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人关节软骨来源CD105+/CD166+间充质干细胞的分选及其多向分化特性的鉴定
Immunomagnetic sorting and differentiation of mesenchymal stem cells in human articular cartilage
- 陆军军医大学学报 2009年31卷第21期 页码：2041-2045
- 作者机构：
- 作者简介：
- 基金信息：
DOI：
中图分类号： R322.72;R329-33;R329.21-
纸质出版：2009
- 稿件说明：
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常红星,杨柳,李忠,陈光兴,刘军,文亚名,戴刚.人关节软骨来源CD105+/CD166+间充质干细胞的分选及其多向分化特性的鉴定[J].陆军军医大学学报(原第三军医大学学报),2009,31(21):2041-2045.
CHANG Hong-xing,YANG Liu,LI Zhong,CHEN Guang-xing,LIU Jun,WEN Ya-ming,DAI Gang.Immunomagnetic sorting and differentiation of mesenchymal stem cells in human articular cartilage[J].J Amry Med Univ (J Third Mil Med Univ),2009,31(21):2041-2045.
常红星,杨柳,李忠,陈光兴,刘军,文亚名,戴刚.人关节软骨来源CD105+/CD166+间充质干细胞的分选及其多向分化特性的鉴定[J].陆军军医大学学报(原第三军医大学学报),2009,31(21):2041-2045. DOI：
CHANG Hong-xing,YANG Liu,LI Zhong,CHEN Guang-xing,LIU Jun,WEN Ya-ming,DAI Gang.Immunomagnetic sorting and differentiation of mesenchymal stem cells in human articular cartilage[J].J Amry Med Univ (J Third Mil Med Univ),2009,31(21):2041-2045. DOI：
摘要
目的 建立免疫磁珠技术高效分选人关节软骨CD166
+
/CD105
+
间充质干细胞（MSCs）的方法，并鉴定其多向分化特性。 方法 酶消化法获取5例手术切除的膝关节软骨组织细胞，并行原代(P
0
代)和传代（P
2
、P
4
、P
6
代）培养；流式细胞仪检测其中CD166
+
/CD105
+
细胞百分比。比较免疫磁珠连续法与间断法分选P
4
代培养细胞中CD166
+
/CD105
+
细胞的所需时间、分选细胞活度及其百分比和收获率。对连续法分选CD166
+
/CD105
+
细胞进行成骨、成软骨、成脂肪诱导，Gomori钙钴法碱性磷酸酶（ALP）、Ⅱ型胶原免疫组化、油红O染色检测其分化特性。 结果 人关节软骨组织分离细胞和原代培养细胞中的CD105
+
/CD166
+
细胞百分比较低[（1.13±0.68）%、（5.16±3.59）%
]
，传代培养细胞中的CD105
+
/CD166
+
细胞百分比随传代次数增加而明显增高[P
2
、P
4
、P
6
代培养细胞中CD166
+
/CD105
+
细胞百分比分别为(12.68±2.59)%、（17.81±3.80）%、（19.36±3.93）%
]
（P0.05），传代培养至P
4
代，其CD10
+
/CD166
+
细胞百分比与P
6
代[（19.36±3.937）%
]
比较差异不显著（P0.05）。免疫磁珠连续法分选P
4
代培养细胞中CD105
+
/CD166
+
细胞的百分比高于间断分选法[（93.8±3.95）% vs （90.1±2.43）%，P＜0.05
]
，且分选时间显著缩短[（6.8±0.40）h vs （1200±10.21） h， P＜0.01），而细胞收获率 [(62.0±7.5）% vs (68.5±7.0）%
]
和细胞活度[（93.8±1.48）% vs （94.6±1.14）%
]
差异不显著（P＞0.05）。连续法分选CD166
+
/CD105
+
细胞成骨诱导后ALP染色阳性；成软骨诱导前Ⅱ型胶原弱阳性，诱导后Ⅱ型胶原阳性；成脂肪诱导后，细胞内可见脂肪滴，油红O染色阳性。 结论 人关节软骨P
4
、P
6
代培养细胞中的MSCs比例较高。免疫磁珠连续法能够高效分选人关节软骨MSCs，所分选的MSCs具有成骨、成软骨及成脂肪分化功能。
Abstract
Objective To establish a simple and feasible method of immunomagnetic separation of CD166
+
/CD105
+
mesenchymal stem cells (MSCs) from human articular cartilage. Methods The CD166
+
/CD105
+
MSCs in chondrocytes were isolated and cultured from 5 tissue samples of human knee articular cartilage with primary osteoarthritis (OA). The percentage of double positive cells were analyzed by flow cytometry just after digested
and in passages 0
2
4 and 6. Two techniques including the Alsalameh two stage method and our continuous process were carried out. The 2 techniques of immunomagnetic separation of CD166
+
/CD105
+
MSCs from the passage 4 chondrocytes were evaluate by separation time
cell activity
CD166
+
/CD105
+
MSCs ratio and its cell harvesting ratio. The potential of CD105
+
/CD166
+
cells to differentiate to adipogenic
osteogenic
and chondrogenic lineages was analyzed after immunomagnetic selection by histochemistry and immunohistochemistry. Results The CD105
+
/CD166
+
MSCs ratios in the chondrocytes just after digested
passage 0
2 and 4 were （1.13±0.68）%
（5.16±3.59）%
（12.68±2.59）% and（17.81±3.80）% respectively(P＜0.05); No significant difference was seen between that of the passage 4 and passage 6 [（17.81±3.80）% vs （19.36±3.93）%
P＞0.05
]
. The separation time and CD166
+
/CD105
+
MSCs ratio of the Alsalameh two stage method and the our continuous process were (6.8±0.40) h and (120.0±10.21) h
and (93.8±3.95)% and (90.1±2.43)% respectively (P0.05
0.01). The cell activity and CD166
+
/CD105
+
MSC harvesting ratio were (93.8±1.48)%
(94.6±1.14)% and (62.0±7.5)%
(68.5±7.0)% respectively (P0.05)
. Purified cartilage derived CD105
+
/CD166
+
cells did not express the markers of differentiated chondrocytes. However
they expressed type Ⅱ collagen after differentiation. Differentiation to adipocytes occurred in CD105
+
/CD166
+
cells culture
as indicated by characteristic cell morphology and oil red O staining of lipid vacuoles. Osteogenesis was observed in isolated CD105
+
/CD166
+
cells cultured in the presence of osteogenic supplements. Conclusion The CD105
+
/CD166
+
MSCs in the chondrocytes cultured from human knee articular cartilage samples covers most after chondrocytes at passage 4 and 6. Our continuous process of immunomagnetic separation of CD105
+
/CD166
+
MSCs in the chondrocytes culture is an effective
convenient and feasible method. And the cultured CD105
+
/CD166
+
cells maintain the potential to differentiate to adipogenic
osteogenic
and chondrogenic lineages.
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