来源于骨髓_外周血与脐带血的间充质干细胞生物学特性比较

中国组织工程研究与临床康复 第13卷 第45期 2009–11–05出版

Journal of Clinical Rehabilitative Tissue Engineering Research November 5, 2009 Vol.13, No.45

P .O.Box 1200,Shenyang 110004 kf23385083@ 8966

1

Department of Hematology, 2

Department of Blood Transfusion, Navy General Hospital of Chinese PLA, Beijing 100048, China

Huang You-zhang, Associate chief technician, Department of Hematology, Navy General Hospital of Chinese PLA, Beijing 100048, China

huangyouzhang08@

Supported by: the Scientific Research Plan of Ministry of Health and Navy Logistics Department,No. 200828203*

Received: 2009-06-09 Accepted: 2009-09-01

解放军海军总医院，1血液科，2

输血科，北京市100048

黄友章，男，1956年生，湖南省长沙市人，汉族，1992年中国科技大学毕业，副主任技师，主要从事血液学方面的研究。huangyouzhang 08@yahoo.

海军后勤部卫生部科研计划课题(200828203)*

中图分类号:R394.2 文献标识码:B

文章编号:1673-8225 (2009)45-08966-05

收稿日期：2009-06-09

修回日期：2009-09-01(20090609014/ ZS ·Q)

来源于骨髓、外周血与脐带血的间充质干细胞生物学特性比较*

黄友章1，沈建良1，宫立众1，尹文杰1，刘 毅1，成 海2，郑培浩1，岑 坚1

Comparison of biological characteristics of mesenchymal stem cells derived from bone marrow,

peripheral blood and cord blood

Huang You-zhang 1, Shen Jian-liang 1, Gong Li-zhong 1, Yin Wen-jie 1, Liu Yi 1, Cheng Hai 2, Zheng Pei-hao 1, Cen Jian 1

Abstract

BACKGROUND: Mesenchymal stem cells (MSCs) exist in human tissues. Presently, cell source is single; culture method has great differences; obtained results are not consistent. Thus, it cannot verfy that isolated and cultured cells are identical cells, which is difficult to compare.

OBJECTIVE: To compare the biological features of MSCs derived form bone marrow (BM), perpheral blood (PB) and cord blood (CB) under in vitro culture conditions.

DESIGN, TIME AND SETTING: The cytological in vitro controlled study was performed at the Department of Hematology, Navy General Hospital of Chinese PLA from June 2007 to December 2008.

MATERIALS: A total of 10 donors of hemopoietic stem cell transplantation at the Department of Hematology, Navy General

Hospital of Chinese PLA were selected. MB and PB cells were obtained from the same donor, and cell volumes were respectively 20 mL and 2 mL. CB cells (30 mL) were obtained from healthy primipara at the Department of Obstetrics, Navy General Hospital of Chinese PLA.

METHODS: MSCs were obtained from BM, PB and CB by Percoll density gradient + adherence method, and then incubated in DMEM/F12 medium containing 10% fetal bovine serum. When 80%-90% confluency, cells were digested in trypsin-EDTA and made into 5×108/L cell suspension as P 0. Above-described operation was performed as P 1, and the rest may be deduced by analogy as P 2-P 5.

MAIN OUTCOME MEASURES: The following parameters were measured: cell growth morphology; results of Wright-Giemsa staining; results of cytochemistry; cell proliferation amount; cell surface markers using flow cytometry.

RESULTS: Time of adherence, time to 50% confluency and time to 80% confluency of BMSCs were earlier comarped with the PBMSCs and UCMSCs. Adherent cells from BM grew in whirpool-like type, while CB and PB did not at 5-7 days. Majority of

aderent cells from BM were fibroblast-like cells, and small parts were endothelioid cells. Aderent cells from PB and CB at the fifth generation contained more endothelioid cells and mononuclear and macrophage-like cells besides fibroblast-like cells. PAS stain, Sudan black B stain, alkaline phosphatase (AKP) staining of adherent cells from BM, PB and CB were negative from P 1 to P 5. Compared with P0 cells, number of BMMSCs till P 5 was significantly more in PBMSCs and UCMSCs (P < 0.05). Positive rates of CD29, CD44, CD90, CD71, CD105, CD166 and HLA-ABC were 55.9%-92.8% at P0 to P5, but ≤6% following BMMSCs were incubated; 19.7%-33.4% at P0 to P5, but ≤10% following PBMSCs were incubated; 35.4%-93.2% at P 0 to P 5, but ≤20%

following CBMSCs were incubated. Positive rates of CD34, CD45 and HLA-DR were low in BM-, PB- and CB-MSCs. Positive rates of CD14 and CD31 were low in BMMSCs; 12.1%-28.3% in PBMSCs, and 8.1%-21.3% in CBMSCs.

CONCLUSION: MSCs can be attained from BM, PB and CB. Quantities of MSCs form BM are the highest, with single component, followed by CBMSCs and PBMSCs, with multiple components.

Huang YZ, Shen JL, Gong LZ, Yin WJ, Liu Y, Cheng H, Zheng PH, Cen parison of biological characteristics of mesenchymal stem cells derived from bone marrow, peripheral blood and cord blood.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu. 2009;13(45): 8966-8970. [ ]

摘要

背景：间充质干细胞存在于人体多种组织，目前研究报道所用细胞来源单一，培养方法差异大，得出的结果不一致，不能证明分离、培养出的细胞是相同的细胞，难以比较。

目的：课题提出在体外培养条件下可对同一个体来源的骨髓、外周血来源间充质干细胞及不同个体脐带血来源间充质干细胞的生物学特性进行比较的理论假设。

设计、时间及地点：细胞学体外对照观察，于2007-06/2008-12在解放军海军总医院血液科完成。

材料：解放军海军总医院血液科造血干细胞移植供者10例，取同一供者的骨髓、外周血细胞，细胞体积分别为20 mL 与2 mL 。解放军海军总医院产科10例健康初产妇脐带血细胞，细胞体积为30 mL 。

方法：采用Percoll 密度梯度+贴壁法分离骨髓、外周血、脐带血来源的间充质干细胞，加入含体积分数为10%胎牛血清的DMEM/F12培养基。当细胞生长汇合至80%~90%时胰蛋白酶-EDTA 消化，制成5×108

L -1

细胞悬液，计为P 0。重复上述操作，即为P 1，依此类推P 2~P 5。

主要观察指标：细胞生长形态观察，细胞瑞氏-吉姆萨染色结果，细胞化学染色结果，细胞增殖数量，流式细胞仪检测细胞表面标记。

结果：①骨髓间充质干细胞贴壁时间、汇合至50%时间、汇合至80%时间均早于外周血、脐带血间充质干细胞；骨髓间充质干细胞培养5~7 d 时呈漩涡状生长，而外周血、脐带血间充质干细胞无此生长特性。②初始培养和传代培养后，骨髓间充质干细胞多为成纤维样细胞，仅有少量内皮样细胞；而传至第5代的外周血、脐带血间充质干细胞除有成纤维样细胞外，还有较多的内皮样细胞和单核-巨噬样细胞。③P 1~P 5骨髓、外周血及脐带血来源间充质干细胞PAS 染色、碱性磷酸酶染色、苏丹黑B 染色均呈阴性。④与P 0细胞比较，传代培养至P 5后骨髓间充质干细胞增殖数量明显多于外周血、脐带血间充质干细胞(P < 0.05)。⑤CD29，CD44，CD90，CD71，CD105，CD166和HLA-ABC 阳性率，骨髓间充质干细胞接种后≤6%，

基础医学