| 人脐带来源Muse细胞培养、筛选、鉴定及其移植治疗大鼠脊髓损伤 |
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Received:March 12, 2019
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| 作者 | Author | 单位 | Unit | E-Mail |
| 冷子宽 |
LENG Zi kuan |
西安交通大学第二附属医院骨科, 陕西 西安 710004
郑州大学第一附属医院骨科, 河南 郑州 450052 |
Department of Orthopaedics, the Second of Affiliated Hospital of Xi'an Jiaotong University, Xi'an 710004, Shaanxi, China |
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| 高正超 |
GAO Zheng chao |
西安交通大学第二附属医院骨科, 陕西 西安 710004 |
Department of Orthopaedics, the Second of Affiliated Hospital of Xi'an Jiaotong University, Xi'an 710004, Shaanxi, China |
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| 贺西京 |
HE Xi jing |
西安交通大学第二附属医院骨科, 陕西 西安 710004 |
Department of Orthopaedics, the Second of Affiliated Hospital of Xi'an Jiaotong University, Xi'an 710004, Shaanxi, China |
xijing_h@vip.tom.com |
| 赵英杰 |
ZHAO Ying jie |
西安交通大学第二附属医院骨科, 陕西 西安 710004 |
Department of Orthopaedics, the Second of Affiliated Hospital of Xi'an Jiaotong University, Xi'an 710004, Shaanxi, China |
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| 孙丽君 |
SUN Li jun |
西安交通大学第二附属医院骨科, 陕西 西安 710004 |
Department of Orthopaedics, the Second of Affiliated Hospital of Xi'an Jiaotong University, Xi'an 710004, Shaanxi, China |
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| 翟静静 |
ZHAI Jing jing |
西安交通大学第二附属医院骨科, 陕西 西安 710004 |
Department of Orthopaedics, the Second of Affiliated Hospital of Xi'an Jiaotong University, Xi'an 710004, Shaanxi, China |
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| 许建中 |
XU Jian zhong |
郑州大学第一附属医院骨科, 河南 郑州 450052 |
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| 期刊信息：《中国骨伤》2019年32卷，第4期，第327-334页 |
| DOI：10.3969/j.issn.1003-0034.2019.04.007 |
| 基金项目:国家自然科学基金资助项目（编号：81571209） |
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目的：应用免疫磁珠筛选方法从人脐带华通胶间充质基质细胞中特异性地分选多系分化持续应激细胞（multilineage-differentiating stress-enduring，Muse），并探讨Muse细胞移植治疗大鼠亚急性脊髓损伤的安全性和有效性。
方法：将捐赠的脐带华通胶剥离剪碎、胶原酶/胰酶消化、密度梯度离心获得足量间充质基质细胞，应用免疫磁珠特异性筛选SSEA3+的Muse细胞，并使用流式细胞仪及细胞免疫组织化学方法鉴定。体内实验：使用NYU-Ⅲ代大鼠脊髓损伤打击仪建立垂直打击脊髓损伤模型，分为A组（假手术组）、B组（对照组）、C组（Non-Muse细胞移植组）和D组（Muse细胞移植组）。A组大鼠接受标准化的椎板切除手术，但不进行脊髓打击；B、C、D组大鼠均接受标准化的脊髓打击损伤，10 g撞击杆于12.5 mm高度自由落下。脊髓损伤造模后2周进行细胞移植，A组大鼠仅打开伤口不接受移植操作，B、C、D组大鼠分别接受PBS注射、Non-Muse细胞移植、Muse细胞移植，每只大鼠脊髓接受四点注射法，共4×105个细胞。自脊髓损伤造模术后1 d及1、2、3、4、5、6周分别进行大鼠BBB运动功能评分，造模术后6周（即细胞移植后4周）处死动物，用免疫荧光染色研究大鼠脊髓内移植细胞存活、迁移和分化情况。
结果：流式细胞仪显示脐带间充质基质细胞CD105+、CD90+和CD73+的百分比均达到99.5%以上，且CD45、CD14表达均低于0.5%，而SSEA3阳性率仅有1.46%。MACS特异性筛选后的细胞群有92.0%同时表达SSEA3和CD105，且免疫组织化学显示SSEA3呈典型的膜染色、形态特别。动物实验：A组动物在各个时间点的BBB评分均为21。单因素方差分析及LSD检验BBB评分情况：细胞移植后6周，C、D组与B组比较差异均有统计学意义（P=0.004，0.002），C组和D组比较差异无统计学意义。进一步组内配对t检验表明，C组第4周与第3周比较差异有统计学意义（P=0.005）；D组第4周与第3周比较差异有统计学意义（P=0.016），且第6周与第5周比较差异有统计学意义（P=0.034），说明大鼠运动功能持续改善。脊髓免疫组织化学染色显示，Muse细胞和Non-Muse细胞均由注射点向损伤区迁移，但Muse细胞存活数量明显多于Non-Muse细胞，且在损伤区均匀分布。
结论：特异性免疫磁珠筛选是获得高纯度Muse细胞的良好高效的方法，Muse细胞可在大鼠损伤脊髓内大量存活4周并向损伤区迁移，且能持续改善大鼠运动功能，Muse细胞有望成为治疗脊髓损伤的一种新的种子细胞。 |
| [关键词]:脐带 细胞培养技术 细胞移植 脊髓损伤 |
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| Cultivation,screening,identification and transplantation of Muse cell from human umbilical cord-derived for spinal cord injury in rats |
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Abstract:
Objective:To investigate multilineage-differentiating stress-enduring (Muse) by immunomagnetic bead screening from Wharton's jelly mesenchymal stromal cells(WJ-MSCs),and explore transplantation of Muse cell for safety and effectivensess of sub acute cord injury in rats.
