小鼠输卵管上皮细胞的原代培养及纯化方法研究

Study on Mouse Oviductal Epithelial Primary Cell Culture and Its Purification

建立小鼠输卵管上皮细胞原代培养及纯化方法。小鼠输卵管上皮细胞原代培养采用组织块培养法和酶消化法。酶消化法于37℃分为4个处理组进行。处理1以2.5 g/L胰酶+0.4 g/L EDTA消化5、102、0 min;处理2以0.5 g/L胰酶+0.08 g/L EDTA消化35、507、5 min;处理3以0.3 g/L的Ⅰ型胶原酶消化60、902、40 min;处理4以2.5 g/L胰酶+0.4 g/L EDTA和0.3 g/LⅠ型胶原酶(1∶2)消化60 min,150 min。原代细胞纯化采用差速贴壁和反复差速贴壁法。传代采用胰酶两步消化法进一步纯化输卵管上皮细胞。组织块法原代培养成纤维细胞生长优势明显,传代纯化细胞效果不佳。用酶消化法原代培养时,处理1效果不理想;处理2采用3个消化时间时均取得比较理想的结果;处理3消化240 min时结果较好;处理4消化150 min效果较好。原代细胞纯化,反复差速贴壁得到的上皮细胞较纯,进一步传代纯化细胞取得了理想的结果。小鼠输卵管上皮细胞原代培养采用酶消化法结合反复差速贴壁分离纯化细胞,传代采用两步消化法,可以成功地进行小鼠输卵管上皮细胞的分离纯化培养。

To obtain a good method of primary culture of oviductal epithelial cells （OECs） and find a way of purification based on the model of mouse. Two methods of primary culture of OECs, tissue explants and enzymic digestion were used. The latter one was divided into 4 treatments that all were disposaled under 37 ℃. The diced tissue blocks were digested for 5 min, 10 min and 20 min with 2.5 g/L trypsin＋0.4 g/L EDTA in treatment 1, for 35 min, 50 min and 75 rain with 0.5 g/L trypsin＋0.08 g/L EDTA in treatment 2, for 60 min, 90 min and 240 min with 0.3 g/L coliagenase Ⅰ in treatment 3, for 60 rain and 150 min with 0.3 g/L collagenase Ⅰ and 2.5 g/I. trypsin＋0.4 g/L EI）TA （2 ： 1） in treatment,4 respectively. As for purification of OECs, the ways of different speed of cell attachment （DSCA） and re-DSCA were adopted in primary culture. On the other hand, two-step digestion was adopted to purify OECs in tissue explant subculture. Fibroblasts were mixed with OECs in prima ry cell culture. Furthermore, fibroblasts were superior to growth, which greatly affected subculture. As to enzymic digestion, treatment 1 had a result with a low attachment efficiency; Treatment 2 gained relatively good results. Treatment 3 only in digestion for 240 min obtained ideal result. It was better in digestion for 150 min in treatment 4. In primary culture, more OECs with less fibroblasts were gained by adopting re DSCA. The effect was good by adopting two-step digestion in suhcuhure. It is advised that enzyme digestion as well as re-DSCA during process of primary cell culture and two-step digestion during process of passage could be adopted to purify OECs successfully.