| Objective:The recent study indicates that the cultivating adrenal medullary cells can secrete catecholamine, which is related to the analgesia. The antinociceptive effects of adrenal medullary cells implanted into spinal subarachroid space can be enhanced. The implanting cells, like a "biological pump", can secrete inner analgesic substance constantly. The cells taken from human adrenal gland were too few to supply the clinic requirment. It is necessary to find animal cells to replace human cells for xenotransplantation. To solve the problem, the bovine adrenal chromaffm cells (BCC) were studied in the experiment. According to characters of BCC, the cultivation of BCC was established. The function of cultivating cells was evaluated with immunocytochemistry and high-performance liquid chromatography (HPLC). The proper time of cell implantation was determined, according to the cells' best functional state. The effects of drugs on the secretion of catecholamine were investigated also. In order to explore the protection method of the cells, the technology of freezing and recovery was used.MethodsThe bovine chromafFin cells were isolated and purified by collagenase digestion Percoll density gradient sedimentation centrifugation and twice inoculation. The surviving rate was identified with trypan blue staining. The purity was identified with dopamine beta hydroxylase (D β H) immunocytochemical staining. The concentration of catecholamine was assaied with HPLC. The bovine chromafFin cells were cultured in vitro for 11 days. At a day intervals, the cell suspension werefetched respectively. The functional state of BCC was assessed by morphological observation, volume of chromaffin particle and catecholamine. The drugs stimulating tests consisted of four groups. Group A is stimulated by Ach, group B, C and D stimulated by huperzine A, Dex and high K+ .Each group consisted of 4 parts. The first part was control , and others were added to different volume of drugs. The cells were incubated for 20 min at 37℃, 5%C02 after adding the drugs. Then the cells suspension was centrifugated at 250 g for 3 min. The suspension added 50 μ 1 HCLO4 was centrifugated at 4℃, 1600g for 3 min and kept at -20℃.The freezing course of BCC went through 4℃, -20 ℃ and liquid nitrogen three steps. The recovery course of BCC was melt at 37℃ water and completed within 2 min.Results1 cultivation and identification of bovine adrenal medullary cells:(1) Cellular growth and shape: On the 1st day of cultivation, the cellular body was slightly dilation, cytoplasma was transparent and cellular bulk was slightly bigger. On the 3rd and 5th day, cellular structure was intact, the boundary was regular and the chromaffin granules were clear. But on the ninth and eleventh day, a few cells were out of shape, the dioptre decreased.(2) Immunocytochemical staining: The percentage of D H positive cells was 63% on the 1st day, it rose and reached 88% and 90% on the 3rd 5th day, then it fell to 78% on the 7th day, and on the llth day, it was only 60%. The number of positive cells reached the peak on 3rd and 5th day.(3) Contentration of catecholamine: NE E and DA are the main constituents of catecholamine. The contentration of catecholamine increased from 46.85 ?3.51ng/106cells on the 1st day to 68.56 ?.86ng/106 cells on the 5th day. From the 7th day, it fell quickly and reached the bottom 44.41+2.76 ng/106cells on the llth day. The level of NE -, E and DA on the 3rd day and the5th day were significantly higher than that of the 1st day and the 7th day (p<0.01);The amount of catecholamine had no remarkable difference on 3rd and 5th day (p>0.05).2 The effect of drugs stimulation on the secretion of catecholamine(1) stimulated by Ach , K+ The level of catecholamine secretion were higher than that of control group (p<0.01); The increase of the level of catecholamine was corresponded with the concentration of the drugs.(2) stimulated by huperzine A There were the same effi...
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