中国组织工程研究

中国组织工程研究 ›› 2019, Vol. 23 ›› Issue (17): 2630-2636.doi: 10.3969/j.issn.2095-4344.1716

• 脐带脐血干细胞 umbilical cord blood stem cells • 上一篇    下一篇

低氧预处理人脐带间充质干细胞促进其源性外泌体对心肌梗死后心肌损伤的修复

张 蘋1，郭 莹1，高亚杰1，王振东1，李佰一1，张晓敏1，牛玉虎1，刘志贞1，马丽辉2，牛 勃3，郭 睿1   

  1. 1山西医科大学生物化学与分子生物学教研室，山西省太原市 030001；2海南省三亚市人民医院，海南省三亚市 572000；3首都儿科研究所生物技术研究室，北京市 100020
  • 修回日期:2019-01-14 出版日期:2019-06-18 发布日期:2019-06-18
  • 通讯作者: 郭睿，教授，硕士生导师，山西医科大学生物化学与分子生物学教研室，山西省太原市 030001； 并列通讯作者：牛勃，教授，博士生导师，首都儿科研究所生物技术研究室，北京市 100020
  • 作者简介:张蘋，女，1988年生，陕西省泾阳县人，汉族，山西医科大学在读硕士，主要从事干细胞与生物诊疗研究。
  • 基金资助:

    国家自然科学基金(81370312)，项目负责人：牛勃；山西省回国留学人员科研资助项目(2016-051)，项目负责人：刘志贞；三亚市医疗卫生科技创新项目(2014 YW01)，项目负责人：马丽辉

Exosomes derived from human umbilical cord mesenchymal stem cells promote myocardial repair after myocardial infarction under hypoxia

Zhang Pin1, Guo Ying1, Gao Yajie1, Wang Zhendong1, Li Baiyi1, Zhang Xiaomin1, Niu Yuhu1, Liu Zhizhen1, Ma Lihui2, Niu Bo3, Guo Rui1   

  1. 1Department of Biochemistry and Molecular Biology, Shanxi Medical University, Taiyuan 030001, Shanxi Province, China; 2People’s Hospital of Sanya City, Sanya 572000, Hainan Province, China; 3Department of Biotechnology, Capital Institute of Pediatrics, Beijing 100020, China
  • Revised:2019-01-14 Online:2019-06-18 Published:2019-06-18
  • Contact: Guo Rui, Professor, Master’s supervisor, Department of Biochemistry and Molecular Biology, Shanxi Medical University, Taiyuan 030001, Shanxi Province, China; Niu Bo, Professor, Doctoral supervisor, Department of Biotechnology, Capital Institute of Pediatrics, Beijing 100020, China
  • About author:Zhang Pin, Master candidate, Department of Biochemistry and Molecular Biology, Shanxi Medical University, Taiyuan 030001, Shanxi Province, China
  • Supported by:

    the National Natural Science Foundation of China, No. 81370312 (to NB); Research Funding Project for Returned Overseas Students in Shanxi Province, No. 2016-051 (to LZZ); the Medical and Health Science and Technology Innovation Project of Sanya, No. 2014 YW01 (to MLH)

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摘要：

文章快速阅读：

文题释义：
低氧预处理：传统细胞培养的氧体积分数为21%，而在体内根据组织局部血供和代谢的不同，氧体积分数为3%-5%。适当的低氧预处理可以减少细胞内氧自由基的产生，提高细胞代谢效能。
外泌体：细胞通过胞吞作用将外源性抗原吞入细胞内形成早期胞内体后，随着早期胞内体囊膜向内出芽形成管腔状囊泡，并选择性地分拣胞质内的蛋白质及脂质成分，形成晚期胞内体，即具有动态亚细胞结构的多囊泡体，一部分多囊泡体进入溶酶体途径降解，一部分与细胞膜融合释放到胞外，即形成外泌体。

 

