CM-DiI标记人骨髓间充质干细胞并检测其移植后产生脑源性神经营养因子的实验研究

doi:10.3969/j.issn.1006-7795.2017.03.019

首都医科大学学报 ›› 2017, Vol. 38 ›› Issue (3): 423-430.doi: 10.3969/j.issn.1006-7795.2017.03.019

CM-DiI标记人骨髓间充质干细胞并检测其移植后产生脑源性神经营养因子的实验研究

赵春松^1,2, 邹海强³, 李晓波⁴, 闫晓明⁵, 关云谦^1,2, 张愚^1,2

1. 首都医科大学宣武医院细胞生物室, 北京 100053;
2. 教育部神经变性病重点实验室, 北京 100053;
3. 广州军区广州总医院神经内科, 广州 510010;
4. 苏北人民医院神经内科, 江苏扬州 225001;
5. 首都医科大学宣武医院功能神经外科, 北京 100053

收稿日期:2016-10-17 出版日期:2017-05-21 发布日期:2017-06-14
通讯作者: 关云谦 E-mail:guanyunqian@aliyun.com
基金资助:
国家自然科学基金（81371377），北京市科委健康培育项目（Z111107067311033），北京市科技新星项目（2009B22）。

CM-DiI labeling human bone marrow mesenchymal stem cells and was used in identifying BNDF expressing cell after transplantation

Zhao Chunsong^1,2, Zou Haiqiang³, Li Xiaobo⁴, Yan Xiaoming⁵, Guan Yunqian^1,2, Zhang Yu^1,2

1. Department of Cell Biology, Xuanwu Hospital, Capital Medical University, Beijing 100053, China;
2. Key Laboratory of Neurodegeneration, Ministry of Education, Beijing 100053, China;
3. Department of Neurology, The General Hospital of Guangzhou Military Command in Guangzhou, Guangzhou 510010, China;
4. Department of Neurosurgery, Northern Jiangsu People's Hospital, Yangzhou 225001, Jiangsu Province, China;
5. Department of Function Neurosurgery, Xuanwu Hospital, Capital Medical University, Beijing 100053, China

Received:2016-10-17 Online:2017-05-21 Published:2017-06-14
Supported by:
This study was supported by National Natural Science foundation of China (81371377), Beijing Municipal Science and Technology Commission (Z111107067311033),Beijing Nova Program of Science and Technology (2009B22).

摘要/Abstract

摘要： 目的观察荧光染料（CM-DiI）标记人骨髓间充质干细胞（bone marrow mesenchymal stem cells，BM-MSC）后，是否会脱落造成周围细胞被污染，标记细胞经静脉移植入脑梗死大鼠后是否会造成宿主脑内神经元被误染色；以及是否可以鉴定人BM-MSC产生脑源性神经营养因子（brain derived neurotrophic factor，BDNF）。方法应用不同浓度CM-DiI分别标记培养的人BM-MSC，观察标记效率。将转染绿色荧光蛋白（green fluorescence protein，GFP）的人胚肾293T细胞和CM-DiI标记的人BM-MSC细胞共培养，7 d之后观察CM-DiI的红色荧光和293T细胞是否双标记。将CM-DiI标记的人BM-MSC经静脉移植入脑梗死大鼠体内，观察植入细胞的红色荧光是否会沾染宿主细胞以及和BDNF免疫荧光的双标记。结果不同浓度CM-DiI（1 000、200、100、20 nmol/L）均可成功标记人BM-MSC，其中1 000、200 nmol/L浓度标记24 h后观察标记效率均达到98%以上，比100、20 nmol/L标记的效率明显升高（P<0.01）。CM-DiI标记后的人BM-MSC与GFP标记的293T细胞共培养，7 d之内没有发现红色荧光沾染绿色的293T细胞。移植后第3天，静脉植入脑梗死大鼠的标记细胞的红色荧光没有沾染宿主脑内神经元，而且可以和BDNF的绿色荧光形成双标记。结论 CM-DiI可以高效地标记人BM-MSC。采用200 nmol/L浓度标记的人BM-MSC细胞在体外培养和移植入脑梗死大鼠后都未发现对周围细胞造成污染，植入细胞的CM-DiI荧光可以鉴定产生BDNF的人BM-MSC。

