【摘要】 本实验研究了山羊皮肤组织细胞的分离、培养、纯化方法和生长特征，获得了传代培养的山羊皮肤上皮样细胞与成纤维样细胞。组织块贴附培养和分散单细胞接种培养均能获得原代山羊皮肤细胞。用150IU／mL胶原酶ⅠTCM199液（10％NCS）37℃下培养消化10h，可较好地分解组织块，产生接种浓度的分散单细胞。消化培养结束后吸管吹打组织决解离的细胞及每隔3～3．5h重复3次吹打收集的细胞的生存力明显高于消化后直接从组织块游离出的细胞。山羊皮肤细胞在体外培养时贴壁生长，其原代或早期传代培养物由上皮样细胞与成纤维样细胞组成。混合生长的细胞用0．25％胰蛋白酶液37℃下消化处理6～8min，脱壁悬浮者主要为成纤维样细胞，再加酶消化5min所回收的细胞绝大多数为上皮样细胞。据此可在传代过程中将二者逐步分离纯化。和PBS相比较，用Hanks′液溶解胰蛋白酶作为消化液所回收的细胞传代后有更多的存活。在消化传代时分步收集解离细胞、及时中和酶活性，可降低胰蛋白酶对细胞的损伤作用。更多还原

【Abstract】 The present experiment studied the dissociation, culture and purification methods of goat skin cells and their growth character. Primary culture of goat skin cells could obtain from explanting monodisperion cells. Collegenase Ⅰ was an appropriate enzyme to dissociate goat skin tissue. Goat skin tissue fragments (1 mm3 ) incubated in collegenase Ⅰ solution (100～150 IU/ml, in 10% NCS TCM 199) 10 h at 37℃, could produce enough dispersed cells to explant. Three methods were used to collect cel1s from collegenase Ⅰ treated fragments: Incubated 10 h at 37℃, then collected the dissociated celIs; Incubated 10 h at 37℃, then gently pipetted pieces up and down, collected the dispersed cells. During the incubation, pipetted and collected cells every 3～3.5 h,repeated three times. The viability of cells from method B and C were better than those from method A. Primary culture of goat skin cells could also derived by outgrowth of migrating cell from fragment of tissue. It could be find that, cells migrated from the explants at day 3～4 after explantation, and 14～20 days late, the cell monolayer confulenced and covered all the bottle surface. Fibroblasts and Epithelial cells grew together in the primary and early passages culture. Their sensibility to trypsin were different. Compound cultures treated by 0.25 % trypsin Hanks’ buffer 6min at 37℃, the dispersed cells were mainly fibroblasts; and the majority cells collected from more 5 min trypsinization were epithelial cells. To explant them respectiveIy could produce pure fibroblasts and epithelial cells culture.The buffers of trypsin effected the growth potential and subsequent attahment oftrypsinized goat skin cells, Hanks’ buffer was better than PBS. Over long trypsinisation would affect cells’ viabiIity, so harvest cells during trypsinization and neutralize trypsin activity immediately, could reduce its harmful effects on cells.更多还原