诊断学理论与实践 ›› 2022, Vol. 21 ›› Issue (06): 691-696.doi: 10.16150/j.1671-2870.2022.06.005

• 论著 • 上一篇    下一篇

脂联素基因修饰内皮祖细胞移植对缺血性脑卒中小鼠神经保护作用的研究

沈琳辉1, 王书鸿2, 缪婕1()   

  1. 1.上海交通大学医学院附属瑞金医院老年病科,上海 200025
    2.同济大学附属东方医院神经内科,上海 200123
  • 收稿日期:2023-02-27 出版日期:2022-12-25 发布日期:2023-04-23
  • 通讯作者: 缪婕 E-mail:mj11453@rjh.com.cn
  • 基金资助:
    国家自然科学基金(青年)项目(81300725)

Transplantation of adiponectin-transduced endothelial progenitor cells improves neurobehavioral outcomes following transient middle cerebral artery occlusion

SHEN Linhui1, WANG Shuhong2, MIAO Jie1()   

  1. 1. Department of Geriatrics, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China
    2. Department of Neurology, Shanghai East Hospital, School of Medicine, Tongji University, Shanghai 200123, China
  • Received:2023-02-27 Online:2022-12-25 Published:2023-04-23
  • Contact: MIAO Jie E-mail:mj11453@rjh.com.cn

摘要:

目的:观察脂联素(adiponectin,APN)修饰的内皮祖细胞(endothelial progenitor cell, EPC)对缺血性脑卒中模型小鼠脑组织修复和神经功能恢复的作用。方法:构建表达APN基因的慢病毒并转染EPC,将过表达APN的EPC移植至短暂性大脑中动脉缺血(transient middle cerebral artery occlusion, tMCAO)模型小鼠脑内,即为EPC-APN组,另设EPC组[移植EPC-绿色荧光蛋白(green fluorescent protein, GFP)]及空白对照组(磷酸盐缓冲液处理)。在tMCAO术后1、7、14 d,用焦油紫染色测量梗死面积,并对小鼠进行神经行为学评分;观察tMCAO术后14 d在梗死周边区检测血管新生情况;采用TUNEL检测内源性细胞的凋亡情况。采用蛋白印迹法检测小鼠血管内皮细胞生长因子(vascular endothelial growth factor, VEGF)。结果:成功制备过表达APN的EPC。EPC-APN组小鼠在脑缺血后第14天的脑萎缩体积百分比,较EPC组和空白对照组相比显著减少(7.2%±0.9%比11.6%±1.2%比16.2%±2.1%,P<0.01)。EPC-APN组小鼠的内源性细胞凋亡数目百分比与EPC组及空白对照组相比明显降低(1.0%±0.1%/mm2比2.1%±0.2%/mm2比11.2%±3.2%/mm2P<0.05);EPC-APN组小鼠的神经行为学评分[改良神经损害严重程度评分(modified neurological severity score, mNSS)]更低[(3.2±0.3)分比(4.5±0.3)分比(5.8±0.4)分,P<0.05];EPC-APN组小鼠脑内的血管新生数目显著增多[(6.2±0.8)个/mm2比(1.8±0.2)个/mm2比(0.2±0.1)个/mm2P<0.01)。EPC-APN组VEGF蛋白的表达水平较EPC组、空白对照组增加,差异有统计学意义(P<0.05)。结果:移植经APN基因修饰的EPS,能减轻小鼠的缺血性脑损伤,改善其神经功能。

关键词: 脂联素, 内皮祖细胞, 脑缺血, 血管新生

Abstract:

Objective: To observe the effect of APN-modified EPCs on brain tissue repair and neurological function recovery in ischemic stroke model mice. Methods: Construction of lentiviruses transfected with APN genes. Genetically modified EPCs overexpressing APN(EPC-APN) or green fluorescent protein (EPC-GFP) were transplanted into a mice model of transient middle cerebral artery occlusion (tMCAO). The EPC-APN transplantation group, EPC-green fluorescent protein (GFP) transplantation control group and phosphate buffer (PBS) control treatment group were established. After transplantation, the enhanced GFP-expressing cells were found at the peri-infact area of brain. At 1, 7 and 14 days after tMCAO, the infarct volume was measured by using cresyl violet. Angiogenesis in the perifocal region was quantified on the 14th day. The apoptosis of endogenous cells was evaluated using TUNEL assay. Expression of vascular endothelial growth factor(VEGF)was detected using Western blot. Results: By constructing APN gene lentiviral vectors to transfect to EPCs, EPCs overexpressing APN were successfully prepared. The volume of brain atrophy in the EPC-APN transplantation group on on the 14th day after cerebral ischemia was significantly reduced compared with that in the EPC-GFP transplantation control group and the PBS control treatment group (7.2%±0.9% vs 11.6%±1.2% vs 16.2%±2.1%, P<0.01). Compared with the control group, the number of apoptosis of endogenous cells in the EPC-APN transplantation group was significantly reduced (1.0%±0.1%/mm2 vs 2.1%±0.2%/mm2 vs 11.2%±3.2%/mm2, P<0.05). Mice in the EPC-APN transplant group had lower neurobehavioral mNSS scores (3.2±0.3 vs 4.5±0.3 vs 5.8±0.5, P<0.05). The number of angiogenesis in the brain of the EPC-APN transplantation group increased significantly[(6.2±0.8)/mm2 vs(1.8±0.2)/mm2 vs (0.2±0.1)/mm2, P<0.01]. Western blot analysis showed that the VEGF expression in EPC-APN group was up-regulated compared with EPC-GFP group (P<0.05). Conclusions: EPC-APN transplantation can reduce ischemic brain injury and improve neurobehavioral outcomes.

Key words: Adiponectin, End othelial progenitor cell, Ischemic stroke, Angiogenesis

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