大鼠骨髓间充质干细胞分离鉴定方法及生物学特性

doi:10.3969/j.issn.2095-4344.2017.09.009

摘要/Abstract

摘要：

文章快速阅读：

文题释义：
CD34分子：是高度糖基化的Ⅰ型跨膜糖蛋白，选择性地表达于人类及其他哺乳动物造血干/祖细胞表面，并随细胞的成熟逐渐减弱至消失。目前已有愈来愈多的研究结果表明CD34分子在介导细胞间黏附作用中发挥着重要作用，可以参与造血干细胞的运输、定植，参与炎症反应以及淋巴细胞的归巢。
CD44分子：参与细胞-细胞、细胞-基质间的相互作用，主要参与异质性黏附，在调控细胞生长、生存、分化和迁移中发挥重要作用，同时作为受体协同分子，CD44还可以与多种分子，包括细胞外基质、生长因子、基质金属蛋白酶等相互作用，参与肿瘤细胞增殖、迁移等的调控。

摘要
背景：如何能简便高效地获得均质性的骨髓间充质干细胞群是软骨组织工程研究的重点。
目的：分离培养大鼠骨髓间充质干细胞，观察其生物学特性。
方法：抽取30只SD大鼠股骨以及胫骨骨髓，采用全骨髓贴壁培养法体外分离培养骨髓间充质干细胞，流式细胞仪测定细胞表面标记物的表达，细胞计数法观察细胞生长增殖能力，MTT法检测细胞存活率。
结果与结论：①刚分离培养的细胞呈圆形、椭圆形，部分细胞呈三角形，接种12-15 d后，细胞开始融合，传代后细胞增殖速度加快，传代2 h后细胞均匀分布在瓶底；②随着传代次数的增加，细胞增殖能力出现下降趋势，第1-5代细胞存活率均超过95%，显著高于第6代和第7代(P < 0.05)；③骨髓间充质干细胞表面CD34呈阴性表达，CD44呈阳性表达；④结果表明，采用全骨髓贴壁法可有效分离大鼠骨髓间充质干细胞，获得的细胞生长稳定，增殖能力强，可选用前5代细胞作为软骨组织工程的种子细胞。

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

ORCID: 0000-0002-2309-8579(曹华)

关键词: 干细胞, 骨髓干细胞, 骨髓间充质干细胞, 全骨髓贴壁培养, 生物学特性, 流式细胞仪, 标记物, 增殖能力

Abstract:

BACKGROUND: How to effectively harvest bone marrow mesenchymal stem cells with homogeneity is crucial for cartilage tissue engineering research.
OBJECTIVE: To isolate and culture rat bone marrow mesenchymal stem cells and to observe biological characteristics of the cells.
METHODS: Bone marrow mesenchymal stem cells were isolated from the femur and tibia of 30 Sprague-Dawley rats using the whole bone marrow adhesion method. Cell surface markers were identified using flow cytometry, cell proliferation ability was observed through cell counting, and cell survival rate was determined by MTT assay.
RESULTS AND CONCLUSION: The harvested cells were mostly round and oval, which partially showed a triangular shape. Cell fusion appeared after 12-15 days of inoculation. The proliferation of passaged cells was accelerated. Two hours after passage, the cells evenly covered the bottom of the culture dish. With the increasing of cell generations, the cell proliferation ability was gradually decreased. The survival rates of cells at passages 1-5 were over 95%, significantly higher than those at passages 6 and 7 (P < 0.05). The harvested cells were negative for CD34, but positive for CD44. To conclude, the whole bone marrow adhesion method is effective to isolate rat bone marrow mesenchymal stem cells with stable growth and strong proliferation ability. Passage 5 cells can be selected as seed cells for cartilage tissue engineering.

Key words: Bone Marrow, Mesenchymal Stem Cells, Cell Proliferation, Tissue Engineering

中图分类号:

R394.2

曹华，高建华，刘小龙，林思思. 大鼠骨髓间充质干细胞分离鉴定方法及生物学特性[J]. 中国组织工程研究, 2017, 21(9): 1357-1361.

Cao Hua, Gao Jian-hua, Liu Xiao-long, Lin Si-si. Isolation, identification and biological characteristics of rat bone marrow mesenchymal stem cells[J]. Chinese Journal of Tissue Engineering Research, 2017, 21(9): 1357-1361.

图/表（结果） 1

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引言

材料方法

1.1 设计 细胞学观察实验。

1.2 时间及地点 于2013年10月至2015年10月在广西医科大学第一附属医院完成。

1.3 材料 健康SD大鼠30只，3-6个月龄，雌雄随机，SPF级，体质量为0.13-0.35 kg，平均(0.34±0.02) kg，由广西医科大学第一附属医院动物实验中心提供，严格遵守相关标准饲养。对SD大鼠的处理符合《关于善待实验动物的指导性意见》中相关规则^[19-20]。

1.4 实验方法

1.4.1 骨髓间充质干细胞分离、培养 取30只SD大鼠，给予2%戊巴比妥钠腹腔注射麻醉后处死，体积分数为75%乙醇浸泡10 min，在无菌条件下取出股骨以及胫骨，PBS冲洗3次^[21-22]，用含有青霉素-链霉素的L-DMEM培养基进行反复吹打，制备骨髓单细胞悬液，1 000 r/min离心5 min，去除上层清液，以细胞浓度为1×10⁹ L^-¹接种于25 cm²培养瓶，放入培养箱中进行培养^[23]。培养3 d后进行全量换液，待细胞贴壁融合80%左右时进行传代培养，每2 d换液1次，在倒置显微镜下观察细胞生长情况^[24-25]。

