CAJ | 학술논문

本试验旨在培养功能性奶牛乳腺上皮细胞系，为奶牛泌乳调控和奶牛乳房炎发病机制研究提供功能性的细胞模型。采用组织块细胞培养法来分离纯化并鉴定奶牛乳腺上皮细胞；采用有限稀释法单克隆奶牛乳腺上皮细胞；采用噻唑蓝( MTT)法来分析奶牛乳腺上皮细胞的生长曲线是否为正常的“S”形；观察细胞角蛋白18免疫荧光来证明所培养的细胞为上皮型；选择培养至10和20代的奶牛乳腺上皮细胞进行染色体核型分析。结果表明：1)利用组织块细胞分离法能够成功获得奶牛乳腺上皮细胞并传至20代。2)培养5~6 d成纤维细胞迅速增殖且周围分裂出少量的上皮细胞。培养8d奶牛乳腺上皮细胞迅速增殖，形成岛屿状集落，呈单层“鹅卵石”和“铺路石”形态生长。3)奶牛乳腺上皮细胞角蛋白18鉴定为阳性。4)培养至10和20代的奶牛乳腺上皮细胞染色体数为60条，具有正常的细胞二倍体核型。综上所述，采用组织块培养细胞能够获得具有稳定性、功能性的奶牛乳腺上皮细胞，但不是永生化细胞。
본시험지재배양공능성내우유선상피세포계，위내우비유조공화내우유방염발병궤제연구제공공능성적세포모형。채용조직괴세포배양법래분리순화병감정내우유선상피세포；채용유한희석법단극륭내우유선상피세포；채용새서람( MTT)법래분석내우유선상피세포적생장곡선시부위정상적“S”형；관찰세포각단백18면역형광래증명소배양적세포위상피형；선택배양지10화20대적내우유선상피세포진행염색체핵형분석。결과표명：1)이용조직괴세포분리법능구성공획득내우유선상피세포병전지20대。2)배양5~6 d성섬유세포신속증식차주위분렬출소량적상피세포。배양8d내우유선상피세포신속증식，형성도서상집락，정단층“아란석”화“포로석”형태생장。3)내우유선상피세포각단백18감정위양성。4)배양지10화20대적내우유선상피세포염색체수위60조，구유정상적세포이배체핵형。종상소술，채용조직괴배양세포능구획득구유은정성、공능성적내우유선상피세포，단불시영생화세포。
To establish a functional model of bovine mammary epithelial cell line for the study of lactation reg-ulation, pathogenesis of mastitis, this research applied tissue culture to isolate and culture bovine mammary epi-thelial cells; the limiting dilution method to purify bovine mammary epithelial cells; methyl thiazolyl tetrazoli-um ( MTT) method was used to identify whether the growth curve of bovine mammary epithelial cells was‘S’ type or not;cytokeratin 18 immunofluorescence was observed to prove that the cells were epithelial type;kary-otype analysis was carried out on bovine mammary epithelial cells in passages 10 and 20. The results showed as follows:1) bovine mammary epithelial cells could be successfully cultured and passaged 20 generations by tis-sue culture method. 2) At 5 to 6 days of cultivation, the fibroblast cells proliferated rapidly and a small amount of epithelial cells divided. At 8 days of cultivation, bovine mammary epithelial cells proliferated rapidly and formed island-like colonies, and grew like‘cobblestone’ and‘slabstone’ as monolayer. 3) Bovine mammary epithelial cells showed positive reaction against cytokeratine 18 antigen. 4) At passages 10 and 20 of cultiva-tion, the cell chromosome number was 60 and the cells had normal diploid karyotype. In conclusion, tissue culture cells are able to obtain stable, functional bovine mammary epithelial cells, but are not immortalized cells.