中国组织工程研究

中国组织工程研究 ›› 2017, Vol. 21 ›› Issue (5): 724-729.doi: 10.3969/j.issn.2095-4344.2017.05.012

• 干细胞移植 stem cell transplantation • 上一篇    下一篇

补阳还五汤联合骨髓间充质干细胞移植对大脑中动脉栓塞模型整合素相关因子的影响

张运克1，2，杨俊红2，高  峰3，段枫阳2   

  1. 1河南中医药大学，河南省郑州市  450056
    2河南中医药大学第一附属医院，河南省郑州市  450000
    3河南省医药科学研究院，河南省郑州市  450008
  • 出版日期:2017-02-18 发布日期:2017-03-20
  • 作者简介:张运克，男，1966年生，河南省平顶山市人，汉族，2002年黑龙江中医药大学毕业，博士，主任医师，主要从事中医药防治神经系统疾病的研究。
  • 基金资助:

    国家自然科学基金课题(81072799/H2708)；河南省高校科技创新团队项目(16IRTSTHN022)

Effect of Buyang Huanwu decoction combined with bone marrow mesenchymal stem cell transplantation on expression of integrin in a rat model of middle cerebral artery occlusion

Zhang Yun-ke1, 2, Yang Jun-hong2, Gao Feng3, Duan Feng-yang2   

  1. 1Henan University of Traditional Chinese Medicine, Zhenzhou 450008，Henan Province, China
    2First Affiliated Hospital of Henan University of Traditional Chinese Medicine, Zhenzhou 450000, Henan Province, China
    3Henan Academy of Medical Pharmaceutical Sciences, Zhengzhou 450008, Henan Province, China
  • Online:2017-02-18 Published:2017-03-20
  • About author:Zhang Yun-ke, M.D., Chief physician, Henan University of Traditional Chinese Medicine，Zhenzhou 450008，Henan Province, China; First Affiliated Hospital of Henan University of Traditional Chinese Medicine, Zhenzhou 450000, Henan Province, China
  • Supported by:

    the National Natural Science Foundation of China, No. 81072799/H2708; the Scientific Innovation Team Project in Henan Provincial Universities, No. 16IRTSTHN022

PDF

263

被引次数

12

摘要：

文章快速阅读：

文题释义：
补阳还五汤治疗脑血管病：补阳还五汤在临床上被广泛用于缺血性脑血管疾病及其后遗症的治疗，针对急、慢期患者辨证为气虚血瘀证型有显著疗效；也可治疗出血性脑卒中后的血管新生，补阳还五汤可通过调节脑出血后新生血管整合素α1β1和α1的表达，参与维持新生血管的稳定，促进损伤区微血管系统重建，实现其益气活血的作用，修复损伤组织。
间充质干细胞治疗脑血管病：间充质干细胞对造血干细胞起支持营养的作用，还具有定向或多向分化的潜能，在适宜的条件下可分化为神经细胞等。Kopen 等将间充质干细胞注射入新生小鼠侧脑室后，发现间充质干细胞经前脑和小脑到达大脑，促进血管的新生。国内有许多应用中药联合间充质干细胞对脑缺血动物模型起到脑保护作用，促进血管新生的报道。

 

