胎盘源间充质干细胞分离提取的新方法

doi:10.3969/j.issn.2095-4344.2015.10.024

摘要/Abstract

摘要：

背景：胎盘是获取间充质干细胞的理想来源，在干细胞治疗和再生医学中具有潜在广泛的用途，建立高效分离提取方法有助于其存储和临床应用。

目的：分析组织绞碎加酶液消化法培养人胎盘间充质干细胞的生物学特性。

方法：采用组织绞碎加酶液消化法分离、富集胎盘组织中间充质干细胞，应用流式细胞术检测细胞表面标记，MTT法分析增殖能力，并采用诱导剂定向诱导胎盘间充质干细胞成脂、成骨和成软骨分化。

结果与结论：胎盘组织来源间充质干细胞在体外可稳定长期传代培养，应用流式细胞仪检测结果显示 CD73、CD90、CD105表达阳性，CD11b、CD19、CD34、CD45、HLA-DR表达阴性。胎盘间充质干细胞增殖活跃，第3-5天进入对数生长期，第6天进入平台期。经诱导后，胎盘间充质干细胞具有向脂肪细胞、骨细胞、软骨细胞分化的潜能。结果表明联合利用组织绞碎和酶液消化法可高效获取胎盘间充质干细胞，在体外培养过程中生长稳定，增殖能力强，可长期传代。

1库：

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

全文链接：

关键词: 干细胞, 培养, 胎盘间充质干细胞, 组织绞碎, 酶消化, 细胞培养

Abstract:

BACKGROUND: Placenta is a valuable source of mesenchymal stem cells for stem cell therapy and future application in the field of regenerative medicine. However, conventional methods cannot acquire a large amount of purified human placenta-derived mesenchymal stem cells. Here, we present a new method for isolating human placenta-derived mesenchymal stem cells suitable for banking strategies and for future clinical applications.

OBJECTIVE: To analyze the biological characteristics of human placenta-derived mesenchymal stem cells cultured by tissue dissociating and collagenase digestion.

METHODS: Human placenta-derived mesenchymal stem cells were obtained from human placenta by tissue dissociating and collagenase digestion method. Immunophenotype was analyzed by flow cytometry. Growth curve was determined by MTT assay, and differentiation ability was evaluated by in vitro adipogenic, osteogenic and chondrogenic induction as well.

RESULTS AND CONCLUSION: Human placenta-derived mesenchymal stem cells could be passaged stably in vitro. Furthermore, the cells expressed CD73, CD90, CD105, but were negative for the markers of CD11b, CD19, CD34, CD45, and HLA-DR. Human placenta-derived mesenchymal stem cells proliferated actively and began to grow logarithmically at days 3-5 followed by a plateau period at day 6. In addition, the isolated cells could be induced into adipocytes, osteocytes, chondrocytes in vitro. In a word, the results of this study demonstrated that the tissue dissociating and collagenase digestion method is an efficient method for obtaining a large amount of human placenta-derived mesenchymal stem cells that can be stably cultured in vitro and have strong proliferative ability.

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Key words: Placenta, Mesenchymal Stem Cells, Cell Culture Techniques

中图分类号:

R394.2

刘洋，李艳琪，王洪一，吴晓冰，荆永光，徐潇，姚尧，张宇，吴祖泽，靳继德. 胎盘源间充质干细胞分离提取的新方法[J]. 中国组织工程研究, 2015, 19(10): 1608-1612.

Liu Yang, Li Yan-qi, Wang Hong-yi, Wu Xiao-bing, Jing Yong-guang, Xu Xiao, Yao Yao, Zhang Yu, Chu-Tse Wu, Jin Ji-de. A new method to isolate mesenchymal stem cells from human placenta[J]. Chinese Journal of Tissue Engineering Research, 2015, 19(10): 1608-1612.

