氨酸-藻酸钠微囊化牛视网膜色素上皮细胞多巴胺分泌特性的研究

目的 检测体外培养的牛视网膜色素上皮（RPE）细胞多巴胺分泌特点，并观察多次传代及微囊包裹对细胞存活率和多巴胺分泌的影响。方法 酶消化法原代培养牛RPE细胞。纯化、鉴定后观察细胞的生长曲线。用高压静电成囊装置制备藻酸钠-多聚赖氨酸-藻酸钠（APA）微囊化细胞。台盼蓝染色法检测囊内细胞存活率。高效液相色谱电化学法检测多巴胺含量。结果 生长曲线显示第1～4天为其指数生长期。多巴胺分泌在2h开始检测到，48h达高峰以后缓慢下降并趋稳定。多次传代后多巴胺分泌量差异无统计学意义。微囊化细胞多巴胺分泌量在第1～4天与非微囊化组比较显著降低（P〈0．01），但在第5～8天差异无统计学意义。微囊内细胞总数在观察的21d内逐渐增加，虽然囊内存活率逐渐降低，但囊内活细胞数及多巴胺分泌量在第14、21天趋于稳定。结论 牛RPE细胞体外生长旺盛，易于纯化和传代。它能够持续分泌多巴胺且不受多次传代及微囊包裹的影响。体外培养21d后仍检测到囊内细胞的存活及多巴胺的分泌。牛RPE细胞是一种具有一定前景的帕金森病细胞移植的供体。

The study on the features of dopamine secretion in microencapsulated cells of bovine retinal pigmentary epithelial cells in vitro

ObjectiveTo detect the features of dopamine secretion of cultured bovine retinal pigmentary epithelial(RPE) cells. The level of dopamine and survival rate after passage and microencapsulation were also observed. MethodsThe primary culture of bovine RPE cells was made using enzyme digestion. After purification and identification the growth curve of the cell was observed. Alginate-polylysine-alginate(APA) microencapsulated cells were made with a high voltage electostatic system. The activity of the cells in the mirocapsule was investigated by trypan blue staining. The secretion of dopamine was determined by high performance liquid chromatography (HPLC) assay. ResultsThe cell had high purity of immunocytochemistry. The growth curve showed that the exponential growth occurred at the first 1~4 days. Dopamine content was first detected at the time point of 2 hour, and arrived at the peak at about 48 hour of the cultivation. The secretion of dopamine was not different between passages. Dopamine secretion was dramatically decreased in the first 4 days after microencapsulation (P<0.01) ,but showed no difference at the time point of 5~8 days between the two groups. Though the survival rate of the cells in microcapsules was decreased gradually (P<0.05), the sum of the living cell and dopamine secretion all went to a stable level at the period of observational time. ConclusionsBovine RPE cells grow actively in vitro, and were easy to be purified. It can secret dopamine constantly. The passage culture and the APA miroencapsulation do not affect the process. Bovine RPE cells are a prospect source in cell transplantation therapy of Parkinson's disease.