Methods:Donated Wharton's Jelly-mesenchymal stromal cells (WJ-MSCs) were successfully derived from a human umbilical cord by a series of procedures namely physical isolation of Wharton's Jelly from cord membrane,collagenase and trypsin treatment and density gradient centrifugation. Magnetic activated cell sorting was performed to specifically select SSEA3+ Muse cells,and flow cytometry and immunocytochemistry were used to identify further. In vivo,spinal cord contusion injury model in rats was induced by NYU-Ⅲ impactor,and were randomly divided and equally into four groups,namely group A (sham),group B (control),group C (Non-Muse cells transplantation) and group D (Muse cells transplantation). Laminectomy was conducted in group A but no spinal cord contusion injury. Laminectomy and cord injury were performed in group B,C and D,10 g trip rod was freely falling down from 12.5 mm. Two weeks later,group B,C and D were received PBS injection,Non-Muse cells transplantation and Muse cells transplantation respectively,four-point injection were performed in each cord with totally 4×105 cells. BBB scores were evaluated on 1 day,1,2,3,4,5 and 6 week after injury. Four weeks after cell transplantation,the rats were sacrificed,and immunohistochemistry were carried out to observe survival,migration and differentiation of the injected cells.
Result:The expression of CD105,CD90 and CD73 were over 99.5% in the derived WJ-MSCs population,but CD45 and CD14 were lower than 0.5%,positive rate of SSEA3+ was 1.46% under flow cytometer,However,after MACS sorting,the percentage of 92.0% Muse cells expressed SSEA3 and CD105,and immunohistochemistry results of SSEA3 showed typically membrane morphology with special processes. In vivo,BBB scores was 21 in group A at different time points. One-way ANOVA and LSD analysis showed that BBB scores in group C and D were significantly higher than that in group B (P=0.004,0.002),but there was no significantly difference between group C and D. Further intra-group paired t test showed that BBB score was significantly higher at 4 weeks than that 3 weeks in group C (P=0.005). However,in group D,BBB scores were significantly higher at 4 and 6 week than those at 3 and 5 weeks,P values were 0.005 and 0.016 respectively. Immunohistochemistry results showed that both Muse cells and Non-Muse cells could survive for 4 weeks in rats and they migrated from the four-point injection to injury site. But there showed more Muse cells survival than Non-Muse cells in the cord.
Conclusion:Immunomagnetic bead screening is efficient to select large number of purified SSEA3+ Muse cells. Muse cells could survive and target-migrate in injured cord to improve BBB scores continuously. Muse cells are a novel kind of seed cells in the spinal cord injury treatment. |
| KEYWORDS:Umbilical cord Cell culture techniques Cell transplantation Spinal cord injuries |
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| 中文格式: | 冷子宽,高正超,贺西京,赵英杰,孙丽君,翟静静,许建中.人脐带来源Muse细胞培养、筛选、鉴定及其移植治疗大鼠脊髓损伤[J].中国骨伤,2019,32(4):327~334 |
| 英文格式: | LENG Zi kuan,GAO Zheng chao,HE Xi jing,ZHAO Ying jie,SUN Li jun,ZHAI Jing jing,XU Jian zhong.Cultivation,screening,identification and transplantation of Muse cell from human umbilical cord-derived for spinal cord injury in rats[J].zhongguo gu shang / China J Orthop Trauma ,2019,32(4):327~334 |
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