1.4.2 骨髓间充质干细胞鉴定 利用流式细胞分析仪检测骨髓间充质干细胞表面标记物的表达，取第3代骨髓间充质干细胞，吹打成单细胞悬液，移入10 mL离心管中，1 000 r/min离心5 min，去除上清液，PBS重悬，重复离心，连续洗涤3次，调整细胞浓度为2×10⁹ L^-¹，200目筛网过滤，备用，取流式细胞仪专用试管4个，每管加入100 μL细胞悬液，加入荧光抗体CD44-APC小鼠抗人单克隆抗体、CD34-PE小鼠抗人单克隆抗体及同型对照抗体各5 μL，孵育15 min，采用流式细胞仪进行测定^[26-27]。

1.4.3 骨髓间充质干细胞增殖能力 观察第1代、第3代、第5代以及第7代骨髓间充质干细胞在培养第1，2，3，4天时的生长情况^[28-29]。

1.4.4 骨髓间充质干细胞活力测定^[30-31] 采用MTT法检测第1-7代骨髓间充质干细胞活力。将细胞浓度为1×10⁸ L^-¹的骨髓间充质干细胞接种于96孔培养板中，加20 μL 5 g/L MTT培养4 h，小心吸弃全部上清液，并加入150 μL二甲基亚砜，充分振荡、溶解，采用酶联检测仪在570 nm波长下测定细胞吸光度，计算存活率，重复测定3次，取平均值。

1.5 主要观察指标 ①骨髓间充质干细胞形态；②骨髓间充质干细胞表面标记物的表达；③骨髓间充质干细胞的增殖能力；④骨髓间充质干细胞存活率。

1.6 统计学分析 采用SPSS 18.0软件处理数据，计量资料采用x(_)±s表示，行t 检验，P < 0.05为差异有显著性意义。

讨论

关节软骨缺损是临床上常见的疾病，手术治疗虽然能够提高临床疗效，但是患者容易产生二次创伤，治疗预后较差^[32]。近年来，软骨组织工程得到飞速发展，但是由于各种因素限制等，该治疗方法难以在基层推广应用。另外，关节软骨缺损患者选择该方法治疗时由于缺乏理想的种子细胞，也使得其应用受到很大限制。自体软骨细胞是目前最为理想的细胞，但是细胞数量有限，长期体外培养容易引起细胞老化，严重者甚至出现细胞生物功能退变，丧失分化能力。异体软骨细胞移植时又会产生免疫排斥问题，导致修复效果不理想^[33]。

骨髓间充质干细胞是一类比较特殊的细胞，该细胞能够在微环境信号的指令下分化为成骨细胞以及软骨细胞，在软骨下骨损伤修复中能够取得理想的效果。由于关节腔内环境相对特殊，机体内的各种细胞因子等能促进骨髓间充质干细胞在特定条件下向软骨细胞分化，从而形成软骨组织^[34-45]。由此看出：骨髓间充质干细胞是软骨组织中比较合适的种子细胞^[46-47]。

目前，骨髓间充质干细胞的分离方法主要有密度梯度离心法、贴壁细胞分离法、流式细胞仪等，这些方法各有优缺点^[48]。贴壁细胞分离法即根据干细胞在低血清培养基中有贴壁优势特性，定期换液除去不贴壁细胞，从而达到纯化及扩增间充质干细胞的目的。密度梯度离心法即根据骨髓中各细胞成分密度的不同，分离单核细胞进行贴壁培养。二者本质上无多大区别。随着对间充质干细胞表面抗原认识的深入，又有人利用免疫方法如流式细胞仪法、免疫磁珠法等对其进行分离纯化。但经过流式或磁珠分选后的细胞出现了增殖缓慢等一些问题，加之成本较高和技术的难度，在某种程度上限制了这些方法的广泛应用。鉴于实验条件，实验采用全骨髓贴壁细胞分离法对骨髓间充质干细胞进行培养。该方法操作相对简单、快速、实用，是一种比较理想的分离、纯化方法。

研究表明，骨髓间充质干细胞表面有多种抗原，如CD34、CD44等。CD34在骨髓成纤维细胞、造血干细胞中表达较多，CD44属于细胞黏附分子，为多功能透明质酸受体，具有透明质酸、胶原蛋白、纤维黏连蛋白等细胞外基质的结合位点，通过其胞质内区域丝氨酸和苏氨酸的磷酸化和去磷酸化，CD44与细胞骨架蛋白相互作用，使骨髓间充质干细胞和细胞外基质蛋白结合。骨髓间充质干细胞通过表达CD44介导细胞间、细胞与细胞外基质间的相互作用，影响自身的生长、增殖和分化，在骨髓微环境中起重要作用。流式细胞仪检测结果显示CD34呈阴性表达，CD44呈阳性表达，由此看出骨髓间充质干细胞是一种不同于造血干细胞的间充质干细胞。

综上所述，实验建立起了体外分离培养骨髓间充质干细胞的方法，初步了解了间充质干细胞的一些生物学特性，为应用骨髓间充质干细胞作为种子细胞，通过组织工程方法修复关节软骨缺损提供了实验基础。中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程