微囊化牛视网膜色素上皮细胞体外BDNF及GDNF分泌特性的研究

Features of BDNF and GDNF secretion in microencapsulated cells of bovine retinal pigment epithelium in vitro

目的检测体外培养的牛视网膜色素上皮细胞（retinal pigment epithelium，RPE）脑源性神经营养因子（brain derived neurotrophic factor，BDNF）及胶质源性神经营养因子（glial cell line-derived neurotrophic factor，GDNF）分泌特点，并观察微囊包裹对细胞存活率及BDNF和GDNF分泌的影响。方法原代培养牛RPE，用高压静电成囊装置制备海藻酸钠-多聚赖氨酸-海藻酸钠微囊化细胞，台盼兰染色法检测囊内细胞活性，ELISA法检测BDNF及GDNF分泌量。结果原代及传代后RPE上清液中在基础状态下就能够检测到BDNF和GDNF，传代后两者的分泌量较原代无显著差异。微囊化细胞BDNF和GDNF分泌量在1-5 d与未微囊化细胞相比显著降低（P〈0.01），而6-8 d两组无显著差异。囊内活细胞数及BDNF和GDNF分泌量在体外培养的第14天至第21天趋于稳定。结论RPE基础状态下就能够分泌少量BDNF和GDNF，且不受传代及微囊包裹的影响。体外培养21 d后仍检测到囊内细胞的存活及BDNF和GDNF的分泌。牛RPE是一种具有一定前景的帕金森病细胞移植的供体。

Objective To detect the features of brain derived neurotrophic factor（BDNF） and Glial cell linederived neurotrophic factor（GDNF） secretion of cultured bovine retinal pigment epithelium（RPE）. The levels of BDNF and GDNF and survival rate after passage and microencapsulation were also observed. Methods The primary culture of bovine RPE was cultured after enzyme digestion. 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 BDNF and GDNF were detected by ELISA technique. Results The secretion of BDNF and GDNF were detected in supernatant under basal condition in primary and passage cells. The secretion of these substances had no difference between primary cells and passages. BDNF and GDNF secretions were dramatically decreased after microencapsulation in the first 5 days（ P 〈 0.01 ） ,but showed no difference at the time point of 6 -8 days between two groups. The sum of living cells in microcapsules and BDNF and GDNF secretion went to a stable level at the period of observational time. Conclusion Bovine RPE can secrete BDNF and GDNF under basal condition in vitro while the passage culture and the APA miroencapsulation do not affect this process. The secretion process and the survival of the cell can last for 21 days in the mirocapsule after culture in vitro. Bovine RPE is a prospect source in cell transplantation therapy for Parkinson＇ s disease.