Cultivation and identification of guinea pig gallbladder smooth muscle cells using an improved tissue adhesion method
吴飞杨 1高伟成 1胡金龙 2钟楷元 3彭俊斌 4袁中旭 2姚佳明 2曹葆强 2钟兴国5
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作者信息
1. 蚌埠医科大学研究生院,蚌埠 233030
2. 安徽省第二人民医院,合肥 230000
3. 齐齐哈尔医学院,齐齐哈尔 161003
4. 安徽理工大学医学院,淮南 232001
5. 蚌埠医科大学研究生院,蚌埠 233030;安徽省第二人民医院,合肥 230000
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摘要
目的 本研究使用组织贴壁法构建一种经济、简单、纯高度的胆囊平滑肌细胞模型,为胆囊疾病相关实验提供材料.方法 从豚鼠腹腔中分离胆囊,沿着纵轴将胆囊剪开,使用组织清洗液多次漂洗胆囊组织.将清洗好的胆囊组织转移到超净工作台,并再次漂洗,将漂洗好的胆囊组织用动物眼科镊去除胆囊的粘膜和浆膜,胆囊组织用维纳斯剪分割成1 mm × 1 mm的小碎块.组织块均匀的铺到皿底,缓慢倒置,在培养箱中贴壁25分钟,加入完全培养基进行培养.在培养基中,采用不同的贴壁时间来提取纯化细胞.选用形态学,免疫荧光,免疫组化和蛋白印迹技术对其进行鉴定.结果 贴壁第7天,组织边缘观察到胆囊平滑肌细胞爬出,第21天时,平滑肌细胞呈"峰-谷"状增长.免疫荧光、免疫组化化学和免疫印迹技术鉴定3代胆囊平滑肌细胞肌动蛋白的表达呈阳性.结论 通过本项实验的完成,有望获得成本低、操作简便、纯度高的细胞模型,为胆囊疾病的发病机制研究提供更好的体外模型.
Abstract
Objective This study used the tissue adhesion method to construct an economical,simple,and highly pure gall-bladder smooth muscle cell model,providing materials for experiments related to gallbladder diseases.Method Separate the gallblad-der from the abdominal cavity of guinea pigs,cut it along the longitudinal axis,and rinse the gallbladder tissue multiple times with tis-sue cleaning solution.Transfer the cleaned gallbladder tissue to an ultra clean workbench and rinse it again.Use animal ophthalmic forceps to remove the mucosa and serosa of the gallbladder,and divide the gallbladder tissue into small pieces of 1 mm × 1 mm using Venus scissors.Spread the tissue blocks evenly at the bottom of the dish,slowly invert them,and stick them to the wall in the incuba-tor for 25 minutes.Add complete culture medium for cultivation.In the culture medium,different attachment times are used to extract and purify cells.Morphology,immunofluorescence,immunohistochemistry,and Western blotting techniques were used to identify it.Result On the 7th day of adhesion,smooth muscle cells of the gallbladder were observed to climb out at the edge of the tissue.On the 21st day,smooth muscle cells showed a"peak valley"growth pattern.Immunofluorescence,immunohistochemistry,and immunoblot-ting techniques identified positive expression of actin in 3rd generation gallbladder smooth muscle cells.Conclusions Through the com-pletion of this experiment,it is expected to obtain a low-cost,easy-to-use,and high-purity cell model,providing a better in vitro model for the study of the pathogenesis of gallbladder diseases.
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