兔阴道平滑肌细胞体外分离与培养

发布时间：2018-10-24 07:46

【摘要】：背景:阴道平滑肌细胞原代培养主要有两种方法:酶消化法和组织块贴壁法。前者培养所需时间短,产量高,但所需组织量较大,且试验中污染机会大,最佳消化时间不易把握;后者虽方法简便、有效,但培养所需时间较长。目的:观察组织块+酶消化法原代培养兔阴道平滑肌细胞时间,并与组织块法比较。设计、时间及地点:体外观察实验,于2005-02/2006-02在南昌大学第一附属医院烧伤研究所及动物实验室完成。材料:四五个月龄雌性新西兰大白兔,体质量2.0～2.5kg。方法:①组织块法原代培养阴道平滑肌细胞:将修剪好的1mm×1mm×1mm组织块均匀地接种于培养皿中,然后放入37℃恒温培养箱中静置培养3.0～4.0h,待组织块贴壁牢固后,再补加含体积分数为0.1胎牛血清的DMEM培养液,使组织块浸于培养液中,37℃恒温静置培养3.0～4.0d,待细胞游出后换液,大约每3天换液一次。②组织块+酶消化法原代培养阴道平滑肌细胞:将组织块用0.1%胶原酶Ⅳ在37℃培养箱中消化0.5～1.0h,至组织块边缘变毛时中止消化,然后将消化好的组织块接种于培养皿上,其余步骤同组织块法。③传代培养:第1次传代时机应根据细胞生长具体情况而定,在细胞达50%～60%融合时即进行传代培养。第2次以后传代,在细胞生长达80%融合时进行传代培养。主要观察指标:①原代培养所需时间。②第3代阴道平滑肌细胞表面结构和超微结构。结果:组织块+酶消化法原代培养细胞萌出时间较组织块法早1.0～2.0d,细胞融合时间较组织块法早3.0～4.0d,细胞可稳定传5～6代。倒置显微镜下观察两种方法所培养细胞呈梭形或多角形,融合时部分区域细胞出现平滑肌细胞特征性生长表现"峰-谷"状结构。透射电镜下观察,第3代阴道平滑肌细胞核呈锯齿状,胞浆内含平滑肌细胞特征性结构肌丝和密体,含有大量高尔基体,粗面内质网扩张,游离核糖体丰富,提示获得合成表型阴道平滑肌细胞比例大。结论:组织块+酶消化法原代培养周期较短,需要注意的是在取材、分离整个操作过程中需保持阴道组织块湿润;胶原酶消化组织块时间不应过长,以组织块周边呈现毛须状为度;此外应在静置状态下培养。
[Abstract]:Background: there are two methods in primary culture of vaginal smooth muscle cells: enzyme digestion and tissue mass adherence. The former has the advantages of short time, high yield, large amount of tissue, large pollution opportunity in the experiment, and the best digestion time is difficult to grasp. The latter method is simple and effective, but it takes a long time to culture. Aim: to observe the time of primary culture of rabbit vaginal smooth muscle cells by tissue mass enzyme digestion and compare with tissue block method. Design, time and place: in vitro observation experiment was completed in the Institute of Burn and Animal Laboratory, first affiliated Hospital of Nanchang University, February 2006, 2005. Materials: female New Zealand white rabbits of four or five months of age, with a body mass of 2. 0 ~ 2. 5 kg. Methods: 1 Primary culture of vaginal smooth muscle cells by tissue block method: the trimmed 1mm 脳 1mm 脳 1mm tissue blocks were evenly inoculated into the culture dish, and then cultured in a 37 鈩，

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