人脐带间充质干细胞分离培养建立及其生物学特性观察

谢扬虎; 李玉云

留言板

尊敬的读者、作者、审稿人, 关于本刊的投稿、审稿、编辑和出版的任何问题, 您可以本页添加留言。我们将尽快给您答复。谢谢您的支持!

姓名
邮箱
手机号码
标题
留言内容
验证码

人脐带间充质干细胞分离培养建立及其生物学特性观察

谢扬虎 , 李玉云

文章导航 > 蚌埠医学院学报 > 2010, 35 > 10: (982-985)

引用本文:

Citation:

人脐带间充质干细胞分离培养建立及其生物学特性观察

谢扬虎¹,
李玉云²

1. 蚌埠医学院临床检验诊断学实验中心, 安徽蚌埠 233030;
2. 蚌埠医学院临床检验诊断学教研室, 安徽蚌埠 233030

作者简介: 谢扬虎(1976-),男,硕士研究生,主管检验师.

基金项目:
安徽省教育厅自然科学研究资助项目(2006KJ392B)
中图分类号: R329.24

Experimental study of humam umbilical cord mesenchymal stem cell isolation,culture and biological characteristics

XIE Yang-hu¹,
LI Yu-yun²

1. Research Center of Clinical Laboratory Science, Bengbu Medical College, Bengbu Anhui 233030, China;
2. Department of Clinical Laboratory, Bengbu Medical College, Bengbu Anhui 233030, China
CLC number: R329.24

摘要: 目的:建立从人的脐带组织中分离和培养脐带间充质干细胞(UC-MSCs)的方法,并探讨UC-MSCs的生物学特性。方法:分别采用原代贴壁培养法和酶消化法(胶原酶Ⅱ和胰酶)分离培养UC-MSCs,瑞氏染色观察细胞形态,流式细胞术检测第3代以后的UC-MSCs表面特异标志物表达,MTT法检测P7细胞增殖情况。UC-MSCs同外周血单个核细胞共培养后,用MTT法测定细胞增殖率,Hoechst与PI双染色,观察UC-MSCs对健康人淋巴细胞增殖的影响。结果:原代贴壁培养法1周左右可见成纤维样细胞从组织块边缘爬出,成簇生长;酶消化法3~5天可见成纤维样细胞均匀生长。传代培养后,UC-MSCs均呈长梭形、旋涡状生长;细胞核大,核仁清晰;免疫表型:CD29阳性率为(95.71±2.23)%,CD31和CD34阳性率分别为(2.47±0.54)%和(3.24±0.34)%;第7代UC-MSCs仍具有较强的分裂增殖能力。UC-MSCs对淋巴细胞的增殖反应具有抑制作用,呈细胞数量剂量依赖性,而淋巴细胞对UC-MSCs的生长未见影响。结论:从人脐带中成功分离培养的细胞具有UC-MSCs生物学特性。
- 干细胞,间充质 /
- 脐带 /
- 分离 /
- 培养 /
- 淋巴细胞
Abstract: Objective:To establish a method for isolation and culture of umbilical cord mesenchymal stem cells (UC-MSCs) from human umbilical cord in vitro,and explore its biological characterstics.Methods:Human UC-MSCs were isolated and expanded in culture with explantation technique and enzyme digestion (collagenase Ⅱand trypsin),respectively;the cell morphology was observed by Wright-staining;after the third passage,the surface markers of UC-MSCs were detected and idntified by flow cytometry,and the seventh passage,the cell proliferation was analyzed by MTT assay.After UC-MSCs were co-cultured with peripheral blood mononuclear cells,the impact on the T cells were observed by Hoechst and PI double staining.Results:About one week after explantation,the fibroblast-like cells sprawled out from the edge of the tissue,growing in cluster;3-5 days after enzyme digestion,cells were watched growing fiber-shaped.After passaged,UC-MSCs showed the status of long,spindle-shaped and circinate growth;they had large nucleus and clear nucleolus.Immunophenotype analysis showed that they had strong positive expression for CD29 (95.71±2.23)%,but week expression for CD31,and CD34 were (2.47±0.54)% and (3.24±0.34)%,respectively;the seventh passage UC-MSCs still had strong proliferation ability;UC-MSCs suppressed T cells proliferation in a dose-dependent manner;lymphocytes did not affect the growth of UC-MSCs.Conclusions:The cells isolated successfully from the umbilical cord have biological characteristics of the mesenchymal stem cell.
- stem cell,mesenchymal /
- umbilical cord /
- isolation /
- culture /
- lymphocyte

