几种成年小鼠胸腺上皮细胞分离和富集方法效果的比较

doi:10.13481/j.1671-587x.20160106

吉林大学学报(医学版) ›› 2016, Vol. 42 ›› Issue (01): 24-30.doi: 10.13481/j.1671-587x.20160106

几种成年小鼠胸腺上皮细胞分离和富集方法效果的比较

齐慧慧¹, 陈冰^1,2, 陈晓娜³, 王瑶琪⁴, 佟青玥¹, 孙丽光¹, 杨永广¹

1. 吉林大学第一医院转化医学研究院免疫学研究所, 吉林长春 130061;
2. 吉林大学中日联谊医院麻醉科, 吉林长春 130033;
3. 吉林大学公共卫生学院营养与食品卫生学教研室, 吉林长春 130021;
4. 吉林大学第一医院甲状腺外科, 吉林长春 130021

收稿日期:2015-09-16 发布日期:2016-01-26
通讯作者: 杨永广,教授,博士研究生导师(Tel:0431-88783477,E-mail:yongg.yang@gmail.com);孙丽光,副教授,硕士研究生导师(Tel:0431-88783477,E-mail:sunliguang1717@163.com) E-mail:yongg.yang@gmail.com;sunliguang1717@163.com
作者简介:齐慧慧(1990-),女,黑龙江省哈尔滨市人,在读医学硕士,主要从事胸腺免疫与衰老方面的研究。
基金资助:
国家自然科学基金资助课题(81202379)

Comparison of effects between adult murine TEC isolation and enrichment methods

QI Huihui¹, CHEN Bing^1,2, CHEN Xiaona³, WANG Yaoqi⁴, TONG Qingyue¹, SUN Liguang¹, YANG Yongguang¹

1. Institute of Immunology, Academy of Translational Medicine, First Hospital, Jilin University, Changchun 130061, China;
2. Department of Anesthesia, China-Japan Union Hospital, Jilin University, Changchun 130033, China;
3. Department of Nutrition and Food Hygiene, School of Public Health, Jilin University, Changchun 130021, China;
4. Department of Thyroid Surgery, First Hospital, Jilin University, Changchun1 30021, China

Received:2015-09-16 Published:2016-01-26

摘要/Abstract

摘要：

目的: 通过比较几种成年小鼠胸腺上皮细胞(TECs)分离和富集方法的效果,寻找一种具有准确体内代表性的高效TECs富集方法。方法: 27只6 ~ 8周龄BABL/c小鼠分为9组,每组3只。1 分离实验分为5个分离组,包括1.0 g·L^-1collagenase Ⅴ组和0.5、0.7、1.0及2.0 U·mL^-1 Liberase^TM组,按照相应的酶工作浓度对胸腺组织进行消化处理;2 富集实验分为4个富集组,包括对照组和免疫磁珠(MACS)、Percoll及Ficoll 富集组,各组用1.0 U·mL^-1Liberase^TM消化胸腺组织后,将所得的单细胞悬液分别用MACS、Percoll和Ficoll法进行细胞富集(对照组除外)。分离组通过流式细胞术检测细胞活力及胸腺基质细胞(TSCs)的产量;富集组通过流式细胞术检测细胞活力、TSCs比例和TSCs中TECs重要标志上皮细胞黏附分子(EpCAM)的表达丰度,检测Percoll富集组和对照组TECs的2个亚群--皮质TECs(cTECs)和髓质TECs(mTECs)比例。结果: 1.0 U·mL^-1 Liberase ^TM组中TSCs的产量明显高于0.5和0.7 U·mL^-1Liberase^TM组(P<0.01),且其细胞活力及消化时间均优于1.0 g·L^-1collagenase Ⅴ组(P<0.01)。3个富集组的细胞活力均较高,但组间比较差异无统计学意义(P>0.05);与对照组比较,3个富集组TECs比例明显增加(P<0.05或 P<0.01);在3个富集组中,Percoll富集组富集的TECs比例高于其他2个富集组(P<0.01);与对照组比较,Percoll富集组cTECs和mTECs的百分比及cTEC/mTEC比值无明显变化(P >0.05)。结论: Liberase ^TM较collagenase Ⅴ更适用于消化成年小鼠胸腺,其最适消化浓度为1.0 U·mL^-1。本研究采用的3种富集方法均可以提高TECs比例,对细胞的破坏均较小;Percoll富集法最有效,在富集后不影响TECs亚群细胞比例,便于后续实验操作。

