摘要
目的针对现有技术的不足,尝试一种活性强、比例高的小鼠肠肌层细胞分离提取方法。方法采用"降阶梯"策略构建解离液组合,逐层解离黏液层、黏膜层、固有层和黏膜下层,再使用消化液消化肌层细胞。其中,降阶梯式解离液是指胰蛋白酶浓度在黏液层、黏膜层和黏膜下层解离液中阶梯式降低;消化液是由胶原酶Ⅰ/Ⅳ、中性蛋白酶与脱氧核糖核酸酶Ⅰ(DNaseⅠ)组成。实验组1、2、3的胰蛋白酶浓度(wt%)梯度分别为:0.50、0.25、0.10;0.50、0.30、0.20;1.00、0.50、0.25。两两比较采用t检验。结果成功分离出肠肌层,并获得了肠肌层细胞,实验组1的肌层暴露比例为(91.00±1.87)%,细胞活性为(79.20±0.49)%。实验组1的肌层暴露比例显著高于实验组2[(91.00±1.87)%比(80.00±3.16)%,t=2.994,P<0.05]和实验组3[(91.00±1.87)%比(83.00±2.55)%,t=2.530,P<0.05];同时实验组1的细胞活性也显著高于实验组2[(79.20±0.49)%比(70.20±0.37)%,t=14.600,P<0.01]和实验组3[(79.20±0.49)%比(62.40±0.40)%,t=26.560,P<0.01]。结论本方法能够提取获得活性强、比例高的肠肌层细胞。
Objective To explore a method for the isolation and extraction of murine gut muscularis cells.MethodsA"descending ladder"dissociation solution was used to dissociate the mucus,mucosa,lamina propria and submucosa.The concentration of trypsin in the mucus,mucosal and submucosal dissociation solutions gradually decreased.The digestion solution for muscularis consisted collagenaseⅠ/Ⅳ,neutral protease and DeoxyribonucleaseⅠ(DNaseⅠ).The trypsin concentration(wt%)gradients of experimental groups 1,2 and 3 were as follows:0.50,0.25,0.10;0.50,0.30,0.20;1.00,0.50,0.25.T-test was used to compare between the groups.ResultsThe muscularis was successfully exposed and the muscularis cells were obtained.The exposure proportion of muscularis was(91.00±1.87)%,and the cell viability was(79.20±0.49)%.The exposure ratio of muscularis in experimental group 1 was significantly higher than that in experimental group 2[(91.00±1.87)%vs.(80.00±3.16)%,t=2.994,P<0.05]and group 3[(91.00±1.87)%vs.(83.00±2.55)%,t=2.530,P<0.05].The cell activity in experimental group 1 was also significantly higher than that in experimental group 2[(79.20±0.49)%vs.(70.20±0.37)%,t=14.600,P<0.01]and group 3[(79.20±0.49)%vs.(62.40±0.40)%,t=26.560,P<0.01].ConclusionThis new method could isolate murine muscularis cells with high viability.
作者
陈凯
胡琼源
管文贤
刘颂
Chen Kai;Hu Qiongyuan;Guan Wenxian;Liu Song(Department of Gastric and Hernia Surgery,Nanjing Drum Tower Hospital,the Afiliated Hospital of Nanjing University Medical School,Nanjing 210008,China)
出处
《中华实验外科杂志》
CAS
2024年第3期468-471,共4页
Chinese Journal of Experimental Surgery
基金
国家自然科学基金(82172645)
江苏省优秀青年基金(BK20200052)
江苏省重点研发计划(BE2022667)。
关键词
肠黏膜
肠肌层
细胞解离
Gut mucosa
Gut muscularis
Cell dissociation
