发情期家兔输卵管上皮细胞和基质细胞的分离培养与鉴定  

Isolation,culture and Identification of rabbit oviductal epithelial cells and stromal cells during oestrus

作　　者：赵永贞[1] 陈秀荔[1] 张彦明[2] 曹斌云[1] 马云[1] 王利心[1] 

机构地区：[1]西北农林科技大学动物科技学院,陕西杨凌712100 [2]西北农林科技大学动物医学院,陕西杨凌712100

出　　处：《西北农林科技大学学报（自然科学版）》2008年第2期27-33,共7页Journal of Northwest A&F University(Natural Science Edition)

基　　金：国家科技支撑计划项目(2006BAD04A00)

摘　　要：【目的】探讨输卵管上皮细胞和基质细胞的体外分离培养方法及在激素刺激下基质细胞对上皮细胞的作用。【方法】通过差速贴壁法体外分离纯化兔输卵管上皮细胞和基质细胞;免疫组织化学染色和免疫荧光染色方法鉴定上皮细胞和基质细胞的类型和纯度;并在无胎牛血清的DMEM/F12培养液和含体积分数15%胎牛血清的DMEM/F12培养液中,分别添加不同浓度的雌激素(17β-E2)和孕激素(P4),比较上皮细胞和基质细胞的增殖情况及其对上皮细胞/基质细胞共培养的影响。【结果】体外成功地分离到兔输卵管上皮细胞和基质细胞;兔输卵管上皮细胞经鼠抗人细胞角蛋白单克降抗体(anti-CK18)免疫组织化学染色后呈阳性,细胞质呈棕色,免疫荧光染色检测其纯度在98%以上;兔输卵管基质细胞经鼠抗人波形蛋白单克降抗体(anti-Vimentin)免疫组织化学染色后呈阳性,细胞质呈棕色,免疫荧光染色检测其纯度在95%以上。17β-E2和P4均能促进兔输卵管上皮细胞和基质细胞的增殖,使其数量明显增加。基质细胞与上皮细胞共培养,上皮细胞增殖较快。【结论】差速贴壁法能够获得纯度较高的输卵管上皮细胞和基质细胞,在17β-E2和P4共同作用下,少量基质细胞能促进上皮细胞生长。[Objective] The research is to study methods for isolation and culture of epithelial ceils and stromal ceils from rabbit oviduct,and stromal contribution to epithelial functions in the stimulation of estrogen. [Method] In vitro rabbit oviductal epithelial ceils and stromal ceils were isolated and purified through differential attachment technique. Immunocytochemical staining and immunofluorescence staining were used to identify types of epithelial ceils and stromal ceils and the purification. Different conentration of 17β-estrogen（17β-E2） and progesterone （P4） were added respectively to the fetal bovine serum-free DMEM/F12 medium and DMEM/F12 mediun including the volume fraction 15% fetal bovine serum,compared with proliferation of epithelial ceils and stromal cells,and whose effected on the cocultured epithelial ceils stromal cessl. [Result] Rabbit oviduct epithelial ceils and stromal cells,immunocytochemistry of rabbit oviductal epithelial cells,the cytoplast show brown（＋）to anti-cytokeratin 18,the purification of epithelial cells reach over 98% through immunofluorescence staining; immunocytochemistry of rabbit oviductal stromal cells, the cytoplast show brown （ ＋ ） to , anti-vimentin, the purification of stromal cells reach over 95% through immunofluorescence staining. 17β-E2 and P4 could accelerate the proliferation of rabbit oviductal epithelial cells and stromal cells,whose quantity distinctly increased. Epithelial cells were co-cultured in stromal cells,epithelial cells proliferate quickly. [Conclusion] This study demonstrates that differential attachment technique can receive more high purity of oviductal epithelial cells and stromal cells. Uncler the combined action of 17β-E2 and P4 ,a small quantity of stromal cells could accelerate the growth of epithilial cells.

关 键 词：输卵管 上皮细胞 基质细胞 共培养 免疫组化和免疫荧光 家兔 

分 类 号：Q954.633.2[生物学—动物学]