微囊化牛嗜铬细胞体外培养的研究

RESEARCH INTO THE MICROENCAPSULATED CULTIVATION OF PHEOCHROMOCYTE BOVIS IN VITRO

目的观察体外培养8天的微囊化牛嗜铬细胞的生长情况、培养液中去甲肾上腺素、甲-脑啡呔水平以及经乙酰胆碱刺激后,去甲肾上腺素和甲-脑啡呔水平的变化情况,以选择细胞移植的适宜时机。方法取8条健康成年牛的双侧肾上腺,经胶原酶消化成单细胞,APA微囊包裹后,体外培养8天,观察细胞的生长情况;放免法检测培养液中去甲肾上腺素、甲-脑啡呔的含量;培养第4天,取细胞悬液5份,分别加入不同浓度的乙酰胆碱予以刺激,检测其中去甲肾上腺素、甲-脑啡呔水平。结果①培养第4天的微囊化牛嗜铬细胞形态结构完整,生长良好;②培养液中去甲肾上腺素、甲-脑啡呔的含量在培养第4天最高,且比第2天明显升高(P<0．05);③培养第4天,经乙酰胆碱刺激后,培养液中去甲肾上腺素、甲-脑啡呔水平较其基础值均有明显增加(P<0．01)。结论在本实验条件下,微囊化牛嗜铬细胞体外培养第4天是进行细胞移植的适宜时机。

Objective The growth condition and secretory function of microencapsulated bovine pheochromocyte cultured in vitro for 8 days was observed in order to find the proper time to transplant. Methods Bilateral adrenal glands were taken from eight healthy bulls. Single pheochromocyte were obtained by retrograde perfusion with a solution of 0.1% collagenase. The ceils were cultured in vitro for eight days after microencapsulated. During culture, the growth condition of the cells was observed microscopically. The culture solution was assessed with radioimmunoassay for determination of noradrenaline（NA） and met - enkephalin（M - ENK） concentrations. On the 4th day, the concentrations were determined again after stimulated by acetylcholine. Results ① The shape, structure and growth condition of microencapsulated bovine pheochromocyte were better; ② The level of NA and M - ENK cultured on the 4th day were significantly increased comparing with that on the 2nd day （ P 〈 0.05） ; ③ On the 4th day, the level of NA and M - ENK were also significantly increased comparing with their baseline after stimulation （ P 〈 0.01 ）. Conclusion On our experimental condition, the 4th day of microencapsulated bovine pheochromocyte cultured in vitro is the proper time to transplant.