摘要
背景：前期研究发现，补阳还五汤联合骨髓间充质干细胞移植可发挥协同抗脑缺血损伤作用。
目的：分析骨髓间充质干细胞移植联合补阳还五汤促进脑缺血血管新生的作用。
方法：将96只SD大鼠随机分为4组，均制作大脑中动脉栓塞模型。联合组于造模前3 d灌胃给予补阳还五汤10 mL/kg，1次/d，造模成功后2 h灌胃1次，以后每12 h灌胃1次；局灶脑缺血2 h再灌注2 h后，干细胞组、联合组向脑室中注入骨髓间充质干细胞悬液，30 min后再注射抗CD34、CD45 抗体；抗体组注射抗CD34、CD45 抗体；模型组注射生理盐水。联合组于再灌注后12，24，36，48 h进行血清αVβ3水平及脑组织免疫组织化学、Q-PCR、免疫印迹检测，其余3组均于再灌注后36 h检测上述指标。
结果与结论：①血清αVβ3水平：抗体组血清αVβ3水平低于模型组(P < 0.05)，干细胞组、联合组高于抗体组(P < 0.05)，且联合组再灌注不同时间点的血清αVβ3水平高于干细胞组(P < 0.01)；②免疫组织化学：抗体组CD34阳性细胞数量少于模型组(P < 0.05)，干细胞组、联合组高于抗体组(P < 0.05)；③Q-PCR与免疫印迹检测：与模型组相比，抗体组、干细胞组pFAK蛋白表达水平降低(P < 0.05)，FAK基因及蛋白表达变化不大；与其他组比较，联合组FAK蛋白(自再灌注后24 h开始)和pFAK蛋白(自再灌注12 h开始)显著升高(P < 0.05，P < 0.01)，且随着再灌注时间延长有逐渐升高趋势；④结果表明：骨髓间充质干细胞移植联合补阳还五汤可逆转抗 CD34+CD45抗体引起的血管内皮细胞数量减少、整合素αVβ3及其下游信号分子表达下降，促进血管新生。

 

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: 0000-0003-1500-1535(张运克)

关键词: 干细胞, 移植, 骨髓间充质干细胞, 补阳还五汤, 整合素, 抗CD34+CD45抗体, 脑中动脉栓塞, 血管新生, 疾病模型, 动物, 大鼠, 国家自然科学基金

Abstract:

BACKGROUND: Previous studies have found that combined use of Buyang Huanwu decoction and bone marrow mesenchymal stem cell (BMSC) transplantation can play a synergic role against cerebral ischemia injury.
OBJECTIVE: To analyze the effect of Buyang Huanwu decoction combined with BMSC transplantation to promote angiogenesis after cerebral ischemia.
METHODS: Ninety-six Sprague-Dawley rats were randomly divided into four groups, and used to make middle cerebral artery occlusion models. In combined group, rats were given intragastrical administration of Buyang Huanwu decoction 10 mL/kg once a day, beginning at 3 days prior to modeling, and then given intragastrical administration of Buyang Huanwu decoction once at 2 hours after modeling, followed by its intragastrical administration every 12 hours. In BMSC and combined groups, BMSC suspension was injected into the rat ventricle after 2-hour cerebral ischemia/2-hour reperfusion, and then 30 minutes later, CD34 and CD45 antibodies were injected. In antibody group, CD34 and CD45 antibodies were injected. In model group, only normal saline was given. Serum αVβ3 level detection, immunohistochemical observation, Q-PCR and western blot tests were performed in the combined group at 12, 24, 36, 48 hours after reperfusion, while these indices were detected in the other three groups at 36 hours after reperfusion.
RESULTS AND CONCLUSION: (1) The level of serum αVβ3 was lower in the antibody group than the model group (P < 0.05), higher in the BMSC and combined groups than the antibody group (P < 0.05), and higher in the combined group than the BMSC group (P < 0.05). (2) Immunohistochemical findings showed that compared with the antibody group, the number of CD34 positive cells was higher in the model, BMSC and combined groups (P < 0.05). (3) Results from the Q-PCR and western blot assay showed that compared with the model group, the pFAK protein expression level was lower in the antibody and BMSC group, but FAK gene expression level had no overt changes; while the protein levels of FAK (24 hours after reperfusion) and pFAK (12 hours after reperfusion) were significantly increased in the combined group than the antibody and BMSC groups (P < 0.05,P < 0.01). Moreover, this increase exhibited a gradually rising trend with the extension of reperfusion time. To conclude, the combined use of BMSC transplantation and Buyang Huanwu decoction can reverse the effect of CD34+CD45 antibodies that lead to the decrease in the number of vascular endothelial cells and levels of integrin αVβ3 and downstream signaling molecules, thereby to promote angiogenesis in the MCAO model.

 

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

Key words: Mesenchymal Stem Cells, Infarction, Middle Cerebral Artery, Drugs, Chinese Herbal, Tissue Engineering

中图分类号: