乳兔心脏成纤维细胞的原代培养及鉴定  

Primary culture and identification of neonatal rabbit cardiac fibroblasts

作　　者：顾建军 朱业 张维 顾翔 GU Jianjun;ZHU Ye;ZHANG Wei;GU Xiang(Department of Cardiology,Subei People's Hospital Affiliated to Yangzhou University Clinical School of Medicine,Yangzhou University,Yangzhou 225001,China)

机构地区：[1]扬州大学临床医学院附属苏北人民医院心内科,江苏扬州225001

出　　处：《中国病理生理杂志》2023年第3期571-576,共6页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No.81800250);中国博士后科学基金资助项目(No.2022M711417)。

摘　　要：目的:探讨并建立乳兔心脏成纤维细胞原代培养方法。方法:选取新出生1 d的新西兰白兔,采用胰蛋白酶和Ⅱ型胶原酶消化心脏组织,差速贴壁法分离和纯化心脏成纤维细胞;使用倒置显微镜观察细胞形态及生长情况,绘制细胞生长曲线;采用CCK-8法检测细胞活力;波形蛋白免疫荧光细胞化学染色法鉴定细胞纯度;Western blot法检测异丙肾上腺素刺激心脏成纤维细胞后Ⅰ型胶原蛋白(ColⅠ)及α-平滑肌肌动蛋白(α-SMA)水平。结果:差速贴壁90 min后在相差显微镜下可见部分成纤维细胞已贴壁生长;3 d后成纤维细胞数量较前增多,形态多样,多为不规则的多边形及扁平形;5 d时成纤维细胞数量显著增多(P<0.05),细胞形态趋于多样性,细胞间完全融合;细胞生长曲线类似“S”形。波形蛋白阳性率可达95%。CCK-8实验结果显示在4~5 d细胞活力最强(P<0.05);给予第2代心脏成纤维细胞异丙肾上腺素刺激48 h后,Col I及α-SMA水平显著增加(P<0.05)。结论:本研究建立了乳兔心脏成纤维细胞原代培养方法。AIM To explore and establish a method for primary culture of neonatal rabbit cardiac fibroblasts.METHODS Cardiac tissues from one-day-old New Zealand white rabbits were digested with trypsin and collagenase type II.Cardiac fibroblasts were isolated and purified with the differential adhesion method.The morphological changes of the cells were observed under inverted microscope,and the growth curve of the cells was plotted.Cell viability was detected by CCK-8 assay,and cellular purity was checked by immunohistochemical staining for vimentin.The protein levels of collagen type I(Col I)andα-smooth muscle actin(α-SMA)induced by isoproterenol were determined by Western blot.RESULTS After 90 min of differential adhesion,some fibroblasts were observed to attach to the plate and start to grow under a contrast microscope.After 3 d of culture,fibroblasts were amplified and exhibited various morphological phenotypes with the majority of irregular polygon and flat shape.After 5 d,the number of fibroblasts was increased significantly(P<0.05),and the cells were confluent with morphological diversity completely.The growth curve presented nearly"S"shape.The positive expression rate of vimentin was 95%.The CCK-8 assay results indicated that the highest cell viability was at 4 and 5 d(P<0.05).The levels of Col I andα-SMA were up-regulated notably at the second generation of cardiac fibroblasts induced by isoproterenol for 48 h(P<0.05).CONCLUSION Primary culture method for neonatal rabbit cardiac fibroblasts is established.

关 键 词：乳兔 原代培养 心脏成纤维细胞 细胞鉴定 

分 类 号：R363[医药卫生—病理学] R-332[医药卫生—基础医学]