摘要
背景：研究表明，外泌体是干细胞治疗心肌梗死的重要作用机制，低氧预处理对人脐带间充质干细胞外泌体的量和内容物都有所影响。
目的：比较常氧和低氧预处理人脐带间充质干细胞源性外泌体对大鼠心肌梗死后心肌损伤修复的影响及可能机制。
方法：①从健康新生儿脐带中分离出人脐带间充质干细胞培养至第4代，待50%-60%融合时，更换为无血清培养基，分别置于传统培养箱和低氧孵箱培养48 h，采用超速离心法从两组细胞培养上清中提取外泌体；②SD雌性大鼠(购于山西医科大学实验动物中心)50只，其中40只建立心肌梗死模型，10只进行假手术处理。从造模成功大鼠中取30只随机分为3组：模型对照组、低氧外泌体组、常氧外泌体组，每组10只。心肌梗死术后1周，再次打开心脏，在变白区即梗死区周边四点注射相同体积外泌体或PBS，每点体积25 μL，每只100 μL。移植后1周，行超声心动图检测，然后取心脏标本进行苏木精-伊红染色和Masson染色，Western blot检测凋亡相关蛋白Cleaved-caspase 3和Bcl-2的表达。
结果与结论：①移植后1周，与模型对照组相比，低氧外泌体组和常氧外泌体组左室射血分数、短轴缩短率明显增加(P < 0.01)，左室舒张末期内径、左室收缩末期内径显著减小(P < 0.01)，其中低氧外泌体组上述指标改善最显著(P < 0.01)；②苏木精-伊红染色结果显示，假手术组细胞排列整齐，模型对照组病变程度最严重，低氧外泌体组病变程度轻于常氧外泌体组；③Masson染色结果显示，低氧外泌体组和常氧外泌体组的胶原容积分数低于模型对照组，以低氧外泌体组减少最为明显(P < 0.01)；④Western blot结果显示，与模型对照组相比，低氧外泌体组和常氧外泌体组心肌组织凋亡相关蛋白Cleaved-caspase 3的表达减少，Bcl-2的表达增加，均以低氧外泌体组改变最为明显(P < 0.01)；⑤以上结果表明，人脐带间充质干细胞经低氧预处理后，其源性外泌体明显促进了SD大鼠心肌梗死后心肌损伤的修复。


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: 0000-0001-6436-7782(张蘋)

关键词: 心肌梗死, 脐带间充质干细胞, 低氧预处理, 外泌体, 间充质干细胞移植, 国家自然科学基金

Abstract:

BACKGROUND: Studies have shown that exosomes are an important mechanism of stem cell therapy for myocardial infarction. Hypoxic preconditioning affects the quantity and content of exosomes from human umbilical cord mesenchymal stem cells.
OBJECTIVE: To compare the effects of exosomes derived from human umbilical cord mesenchymal stem cells pretreated with hypoxia and cultured with normoxia on myocardial repair after myocardial infarction in rats, and to explore the possible mechanisms.
METHODS: (1) Human umbilical cord mesenchymal stem cells isolated from healthy newborn umbilical cord were cultured for fourth generations and identified. Passage 4 human umbilical cord mesenchymal stem cells with fusion degree of 50%-60% were pretreated for 48 hours in combination with serum-free medium and oxygen gradient culture (hypoxia and normoxia). Exosomes were then extracted from two sets of cell culture supernatants by ultracentrifugation. (2) Fifty Sprague-Dawley female rats provided by the Experimental Animal Center of Shanxi Medical University in China were enrolled, 40 of which were used to make animal models of myocardial infarction and the remaining 10 rats were subjected to sham operation. After successful modeling, 30 model rats were randomly assigned into model group, hypoxic exosomes group, normoxic exosomes group (n=10/group). One week after myocardial infarction, the heart was opened again, and the same volume of exosomes or PBS was injected at four points around the infarct area (the whitening area), 25 μL per point, 100 μL for each rat. One week after transplantation, echocardiography was performed to measure cardiac function. After completion of echocardiography, heart samples were taken and pathological sections were prepared for hematoxylin-eosin staining and Masson staining. Western blot assay was used to detect the expression of apoptosis-related proteins Cleaved-caspase 3 and Bcl-2.
RESULTS AND CONCLUSION: (1) One week after exosomes transplantation, the ejection fraction and fractional shortening rate of the left ventricle in hypoxic exosomes and normoxic exosomes groups significantly increased (P < 0.01), while left ventricular end-diastolic diameter and left ventricular end-systolic diameter significantly decreased (P < 0.01). The above indexes improved most significantly in the hypoxic exosomes group (P < 0.01). (2) Hematoxylin-eosin staining results showed that the cells were in alignment in the sham-operated group. Most serious lesions were found in the model group. The hypoxic exosomes group had less lesions than the normoxic exosomes group. (3) Masson staining results showed that the compared with the model group, the collagen volume fraction was decreased significantly in the two exosomes groups, especially in the hypoxic exosomes group (P < 0.01). (4) Western blot results showed that compared with the model group, the expression of Cleaved-caspase-3 decreased while the expression of Bcl-2 increased in the hypoxic and normoxic exosomes groups, and the variation in the protein expression was more obvious in the hypoxic exosomes group than the normoxic exosomes group (P < 0.01). To conclude, these findings reveal that exosomes derived from human umbilical cord mesenchymal stem cells under hypoxia can improve myocardial repair in Sprague-Dawley rats with myocardial infarction.

Key words: myocardial infarction, umbilical cord mesenchymal stem cells, hypoxic preconditioning, exosomes, mesenchymal stem cell transplantation, National Natural Science Foundation of China

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