首页|小鼠肠肌层细胞分离提取方法的建立

小鼠肠肌层细胞分离提取方法的建立

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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(DNase Ⅰ)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.
The separation of murine gut muscularis cells
Objective To explore a method for the isolation and extraction of murine gut muscu-laris cells.Methods A"descending ladder"dissociation solution was used to dissociate the mucus,mu-cosa,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 Deoxyribonuclease Ⅰ(DNase Ⅰ).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.Results The 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 sig-nificantly 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 activi-ty 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].Conclusion This new method could isolate murine muscularis cells with high viability.

Gut mucosaGut muscularisCell dissociation

陈凯、胡琼源、管文贤、刘颂

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南京大学医学院附属鼓楼医院胃与疝外科,南京 210008

肠黏膜 肠肌层 细胞解离

国家自然科学基金江苏省优秀青年基金江苏省重点研发计划

82172645BK20200052BE2022667

2024

中华实验外科杂志
中华医学会

中华实验外科杂志

CSTPCD
影响因子:0.759
ISSN:1001-9030
年,卷(期):2024.41(3)
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