关键词: 间充质干细胞, CM-DiI, 移植, 脑梗死, 脑源性神经营养因子

Abstract: Objective To observe whether the CM-DiI carried by human bone marrow mesenchymal stem cells (BM-MSC) after labeling will contaminate the surrounding cells in vitro and in vivo and be used in identifing brain derived neurotrophic factor (BDNF) production. Methods The human bone marrow derived mesenchymal stem cells were labeled by CM-DiI at different concentrations and the labeling percentages were observed. The CM-DiI labeled human BM-MSCs were co-cultured with green fluorescence protein (GFP) labeled 293T cell for 7 days. Then the contamination of 293T cells by CM-DiI was observed. The cerebral ischemia rat model received CM-DiI labeled human BM-MSCs intravenously transplantation, the overlapping of red fluorescence with host neurons and brain derived neurotrophic factor BDNF in the host brain were observed. Results The human BM-MSCs could be successfully labeled by CM-DiI at 1 000, 200,100, and 20 nmol/L. The concentrations of 1 000 and 200 nmol/L reached more than 98% labeling percentages which were significantly higher than that labeled by 100 and 20 nmol/L(P<0.01). After 7 days co-culture of GFP labeled 293T cells, the CM-DiI labeled human BM-MSCs didn't contaminate the 293T cells. Three days after transplantation, the CM-DiI labeled human BM-MSCs migrated into the infarct area of brain ischemia rats, but didn't bring any red fluorescence into the host cells. CM-DiI labeled human BM-MSCs could be double labeled by BDNF. Conclusion CM-DiI could label human BM-MSCs effectively. The 200 nmol/L CM-DiI labeled human BM-MSCs will not contaminate the surrounding cells both in vitro and in vivo. The CM-DiI (200 nmol/L) labeling can be used in identifying transplanted BM-MSCs which are expressing BDNF.

Key words: mesenchymal stem cells, CM-DiI, transplantation, cerebral infarct, brain derived neurotrophic factor

中图分类号:

赵春松, 邹海强, 李晓波, 闫晓明, 关云谦, 张愚. CM-DiI标记人骨髓间充质干细胞并检测其移植后产生脑源性神经营养因子的实验研究[J]. 首都医科大学学报, 2017, 38(3): 423-430.

Zhao Chunsong, Zou Haiqiang, Li Xiaobo, Yan Xiaoming, Guan Yunqian, Zhang Yu. CM-DiI labeling human bone marrow mesenchymal stem cells and was used in identifying BNDF expressing cell after transplantation[J]. Journal of Capital Medical University, 2017, 38(3): 423-430.

参考文献

[1] Takahashi M, Li T S, Suzuki R, et al. Cytokines produced by bone marrow cells can contribute to functional improvement of the infarcted heart by protecting cardiomyocytes from ischemic injury[J]. Am J Physiol Heart Circ Physiol, 2006, 291(2):H886-H893.
[2] Yavagal D R, Lin B, Raval AP, et al. Efficacy and dose-dependent safety of intra-arterial delivery of mesenchymal stem cells in a rodent stroke model[J]. PLoS One, 2014, 9:e93735.
[3] Karlupia N, Manley N C, Prasad K, et al. Intraarterial transplantation of human umbilical cord blood mononuclear cells is more efficacious and safer compared with umbilical cord mesenchymal stromal cells in a rodent stroke model[J]. Stem Cell Res Ther, 2014, 5(2):45.
[4] Bake S, Selvamani A, Cherry J, et al. Blood brain barrier and neuroinflammation are critical targets of igf-1-mediated neuroprotection in stroke for middle-aged female rats[J]. PloS One, 2014, 9(3):e91427.
[5] Lanfranconi S, Locatelli F, Corti S, et al. Growth factors in ischemic stroke[J]. J Cell Mol Med, 2011, 15(8):1645-1687.
[6] Kurozumi K, Nakamura K, Tamiya T, et al. Mesenchymal stem cells that produce neurotrophic factors reduce ischemic damage in the rat middle cerebral artery occlusion model[J]. Mol Ther, 2005, 11(1):96-104.
[7] Kurozumi K, Nakamura K, Tamiya T, et al. Bdnf gene-modified mesenchymal stem cells promote functional recovery and reduce infarct size in the rat middle cerebral artery occlusion model[J]. Mol Ther, 2004, 9(2):189-197.
[8] Wang J, Yang Z, Liu C, et al. Activated microglia provide a neuroprotective role by balancing glial cell-line derived neurotrophic factor and tumor necrosis factor-alpha secretion after subacute cerebral ischemia[J]. Int J Mol Med, 2013, 31(1):172-178.
[9] Shen L H, Li Y, Chopp M. Astrocytic endogenous glial cell derived neurotrophic factor production is enhanced by bone marrow stromal cell transplantation in the ischemic boundary zone after stroke in adult rats[J]. Glia, 2010, 58(9):1074-1081.
[10] Singleton C D, Casagrande V A. A reliable and sensitive method for fluorescent photoconversion[J]. J Neurosci Methods, 1996, 64(1):47-54.
[11] Capitelli C S, Lopes C S, Alves A C, et al. Opposite effects of bone marrow-derived cells transplantation in mptp-rat model of parkinson's disease:a comparison study of mononuclear and mesenchymal stem cells[J]. Int J Med Sci, 2014, 11(10):1049-1064.
[12] Bernaudin M, Marti H H, Roussel S, et al. A potential role for erythropoietin in focal permanent cerebral ischemia in mice[J]. J Cereb Bood Flow Metab, 1999, 19(6):643-651.
[13] Chen Z, Phillips L K, Gould E, et al. Mhc mismatch inhibits neurogenesis and neuron maturation in stem cell allografts[J]. PloS one, 2011, 6(3):e14787.
[14] Harting M T, Jimenez F, Xue H, et al. Intravenous mesenchymal stem cell therapy for traumatic brain injury[J]. Neurosurg, 2009, 110(6):1189-1197.
[15] Bliss T M, Andres R H, Steinberg G K. Optimizing the success of cell transplantation therapy for stroke[J]. Neurobiol Dis, 2010, 37(2):275-283.
[16] 涂雪松. 干细胞移植治疗缺血性脑血管病实验研究进展[J]. 中国脑血管病杂志, 2014, 11(8):440-445.
[17] Tyndall A, Gratwohl A. Adult stem cell transplantation in autoimmune disease[J]. Curr Opin Hematol, 2009, 16(4):285-291.
[18] Toyoshima A, Yasuhara T, Kameda M, et al. Intra-arterial transplantation of allogeneic mesenchymal stem cells mounts neuroprotective effects in a transient ischemic stroke model in rats:analyses of therapeutic time window and its mechanisms[J]. PloS One, 2015, 10(6):e0127302.

