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

沈雪梅; 李晶; 张刚; 崔宏晓; 刘丽慧; 姚军虎; 徐秀容

doi:10.3969/j.issn.1006-267x.2016.05.020

动物营养学报 >

2016 , Vol. 28 >Issue 5: 1442 - 1449

DOI: https://doi.org/10.3969/j.issn.1006-267x.2016.05.020

实验方法 Experimental Methods

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

沈雪梅 ,
李晶 ,
张刚 ,
崔宏晓 ,
刘丽慧 ,
姚军虎 ,
徐秀容

展开

西北农林科技大学动物科技学院, 杨凌 712100

收稿日期: 2015-12-04

网络出版日期: 2016-05-24

基金资助

陕西省科技攻关项目(2013K02-18)

收起

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

SHEN Xuemei ,
LI Jing ,
ZHANG Gang ,
CUI Hongxiao ,
LIU Lihui ,
YAO Junhu ,
XU Xiurong

Expand

College of Animal Science and Technology, Northwest Agriculture and Forestry University, Yangling 712100, China

Received date: 2015-12-04

Online published: 2016-05-24

Fold

摘要

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。

关键词： 隐窝; 绒毛; 肠上皮细胞; 螯合剂; 家兔

本文引用格式

沈雪梅 , 李晶 , 张刚 , 崔宏晓 , 刘丽慧 , 姚军虎 , 徐秀容 . 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[J]. 动物营养学报, 2016 , 28(5) : 1442 -1449 . DOI: 10.3969/j.issn.1006-267x.2016.05.020

Abstract

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 New Zealand 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 was 7.75% 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% and 93.48%, 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.

Key words： crypt; villus; intestine epithelial cell; chelating agents; rabbits

参考文献

[1] PETERSON L W,ARTIS D.Intestinal epithelial cells:regulators of barrier function and immune homeostasis[J].Nature Reviews Immunology,2014,14(3):141-153.

[2] HORITA N,TSUCHIYA K,HAYASHI R,et al.Fluorescent labelling of intestinal epithelial cells reveals independent long-lived intestinal stem cells in a crypt[J].Biochemical and Biophysical Research Communications,2014,454(4):493-499.

[3] BEDFORD A,CHEN T,HUYNH E,et al.Epidermal growth factor containing culture supernatant enhances intestine development of early-weaned pigs in vivo:potential mechanisms involved[J].Journal of Biotechnology,2015,196-197:9-19.
[4] GRABINGER T,LUKS L,KOSTADINOVA F,et al.Ex vivo culture of intestinal crypt organoids as a model system for assessing cell death induction in intestinal epithelial cells and enteropathy[J].Cell Death & Disease,2014,5(5):e1228.
[5] VINCENT A,KAZMIERCZAK C,DUCHÊNE B,et al.Cryosectioning the intestinal crypt-villus axis:an ex vivo method to study the dynamics of epigenetic modifications from stem cells to differentiated cells[J].Stem Cell Research,2015,14(1):105-113.

[6] BERDASCO M,ESTELLER M.DNA methylation in stem cell renewal and multipotency[J].Stem Cell Research and Therapy,2011,2(5):42.
[7] CLEVERS H.The intestinal crypt,a prototype stem cell compartment[J].Cell,2013,154(2):274-284.

[8] TANCOS Z,NEMES C,POLGAR Z,et al.Generation of rabbit pluripotent stem cell lines[J].Theriogenology,2012,78(8):1774-1786.

[9] BEVINS C L,SALZMAN N H.Paneth cells,antimicrobial peptides and maintenance of intestinal homeostasis[J].Nature Reviews Microbiology,2011,9(5):356-368.

[10] SALZMAN N H.Paneth cell defensins and the regulation of the microbiome:detente at mucosal surfaces[J].Gut Microbes,2010,1(6):401-406.

[11] EVANS G S,FLINT N,SOMERS A S,et al.The development of a method for the preparation of rat intestinal epithelial cell primary cultures[J].Journal of Cell Science,1992,101(Pt 1):219-231.
[12] POTHIER P,HUGON J S.Characterization of isolated villus and crypt cells from the small intestine of the adult mouse[J].Cell and Tissue Research,1980,211(3):405-418.
[13] BJERKNES M,CHENG H.Methods for the isolation of intact epithelium from the mouse intestine[J].The Anatomical Record,1981,199(4):565-574.

[14] AYABE T,SATCHELL D P,WILSON C L,et al.Secretion of microbicidal α-defensins by intestinal Paneth cells in response to bacteria[J].Nature Immunology,2000,1(2):113-118.

[15] FLINT N,COVE F L,EVANS G S.A low-temperature method for the isolation of small-intestinal epithelium along the crypt-villus axis[J].Biochemical Journal,1991,280(2):331-334.

[16] FULLER M K,FAULK D M,SUNDARAM N,et al.Intestinal crypts reproducibly expand in culture[J].Journal of Surgical Research,2012,178(1):48-54.

[17] 宁宇,王锋超,刘登群,等.在体分离小鼠小肠隐窝绒毛上皮细胞的生化完整性研究[J].现代生物医学进展,2009,9(14):2610-2612.
[18] 高博,杨晓农,于学辉,等.家兔GAPDH基因实时荧光定量RT-PCR方法的建立[J].中国畜牧兽医,2010,37(1):69-73.
[19] GROSSMANN J,WALTHER K,ARTINGER M,et al.Progress on isolation and short-term ex-vivo culture of highly purified non-apoptotic human intestinal epithelial cells (IEC)[J].European Journal of Cell Biology,2003,82(5):262-270.

[20] CANO-GAUCI D F,LUALDI J C,OUELLETTE A J,et al.In vitro cDNA amplification from individual intestinal crypts:a novel approach to the study of differential gene expression along the crypt-villus axis[J].Experimental Cell Research,1993,208(2):344-349.

[21] CHOUGULE P,HERLENIUS G,HERNANDEZ N M,et al.Isolation and characterization of human primary enterocytes from small intestine using a novel method[J].Scandinavian Journal of Gastroenterology,2012,47(11):1334-1343.

[22] TAUC H M,TASDOGAN A,PANDUR P.Isolating intestinal stem cells from adult Drosophila midguts by FACS to study stem cell behavior during aging[J].Journal of Visualized Experiments,2014(94):52223.

Options

文章导航

摘要

本文引用格式

Abstract

参考文献

关于本刊

在线期刊

作者中心

相关单位

联系我们

模态框（Modal）标题

摘要

本文引用格式

Abstract

参考文献

关于本刊

在线期刊

作者中心

相关单位

联系我们