[1]	管英华 , 魏晓巍 , 谢扬虎 , 李见 , 王露 , 李玉云 , 张强 . 人脐带间充质干细胞对再生障碍性贫血小鼠造血的影响. 蚌埠医学院学报, 2013, 37(6): 648-651.
[2]	范宇晨 , 郭涛 . 人眼组织小梁网细胞的体外培养及鉴定的新方法. 蚌埠医学院学报, 2020, 45(2): 163-166. doi: 10.13898/j.cnki.issn.1000-2200.2020.02.007
[3]	王海涛 , 王春生 . 大鼠骨髓间充质干细胞对同种异体T淋巴细胞的作用及其机制. 蚌埠医学院学报, 2010, 35(12): 1210-1213.
[4]	许克庆 , 肖玉周 . 生长因子相关的骨髓间充质干细胞成骨诱导研究进展. 蚌埠医学院学报, 2011, 36(4): 426-429.
[5]	郑岚 . 外周血异型淋巴细胞检测的临床意义. 蚌埠医学院学报, 2010, 35(5): 516-517.
[6]	梁华 , 梁友宝 , 李柏青 . 人外周血白介素-22产生细胞在不同淋巴细胞亚群中的比例. 蚌埠医学院学报, 2011, 36(5): 441-444.
[7]	李竟 , 郑照军 , 郑晶 , 郭晖 . 淋巴细胞、C反应蛋白联合外周血中性粒细胞淋巴细胞比值检测在新型冠状病毒肺炎早期诊断中的应用价值. 蚌埠医学院学报, 2020, 45(6): 701-703. doi: 10.13898/j.cnki.issn.1000-2200.2020.06.001
[8]	曹娟 , 王春 , 姜敏 , 范晓红 . 阻塞性睡眠呼吸暂停低通气综合征对患者外周血淋巴细胞免疫表型及C反应蛋白的影响. 蚌埠医学院学报, 2015, 40(11): 1506-1510. doi: 10.13898/j.cnki.issn.1000-2200.2015.11.014
[9]	陈杰 , 李慧慧 , Mohsin Raza kashif , 高淑娴 , 周平 , 郑庆委 , 韦莉 , 申林 , 管俊昌 . 孕鼠给予葡萄球菌肠毒素B对新生子代鼠脾脏淋巴细胞体外增殖的影响. 蚌埠医学院学报, 2018, 43(8): 981-983,988. doi: 10.13898/j.cnki.issn.1000-2200.2018.08.001
[10]	于永涛 , 丁新苑 , 云宗金 . 急性缺血性卒中病人颈动脉斑块特征与危险因素及与中性粒/淋巴细胞比值相关性分析. 蚌埠医学院学报, 2020, 45(8): 1064-1067. doi: 10.13898/j.cnki.issn.1000-2200.2020.08.019
[11]	姚春艳 , 金齐力 , 姜丽娜 , 唐洁 , 李柏青 . 正常人外周血自然杀伤T细胞(CD3⁺ CD16⁺ CD56⁺)比例. 蚌埠医学院学报, 2008, 33(4): 379-382.
[12]	魏晓巍 , 李玉云 , 张强 . 脐带血间充质干细胞的分离培养及诱导表达IDO对免疫介导再生障碍性贫血小鼠的治疗研究. 蚌埠医学院学报, 2012, 36(10): 1147-1150,1161.
[13]	王素侠 , 卜强 , 陈娟玉 . 改良外周血染色体培养效果的评价. 蚌埠医学院学报, 2019, 44(12): 1681-1683. doi: 10.13898/j.cnki.issn.1000-2200.2019.12.028
[14]	冯锡才 , 闵宏林 , 李英欣 , 唐素兰 . 溶脲脲原体两种培养方法比较. 蚌埠医学院学报, 2005, 30(2): 169-169.
[15]	章敏姬 . 2种新生儿脐带结扎方法的临床比较. 蚌埠医学院学报, 2011, 36(4): 363-365.
[16]	郑丽坤 , 张磊 , 付岭 , 刘金梅 , 陈乃耀 , 卢士红 . 中性粒细胞上清液对人脐带间充质干细胞迁移的影响. 蚌埠医学院学报, 2017, 42(7): 845-847,851. doi: 10.13898/j.cnki.issn.1000-2200.2017.07.002
[17]	赵梦蝶 , 查从亮 , 江浩 , 项平 . 人脐带血内皮祖细胞的分离、培养与鉴定. 蚌埠医学院学报, 2016, 41(9): 1121-1124. doi: 10.13898/j.cnki.issn.1000-2200.2016.09.001
[18]	项平 , 黄锦桃 , 撒亚莲 , 刘爱军 , 李海标 . 大鼠骨髓间充质干细胞的培养及鉴定. 蚌埠医学院学报, 2005, 30(1): 1-3.
[19]	赵磊 , 杨永红 , 孙桂君 . 清洁自然干燥法在新生儿脐部护理中的应用. 蚌埠医学院学报, 2013, 37(11): 1503-1505.
[20]	储磊 , 冯友江 . 多次献血对免疫功能的影响分析. 蚌埠医学院学报, 2015, 40(12): 1705-1707. doi: 10.13898/j.cnki.issn.1000-2200.2015.12.033