CM-DiI标记人骨髓间充质干细胞并检测其移植后产生脑源性神经营养因子的实验研究

CM-DiI labeling human bone marrow mesenchymal stem cells and was used in identifying BNDF expressing cell after transplantation

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 15

编辑推荐

Metrics

本文评价

[1]	杨敏, 燕翔, 刘锡娟, 安国, 丁慧荣, 蔡英, 葛会争. 人肺癌细胞原位移植瘤小鼠模型免疫细胞的多色流式细胞术检测方法的建立[J]. 首都医科大学学报, 2023, 44(1): 99-106.
[2]	何颖, 冯传良, 刘进营, 郑慧敏, 江圣杰. 三维微环境手性特征调控间充质干细胞线粒体功能[J]. 首都医科大学学报, 2022, 43(3): 440-445.
[3]	孙丛丛, 吴涵, 周丹娜, 姜玮, 王雯, 杨春林, 马辛, 任艳萍. 脑源性神经营养因子对经颅磁刺激治疗难治性抑郁症的疗效预测作用[J]. 首都医科大学学报, 2022, 43(2): 227-232.
[4]	张慧博, 杨晓旭, 刘欣圆, 顾华, 王双坤, 杨旗. 磁共振影像对脑静脉窦血栓形成的诊断价值研究[J]. 首都医科大学学报, 2022, 43(1): 67-73.
[5]	孙鹏慧, 李莹莹, 刘鑫, 贾学佳, 贾秀琴, 杨旗. 卒中相关性肺炎患者神经功能恶化的影像指标及其预测价值分析[J]. 首都医科大学学报, 2022, 43(1): 74-81.
[6]	胡文莉, 侯登榜, 王粮山, 贾明, 来永强, 侯晓彤, 王红. 心脏手术后脑梗死的危险因素分析[J]. 首都医科大学学报, 2021, 42(6): 909-914.
[7]	朱平, 禹环, 符丽梅, 廖欣, 熊暮珺, 雷阿明, 祝平安, 梁欣荃. 脐血移植治疗难治复发急性白血病的临床分析[J]. 首都医科大学学报, 2021, 42(6): 1041-1045.
[8]	李珊珊, 鲍会漳, 孙意, 王春燕, 贾玫. 降钙素原与骨髓移植后菌血症患者感染菌群特点及预后的相关性[J]. 首都医科大学学报, 2021, 42(5): 726-732.
[9]	程姣姣, 阮祥燕, 杜娟, 金凤羽, 李扬璐, 谷牧青. 卵巢组织冻存移植安全性的研究进展[J]. 首都医科大学学报, 2021, 42(4): 505-510.
[10]	王慧娟, 杨光忠, 菅原, 耿传营, 周慧星, 陈文明. VRD与VCD方案诱导治疗后序贯自体造血干细胞移植对新诊断的多发性骨髓瘤患者的疗效分析[J]. 首都医科大学学报, 2021, 42(4): 615-622.
[11]	孙熙木, 王华光, 赵昕, 吕少诚, 贺强, 刘丽宏. 肝移植患者术后巨细胞病毒感染的危险因素分析[J]. 首都医科大学学报, 2021, 42(4): 664-671.
[12]	阮祥燕, 杨瑜, 卢丹, 杜娟, 程姣姣, 谷牧青, 李扬璐, 金凤羽, 段微, 代荫梅, 鞠蕊, 许新, MatthiasKorell, AlfredO.Mueck. 中国首例乳腺癌患者冻存卵巢组织移植成功报道及文献复习[J]. 首都医科大学学报, 2021, 42(2): 177-182.
[13]	庄虔莹, 王芹, 陈仝, 杨晓葵. 脱氢表雄酮对卵巢储备功能减退患者IVF结局及卵泡液GDF-9、BMP-15的影响[J]. 首都医科大学学报, 2021, 42(2): 188-193.
[14]	王超宇, 隗明, 刘永哲, 陈云霞, 谷丽. 肺内外隐球菌感染病例的临床特征分析[J]. 首都医科大学学报, 2021, 42(2): 328-333.
[15]	杨楠, 丁锚, 黄语悠, 房亚兰, 师文娟, 赵咏梅. 一氧化氮在大鼠短暂性局灶性脑缺血损伤中对神经生长因子和脑源性神经营养因子表达的影响[J]. 首都医科大学学报, 2020, 41(6): 908-913.