关键词: 小鼠, BABL/C, 胸腺上皮细胞, 细胞富集, 流式细胞术

Abstract:

Objective: To compare the effects between adult murine thymic epithelial cells (TECs)isolation and enrichment methods,and to find a high-efficiency TECs enrichment method with accurate invivo representation.Methods: Twenty-seven BABL/c mice (6-8 weeks)were divided into nine groups,and each group contained three mice. ① The isolation experiment contained five isolation groups: collagenase Ⅴ 1.0 g·L^-1 group and 0.5,0.7,1.0,2.0 U·mL^-1 Liberase^TM groups. Thymus digestion was performed according to relevant enzyme working concentrations. ② The enrichment experiment contained four enrichment groups: control,magnetic activated cell sorting (MACS),Percoll and Ficoll enrichment groups;in each group,thymus digestion was performed using the optimal working concentration (1.0 U·mL^-1)of Liberase ^TM and the digests were continued for TECs enrichment by MACS,Percoll,Ficoll respectively (except control group). For isolation experiment,in each group,the cell viability and the yield of thymic stromal cells (TSCs)were examined by flow cytometry. For the enrichment experiment,the cell viability,the percentage of TSCs and the expression of epithelial cell adhesion molecule (EpCAM)in TSCs in each group were examined by flow cytometry. Furthermore,in both Percoll enrichment group and control group,the percentages of cortical TECs (cTECs)and medullary TECs (mTECs)were examined as well.Results: For the isolation experiment,the yield of TSCs in 1.0 U·mL^-1 Liberase^TM group was higher than those in 0.5 and 0.7 U·mL^-1 Liberase^TM groups(P<0.01);the cell viability and digestion time in 1.0 U·mL^-1 Liberase^TM group were superior to 1.0 g·L^-1 collagenase Ⅴ group (P<0.01). The cell viabilities in three enrichment groups were higher,and there were no significant differences between them (P > 0.05).Compared with control group,the percentages of TECs in three enrichment groups were increased (P<0.05 or P<0.01). And among three enrichment groups,the percentage of TECs in Percoll enrichment group was higher than those in other two groups (P<0.01). There were no significant differences in the percentages of cTECs and mTECs as well as the ratios of cTEC to mTEC between control group and Percoll enrichment group (P > 0.05). Conclusion: Liberase ^TM is more suitable for adult murine TECs isolation than collagenase Ⅴ and its optimal digestion concentration is 1.0 U·mL^-1.All the three enrichment methods mentioned above can increase the percentage of TECs,and they all do little harm to the cells;Percoll is the most efficient method for adult murine TECs enrichment among three enrichment methods,and it doesn't affect the ratio of cTEC to mTEC,thereby making it easier for the following study.

Key words: thymic epithelial cells, cell enrichment, flow cytometry

中图分类号:

R-331

齐慧慧, 陈冰, 陈晓娜, 王瑶琪, 佟青玥, 孙丽光, 杨永广. 几种成年小鼠胸腺上皮细胞分离和富集方法效果的比较[J]. 吉林大学学报(医学版), 2016, 42(01): 24-30.

QI Huihui, CHEN Bing, CHEN Xiaona, WANG Yaoqi, TONG Qingyue, SUN Liguang, YANG Yongguang. Comparison of effects between adult murine TEC isolation and enrichment methods[J]. Journal of Jilin University Medicine Edition, 2016, 42(01): 24-30.