点击查看大图

计量

文章访问数: 2452
HTML全文浏览量: 301
PDF下载量: 26
被引次数: 0

人脐带间充质干细胞分离培养建立及其生物学特性观察

谢扬虎¹
李玉云²

作者简介: 谢扬虎(1976-),男,硕士研究生,主管检验师.

1. 蚌埠医学院临床检验诊断学实验中心, 安徽蚌埠 233030;
2. 蚌埠医学院临床检验诊断学教研室, 安徽蚌埠 233030

收稿日期: 2010-04-02

基金项目: 安徽省教育厅自然科学研究资助项目(2006KJ392B)

关键词:

摘要: 目的:建立从人的脐带组织中分离和培养脐带间充质干细胞(UC-MSCs)的方法,并探讨UC-MSCs的生物学特性。方法:分别采用原代贴壁培养法和酶消化法(胶原酶Ⅱ和胰酶)分离培养UC-MSCs,瑞氏染色观察细胞形态,流式细胞术检测第3代以后的UC-MSCs表面特异标志物表达,MTT法检测P7细胞增殖情况。UC-MSCs同外周血单个核细胞共培养后,用MTT法测定细胞增殖率,Hoechst与PI双染色,观察UC-MSCs对健康人淋巴细胞增殖的影响。结果:原代贴壁培养法1周左右可见成纤维样细胞从组织块边缘爬出,成簇生长;酶消化法3~5天可见成纤维样细胞均匀生长。传代培养后,UC-MSCs均呈长梭形、旋涡状生长;细胞核大,核仁清晰;免疫表型:CD29阳性率为(95.71±2.23)%,CD31和CD34阳性率分别为(2.47±0.54)%和(3.24±0.34)%;第7代UC-MSCs仍具有较强的分裂增殖能力。UC-MSCs对淋巴细胞的增殖反应具有抑制作用,呈细胞数量剂量依赖性,而淋巴细胞对UC-MSCs的生长未见影响。结论:从人脐带中成功分离培养的细胞具有UC-MSCs生物学特性。

English Abstract

Experimental study of humam umbilical cord mesenchymal stem cell isolation,culture and biological characteristics

XIE Yang-hu¹
LI Yu-yun²

1. Research Center of Clinical Laboratory Science, Bengbu Medical College, Bengbu Anhui 233030, China;
2. Department of Clinical Laboratory, Bengbu Medical College, Bengbu Anhui 233030, China

Received Date: 2010-04-02

Abstract: Objective:To establish a method for isolation and culture of umbilical cord mesenchymal stem cells (UC-MSCs) from human umbilical cord in vitro,and explore its biological characterstics.Methods:Human UC-MSCs were isolated and expanded in culture with explantation technique and enzyme digestion (collagenase Ⅱand trypsin),respectively;the cell morphology was observed by Wright-staining;after the third passage,the surface markers of UC-MSCs were detected and idntified by flow cytometry,and the seventh passage,the cell proliferation was analyzed by MTT assay.After UC-MSCs were co-cultured with peripheral blood mononuclear cells,the impact on the T cells were observed by Hoechst and PI double staining.Results:About one week after explantation,the fibroblast-like cells sprawled out from the edge of the tissue,growing in cluster;3-5 days after enzyme digestion,cells were watched growing fiber-shaped.After passaged,UC-MSCs showed the status of long,spindle-shaped and circinate growth;they had large nucleus and clear nucleolus.Immunophenotype analysis showed that they had strong positive expression for CD29 (95.71±2.23)%,but week expression for CD31,and CD34 were (2.47±0.54)% and (3.24±0.34)%,respectively;the seventh passage UC-MSCs still had strong proliferation ability;UC-MSCs suppressed T cells proliferation in a dose-dependent manner;lymphocytes did not affect the growth of UC-MSCs.Conclusions:The cells isolated successfully from the umbilical cord have biological characteristics of the mesenchymal stem cell.