首都医科大学学报 ›› 2013, Vol. 34 ›› Issue (3): 391-397.doi: 10.3969/j.issn.1006-7795.2013.03.014

• 基础研究 • 上一篇    下一篇

小鼠骨髓巨噬细胞过继性回输的体内示踪及其在高血压心脏损伤中的应用研究

李玉琳, 吴依娜, 张聪聪, 阚晓玉, 阿希, 赵伟, 王绿娅, 杜杰   

  1. 首都医科大学附属北京安贞医院,北京市心肺血管疾病研究所,心血管重塑相关疾病教育部重点实验室,北京 100029
  • 收稿日期:2013-04-08 出版日期:2013-06-21 发布日期:2013-06-17
  • 通讯作者: 杜杰 E-mail:jiedu@yahoo.com
  • 基金资助:

    国家自然科学基金(81230006;81000069);北京市自然科学基金(2010B031,7132043)。

Adoptive transfer of murine bone-marrow derived macrophages tracing in vivo and application in hypertensive cardiac injury

LI Yulin, WU Yina, ZHANG Congcong, KAN Xiaoyu, A Xi, ZHAO Wei, WANG Lvya, DU Jie   

  1. Beijing Anzhen Hospital, Beijing Institute of Heart,Lung and Vessel Diseases,Capital Medical University,The Key Laboratory of Remodeling Cardiovascular Diseases,Ministry of Education, Beijing 100029,China
  • Received:2013-04-08 Online:2013-06-21 Published:2013-06-17
  • Supported by:

    This study was supported by National Natural Science Foundation of China(81230006;81000069) and Natural Science Foundation of Beijng (2010B031,7132043).

摘要:

目的 通过增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)转基因和CD45.1 两种工具小鼠,进行小鼠骨髓巨噬细胞(bone marrow-derived macrophages,BMM)过继性回输后体内示踪的研究。利用小鼠高血压模型,观察回输BMM对高血压心脏损伤的作用。方法 以EGFP转基因小鼠为供体,分离骨髓细胞,以巨噬细胞集落刺激因子诱导制备骨髓巨噬细胞。尾静脉回输EGFP+BMM到野生型小鼠。受体小鼠分2组:①实验组:每只注射2×106 EGFP+BMM;②对照组:注射等体积磷酸盐缓冲液(PBS)。术后1、7 d观察受体小鼠外周血中EGFP+细胞存活和比例;7 d后收取小鼠心、肾、脾和肝脏组织,观察GFP+细胞的表达。以CD45.1小鼠为供体, CD45.2小鼠为受体,同上进行BMM回输,术后7 d检测受体小鼠外周血中CD45.1+细胞的比例。利用血管紧张素Ⅱ(Ang Ⅱ)灌注建立小鼠高血压模型,同时回输PBS或EGFP+BMM。灌注后7 d,观察EGFP+BMM在心脏的浸润、心脏炎性反应和纤维化。结果 EGFP+BMM回输后1、7 d,外周血中EGFP+细胞比例分别为[1st day:(4.6±0.5)%;7th day:(4.4±0.5)%],对照组外周血中无EGFP+细胞存在。回输第7天,受体小鼠脾脏中有GFP+细胞表达,心、肾、肝中无GFP+细胞表达。CD45.1+BMM回输后第7天,外周血中CD45.1+细胞比例为(4.3%±0.4)%,对照组中无CD45.1+细胞。与对照组相比,AngⅡ灌注促进血压升高、心脏中炎性细胞浸润和胶原沉积。AngⅡ灌注后,回输的EGFP+BMM能浸润到心脏,促进心脏中炎性反应和纤维化,但不影响血压升高。结论 该方法解决了BMM的过继性回输后体内的标记示踪问题,回输BMM可以在受体体内存活,而且证实巨噬细胞回输促进高血压心脏纤维化的发生。

关键词: 骨髓巨噬细胞, 过继性回输, 示踪技术, 高血压

Abstract:

Objective Two types of mice enhanced green fluorescent protein (EGFP) transgenic mice and CD45 type I (CD45.1) mice) to observe whether the adoptive transfusion of murine bone-marrow derived macrophages(BMM)might affect the hypertensive damage to heart in murine hypertension model. Methods After obtaining bone marrow cells from EGFP transgenic mice and inducing cells with macrophage colony-stimulating factor (M-CSF) to BMM, wild-type mice received injection of 2×106 EGFP+BMMs or phosphate-buffered saline (PBS) through tail vein and observed survival proportion of EGFP+cells in peripheral blood at 1st day and 7th day. Then we collected heart, liver and spleen to determine expression of GFP+ cells. Likewise we used CD45.1 BMM to inject into CD45.2 mice and determine the proportion of CD45.1+ cells in peripheral blood. Then angiotensin Ⅱ (Ang Ⅱ) was infused to the mice and EGFP+BMMs or PBS was infused as previously described. After 7 days infiltration of EGFP+BMM, inflammation and fibrosis in heart were determined.Results The proportion of EGFP+cells in peripheral blood in EGFP+BMMs injection group were 1st day:(4.6±0.5)%; 7th day:(4.4±0.5)% while none in control group. After 7 days GFP+cells expressed in spleen while none in liver, kidney or heart. The proportion of CD45.1+cells in peripheral blood in CD45.1+BMMs injection group was (4.3±0.4)% while none in control group. In contrast to control group, AngⅡ infused group had accelerated increased blood pressure, infiltration of inflammatory cells and collagen deposition. Injection of EGFP+BMMs could infiltrate in heart and accelerate inflammation and fibrosis, but did not affect blood pressure. Conclusion This study proved that adoptive transfuse BMMs could survive in recipient and accelerate cardiac fibrosis. We settled the problem of tracing the adoptive transfused BMM in vivo.

Key words: bone-marrow derived macrophages, adoptive transfer, tracing technique, hypertension

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