图/表（结果） 4

2.1 胎盘间充质干细胞的形态胎盘间充质干细胞接种48 h更换培养液时可见细胞呈散在分布的贴壁克隆，长梭形，有胞浆突起，少数呈多角形，传至第3代后的细胞生长速度较快，镜下观察形态均一，平行排列或漩涡状生长，应用吉姆萨染色后可明显见长梭形、纤维样生长形态(图1)。

2.2 细胞免疫表型鉴定结果流式细胞分析结果显示所得细胞高表达CD73(99.91%)、CD90(100%)、CD105(99.14%)，不表达CD34(0.02%)、CD19(0.02%)、CD11b(0.38%)、CD45(0.93%)、HLA-DR(0.18%)(图2)，说明利用组织绞碎加酶液消化法贴壁培养得到的细胞是间充质干细胞。

2.3 人胎盘源间充质干细胞生长曲线 MTT法测得生长曲线可知，细胞传代后前2 d增殖不明显，3-5 d进入对数生长期，第6天开始进入平台期，根据生长曲线计算细胞培养第5天的倍增时间为50 h，见图3。

2.4 人胎盘源间充质干细胞成脂、成骨和成软骨分化能力成脂诱导后，细胞形态逐渐由典型的长梭形变短、变圆，呈椭圆形。诱导10 d后于镜下可见胞浆内出现点状小脂滴，继续诱导培养1周，胞浆内脂滴增多增大。油红O染色可见胞浆内的大量脂滴染成红色(图4A)，说明胎盘源间充质干细胞在体外具有向脂肪细胞分化的潜能。成骨诱导1周左右，细胞逐渐由长梭形变为方形、鳞片形。诱导2周后，细胞外基质分泌增多，胞质中及胞质外出现许多黑色小颗粒， 3周后可见结节状结构。茜素红染色可见红色矿化沉着物(图4B)，进一步观察间充质干细胞软骨分化能力，应用甲苯胺蓝染色检测蛋白多糖表达，镜下可见均一蓝染(图4C)，说明人胎盘源间充质干细胞在体外具有向成脂、成骨和成软骨细胞分化的潜能。

参考文献

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引言

材料方法

设计：细胞学观察实验。

时间及地点：2013年11月至2014年3月在军事医学科学院放射与辐射研究所实验室进行。

材料：足月剖腹产胎儿的胎盘来自解放军307医院，遵照医院规定并经家属书面同意获取。

实验方法：

胎盘源间充质干细胞分离、培养：在无菌条件下获取人胎盘组织，经PBS反复冲洗，去除胎盘血，并剪去残余脐带组织，放置于无菌方盘中待用。组织绞碎联合酶液消化法步骤：①先通过组织绞碎分离机处理胎盘组织，将胎盘打碎最终得到胎盘组织碎片(近似肉糜)。②将胎盘组织碎片置于200 mL无菌离心管中，联合应用0.25%胰蛋白酶和0.1%Ⅰ型胶原酶消化胎盘组织碎片。③37 ℃摇床消化30 min，去除组织留取消化液，经1 500 r/min离心5 min，收集离心获得的细胞沉淀。④将离心后收集到的细胞重悬于含体积分数为10%胎牛血清的α-MEM培养基，在37 ℃，体积分数为5% CO₂培养箱中培养。⑤培养48 h后半量换液，去除未贴壁细胞，以后每三四天换液1次，待间充质干细胞贴壁形成克隆岛后进行消化得到胎盘源的原代间充质干细胞，可继续传代培养或冻存于液氮罐中。

免疫表型鉴定：将接近融合的第3代胎盘源间充质干细胞用胰酶消化，离心洗涤，制备成2×10⁵/管细胞悬液，分别加入单克隆抗人抗体 CD19、CD73、 CD105、HLA-DR、 CD11b、CD34、CD45、CD90避光孵育30 min，加入 1 mL PBS，1 500 r/min离心5 min，弃上清，重复2次；将细胞重悬于500 μL PBS中，用流式细胞仪检测细胞的表面标记。

