改良低温螯合法分离家兔小肠绒毛和隐窝细胞及分离效果鉴定

Separation of Rabbit Intestinal Villus and Crypt Cells Using Improved Cryogenic Chelating Method and Separation Effect Identification

本试验旨在研究家兔小肠绒毛和隐窝细胞的分离方法,为深入研究小肠上皮结构与功能提供体外模型。试验以新西兰白兔为试验材料,在不同螯合剂浓度[5、10、15、20、25 mmol/L乙二胺四乙酸(EDTA)]和螯合温度(4、25℃)下分离小肠绒毛和隐窝细胞,检测所分离细胞团的产量、形态、细胞活率、完整性。结果表明:1)各螯合条件下分离的绒毛和隐窝细胞形态完整,基因组DNA与总RNA完整;2)螯合温度越高且EDTA浓度越大时,细胞富集率越高,但对细胞毒害作用也越强,4℃、5 mmol/L EDTA条件下绒毛和隐窝细胞富集率分别为7.75%和1.01%,绒毛相对完整率和隐窝细胞相对活率分别为91.67%和93.48%;25℃、25 mmol/L EDTA条件下绒毛和隐窝细胞富集率则分别高达17.89%和4.99%,但绒毛相对完整率和隐窝细胞相对活率仅为4.25%和5.17%;3)综合分析后确定,4℃、10 mmol/L EDTA条件下分离效果最佳;4)在最佳条件下所分离隐窝富集物的溶菌酶和α-防御素的相对表达量极显著高于绒毛富集物(P<0.01),显示细胞纯度较高;且隐窝细胞经培养9 h后溶菌酶和α-防御素的相对表达量未发生显著变化(P>0.05),显示细胞依然具有较高活力。由此可见,改良的低温螯合法适用于家兔小肠绒毛和隐窝细胞的高效分离。

This study aimed to investigate the separation method of rabbit intestinal villus and crypt cells,which can be served as the in vitro model for study on the structure and function of the intestinal epithelium.The experiment used NewZealand white rabbits as test material,and separated their intestinal villus and crypt cells under the conditions of different chelating agent concentrations [5,10,15,20 and 25 mmol/L ethylene diamine tetraacetic acid（EDTA） ] and chelating temperatures（4 and 25 ℃）.Then the yield,morphology,cell viability rate and integrity of the separated cells were detected.The results showed as follows：1） under different chelating separation conditions,all of the collected villus and crypt cells remained morphological integrity,and the genomic DNA and total RNA of these villus and crypt cells were intact.2） The higher chelating temperature and EDTA concentration was,the greater cell enrichment rate was obtained,but the toxic effect on cell was stronger.The enrichment rate of villus and crypt cells in group LT ＋ 5 mmol/L EDTA was7.75% and 1.01%,and that in group RT ＋ 25 mmol/L EDTA was 17.89% and 4.99%,respectively;while the relative integrity rate of villus and relative viability rate of crypt cells in the former group were 91.67% and93.48%,and were 4.25% and 5.17% in the latter group,respectively.3） By a comprehensive analysis,the best separation effect was observed under the condition of 4 ℃ and 10 mmol/L EDTA.4） The relative expression levels of lysozyme and α-defensin genes from crypt enrichment separated under the optimal condition were significantly higher than those of from villus enrichment（P〈0.01）,and it indicated that cells in a higher purity.In addition,relative expression levels of lysozyme and α-defensin genes from the crypt cell separated under the optimal condition had no significantly change after cultured for 9 h（P 〉0.05）,and it indicated that cells still had a higher vitality after cultured for 9 h.In conclusion,the improved cryogenic chelating method is suitable for separating rabbit intestinal villus and crypt cells effectively.