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。
BACKGROUND： Vaginal smooth muscle ceils are mainly cultured by enzyme digestion method and explant method. The former method requires a short culture time with a high output, but this method demands a large number of tissues with a high opportunity of pollution, and the optimal digestion time is difficult to control. The latter method is simple and effective, but the culture needs a long time. OBJECTIVE： To observe the culture time of rabbit vaginal smooth muscle cells using explaut ＋ enzyme digestion methods, and to compare with the explant method. DESIGN, TIME AND SETTING： The in vitro observation experiment was performed at the Burn Institute and Animal Laboratory of First Affiliated Hospital, Nanchang University from February 2005 to February 2006. MATERIALS： Female New Zealand rabbits aged 4 5 months, with the body mass of 2.0-2.5 kg were selected for this study. METHODS： Vaginal smooth muscle cells using explant method： 1 mm X 1 mm X 1 mm explants were uniformly incubated in a culture medium, and then placed in a incubator at 37 ℃ for 3.0-4.0 hours. After tissue confluence, tissues were immersed in DMEM containing 0. l volume fraction, stored at 37℃ for 3.0-4.0 days. Following cells grew from the tissue islets, the medium was changed about once every three days. Vaginal smooth muscle cells using explant ＋ enzyme digestion methods： The tissue pieces were digested by 0.1% collagenase type Ⅳ at 37℃ for 0.5-1 hour, and then enzymatic digestion was interrupted when the edge of tissue pieces became coarse. These pieces were cultivated in culture dishes. The remaining steps were the same as the explant method. Serial subculture： The 1st subculture was made when 50%-60% cells were confluent. After the 2rd passage of cells was obtained, subculture was made when 80% cells were confluent. MAIN OUTCOME MEASURES： Time of primary culture; Surface structure and ultrastructure of the 3rd passage of vaginal smooth muscle cells. RESULTS： The eruption time and the confluent time of vaginal smooth muscle cells could be shortened by the explant ＋ collagen digestion methods about 1-2 days and 3-4 days respectively compared with the explant method only. Vaginal smooth muscle cells, which were obtained by the two methods mentioned above, could propagate for 5-6 passages. The attached cells were polygonal or spindle and after confluence could be seen, the＇marked smooth muscle cells ＂Peak-Valley＂ structure in some domains under the inverted microscope. The third passage of smooth muscle cell nuclei were serration; the cytoplasm contained the marked smooth muscle cell structural myofilaments and dense bodies and plenty of golgi bodies; the rough endoplasmic reticalum broadened; the free ribosome were abundant under the transmission electron microscope. This indicated that vaginal smooth muscle cells with synthesis phenotype could be greatly collected. CONCLUSION： Explant ＋ enzyme digestion methods need a short cycle of primary culture. Vaginal explants should be kept moisture during drawing materials, isolation and the whole： The time of enzymatic digestion should not be too long, to the point that palpi pilosi is seen surrounding the explants. Primary culture should remain static state and use of plastic culture dishes.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2008年第46期9090-9094,共5页
Journal of Clinical Rehabilitative Tissue Engineering Research