参考文献

[1] Rinke de Wit TF,Izon DJ,Revilla C,et al. Expression of tyrosine kinase gene in mouse thymic stromal cells[J]. Int Immunol,1996,8(11):1787-1795.

[2] Sun L,Li H,Luo H,et al. Thymic epithelial cell development and its dysfunction in human diseases[J]. Biomed Res Int,2014,2014:206929.

[3] Jain R,Gray DH. Isolation of thymic epithelial cells and analysis by flow cytometry[J]. Curr Protoc Immunol,2014.doi: 10.1002/0471142735.im0326s107.

[4] McLelland BT,Gravano D,Castillo J,et al. Enhanced isolation of adult thymic epithelial cell subsets for multiparameter flow cytometry and gene expression analysis[J]. J Immunol Methods,2011,367(1/2):85-94.

[5] Seach N,Wong K,Hammett M,et al. Purified enzymes improve isolation and characterization of the adult thymic epithelium[J]. J Immunol Methods,2012,385(1/2):23-34.

[6] 李倩如,杜焱,苏艳宾,等. 人CK8&18阳性胸腺上皮细胞对脐血CD34⁺细胞向T细胞分化的影响[J]. 中国免疫学杂志, 2011(3):200-203.

[7] Gray DH,Fletcher AL,Hammett M,et al. Unbiased analysis,enrichment and purification of thymic stromal cells[J]. J Immunol Methods,2008,329(1/2):56-66.

[8] Stoeckle C,Rota IA,Tolosa E,et al. Isolation of myeloid dendritic cells and epithelial cells from human thymus[J]. J Vis Exp: JoVE,2013(79):e50951.

[9] Seach N,Hammett M,Chidgey A. Isolation,characterization,and reaggregate culture of thymic epithelial cells[J]. Methods Mol Biol,2013,945:251-272.

[10] Scoville SD,Keller KA,Cheng S,et al. Rapid column-free enrichment of mononuclear cells from solid tissues[J]. Sci Rep,2015,5:12490.

[11] Irla M,Guenot J,Sealy G,et al. Three-dimensional visualization of the mouse thymus organization in health and immunodeficiency[J]. J Immunol,2013,190(2):586-596.

[12] Kyewski BA,Rouse RV,Kaplan HS. Thymocyte rosettes: multicellular complexes of lymphocytes and bone marrow-derived stromal cells in the mouse thymus[J]. Proceed Natl Acad Sci USA,1982,79(18):5646-5650.

[13] Gray DH,Chidgey AP,Boyd RL. Analysis of thymic stromal cell populations using flow cytometry[J]. J Immunol Methods,2002,260(1/2):15-28.

[14] Anderson MS,Venanzi ES,Klein L,et al. Projection of an immunological self shadow within the thymus by the aire protein[J]. Science,2002,298(5597):1395-1401.

[15] Williams KM,Mella H,Lucas PJ,et al. Single cell analysis of complex thymus stromal cell populations: rapid thymic epithelia preparation characterizes radiation injury[J]. Clin Transl Sci,2009,2(4):279-285.

[16] Xing Y,Hogquist KA. Isolation,identification,and purification of murine thymic epithelial cells[J]. J Vis Exp,2014(90):e51780.

[17] Lierman S,Tilleman K,Cornelissen M,et al. Follicles of various maturation stages react differently to enzymatic isolation: a comparison of different isolation protocols[J]. Reprod Biomed Online,2015,30(2):181-190.