MTT法绘制生长曲线：取第3代胎盘源间充质干细胞，以1×10⁷ L^-¹的细胞浓度接种于96孔板，每孔250 μL，各设6个复孔，在37 ℃、体积分数5% CO₂培养箱中培养。每天在固定时间点取出96孔板，每孔加入20 μL MTT(5 g/L)，37 ℃培养箱中放置4 h；轻轻吸掉培养基上清，然后每孔加入二甲基亚砜150 μL，用酶标仪(波长490 nm)测定各孔吸光度值，以时间为横轴、吸光度值为纵轴绘制生长曲线。

胎盘间充质干细胞成脂、成骨和成软骨分化潜能检测：将第3代胎盘源间充质干细胞以1×10⁷ L^-¹的细胞浓度接种于24孔板内。当细胞达到70%融合时，去掉原培养基，加入预先配好的成脂、成骨和成软骨诱导培养基，每隔3 d换1次液，诱导两三周。当成脂诱导组观察到胞质中出现脂滴时，小心移去培养液，用PBS轻洗2次，40 g/L多聚甲醛固定20 min，油红O染色后于显微镜下观察、拍照。当成骨诱导组镜下观察到有圆形钙化结节形成时用茜素红染色鉴定，软骨诱导应用甲苯胺蓝浸染后镜下观察结果^[18]。

主要观察指标：联合应用组织绞碎分离法和胶原酶消化法获取胎盘源间充质干细胞，对获取的细胞进行形态学观察、增殖能力、细胞表面标志及分化功能的鉴定。

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讨论

间充质干细胞一直为干细胞领域的研究热点，它不仅支持造血系统，而且还可向中胚层和外胚层来源的组织分化，在特定的培养条件下可以分化为骨、脂肪、软骨、肌肉及内皮等组织细胞^[19-24]。

胎盘来源的间充质干细胞易分离培养，增殖迅速，具有分化潜能^[25-27]。与捐献骨髓或采集动员外周血相比，胎盘供者无痛苦，感染机会少，胎盘在临床上为废弃物，其应用和研究不涉及伦理问题。胎盘来源间充质干细胞广义上主要分两种来源：胎盘血间充质干细胞和胎盘组织间充质干细胞。由于胎盘组织具有独特、复杂的解剖结构，其内有大量交错的血管网络及结缔组织，因此传统意义上的分离提取间充质干细胞大部分是来自于胎盘血，该方法是通过选择合适的介质，利用密度梯度离心进行分选，其优点在于获得细胞纯度、活性较好，但由于对胎盘组织处理繁复，分选条件的摸索以及人员操作的不均一性，最终导致各大实验室分选结果不尽相同。因此，建立便捷高效的胎盘源间充质干细胞分离新方法有助于为基础研究提供理想的细胞和基因治疗的载体^[28-30]。

本实验室建立的胎盘源间充质干细胞分离新方法，有别于传统的利用介质梯度离心法或单纯的消化法获取胎盘源间充质干细胞，课题组首先利用组织绞碎机将胎盘打碎成肉糜样，得到胎盘组织碎片之后再联合用胰酶和胶原酶进行短时消化。将绞碎和酶消化二者联合应用可更加高效便捷的获取胎盘源间充质干细胞，并且该方法消化时间短，分离提取过程中对细胞损伤小，为后续实验的进行提供质量保障。经分离培养的胎盘源间充质干细胞，应用流式细胞仪鉴定免疫表型，证明与骨髓源间充质干细胞相似，经吉姆萨染色镜下观察见细胞形态为成纤维细胞样，并呈漩涡状生长，可稳定传代。应用MTT法测定细胞生长曲线，发现胎盘源间充质干细胞增殖活跃，具有较高的分裂速度。通过成脂、成骨、成软骨诱导实验，可以明确这一部分细胞保留自我更新和多向分化潜能，符合干细胞特性。

综上所述，胎盘源间充质干细胞是一种较为安全可靠的间充质干细胞来源，并且通过组织绞碎加酶液消化法可高效获得胎盘源间充质干细胞，该细胞增殖速度较快，成活率较高，具有多向分化的潜能，在体外可实现快速大量扩增，因此，该分离培养方法为胎盘源间充质干细胞基础研究与临床细胞治疗奠定了实验基础。

1库：

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
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