几种成年小鼠胸腺上皮细胞分离和富集方法效果的比较

Comparison of effects between adult murine TEC isolation and enrichment methods

RichHTML

PDF (PC)

赞

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 15

Metrics

本文评价

推荐阅读 0

[1]	赵丹, 曹东慧, 金美善, 吴孟辉, 王玥琦, 杨娜, 周天宇, 张厚君, 姜晶, 曹雪源. β-甘草次酸对炎症相关胃癌的抑制作用及其机制[J]. 吉林大学学报(医学版), 2018, 44(06): 1150-1155.
[2]	王天月, 崔晓燕, 吴骁, 王丹. 磷酸化胞外信号调节激酶在小鼠海马发育过程中的表达及其意义[J]. 吉林大学学报(医学版), 2018, 44(05): 979-982.
[3]	陈瑛波, 宋丹, 吴晋, 景作磊, 刘利平, 姜如娇, 孙捷, 吴英杰. 铁皮石斛对小鼠和胰岛瘤细胞胰岛素抵抗的改善作用[J]. 吉林大学学报(医学版), 2018, 44(04): 709-717.
[4]	范伟, 杨永广, 胡正. 具有人红细胞重建的人源化小鼠模型构建和应用的研究进展[J]. 吉林大学学报(医学版), 2018, 44(04): 859-863.
[5]	张啸环, 段明华, 闫玉礼, 潘新, 李海清, 周长龙, 赵天倚. 人羊膜成纤维细胞的分离和鉴定[J]. 吉林大学学报(医学版), 2018, 44(04): 845-848.
[6]	孙竹梅, 梁伟时, 康佳丽, 孟徐兵, 王建行, 韩淑英. 2型糖尿病db/db小鼠肾脏动态病理特点[J]. 吉林大学学报(医学版), 2018, 44(03): 499-503.
[7]	陈冰, 刘昊川, 李龙云, 赵国庆, 王震, 杨永广, 李凯, 胡正. 人源化小鼠模型构建和应用的研究进展[J]. 吉林大学学报(医学版), 2018, 44(03): 667-674.
[8]	庞春艳, 王慧, 王志华, 冯秀媛, 王永福. 腺病毒介导的“睡美人”转座子与IL-10共表达对NOD小鼠Th17/Treg细胞平衡的影响[J]. 吉林大学学报(医学版), 2018, 44(02): 205-210.
[9]	王建行, 姜妍, 王妍, 吴磊, 穆玉, 韩淑英. 荞麦花叶发酵提取物对2型糖尿病db/db小鼠肾损伤的影响[J]. 吉林大学学报(医学版), 2018, 44(01): 95-100.
[10]	李彩, 魏洁, 甄永占, 代明鹤, 胡刚, 郭立新, 林雅军. 赖氨大黄酸对KK/HlJ糖尿病小鼠胰岛素抵抗的改善作用及其机制[J]. 吉林大学学报(医学版), 2017, 43(06): 1074-1079.
[11]	王维, 刘聪, 蒋岩, 王松萍, 于慧, 敬舒, 庄文越, 王春梅, 陈建光, 李贺. 复方五味子提取物的抗疲劳作用及其机制[J]. 吉林大学学报(医学版), 2017, 43(03): 502-506.
[12]	王芹, 杜利清, 王彦, 徐畅, 李进, 刘强. Rb94基因联合放射治疗对荷瘤裸小鼠食管癌细胞生长的抑制作用[J]. 吉林大学学报(医学版), 2017, 43(02): 220-224.
[13]	李宁, 刘聪, 李佳鸿, 刘畅, 杨鹏, 王春梅, 敬舒, 孙靖辉, 陈建光, 李贺. 五味子淫羊藿混合提取物对东莨菪碱致小鼠学习记忆障碍的改善作用[J]. 吉林大学学报(医学版), 2017, 43(01): 42-46.
[14]	陈阳, 鄂长勇, 关连越, 刘文韬, 王丽娜, 张学文. 四氯化碳诱导的肝硬化小鼠肝部分切除后肝再生模型的建立及评价[J]. 吉林大学学报(医学版), 2016, 42(06): 1243-1248.
[15]	刘超, 任丽莉, 栾治东, 孟智超, 刘乙蒙, 肖建英. 人卵巢cDNA文库中与蛋白激酶Wee1B相互作用的候选分子的筛选及其调控作用[J]. 吉林大学学报(医学版), 2016, 42(05): 866-871.