Liu Jun-feng, DU Zhong-dong, Chen Zhi, Li Shen-tao, Lu Dun-xiang, Li Li
Department of Cardiology, Beijing Children's Hospital, Capital Medical University, Beijing 100045, China.
Zhonghua Yi Xue Za Zhi. 2012 Jun 12;92(22):1560-4.
To assess whether the occurrence of coronary artery lesion was correlated with the changes of endothelial progenitor cell (EPC) number and function in murine model of Kawasaki disease (KD).
Lactobacillus casei cell wall extract (LCWE) was prepared and then C57BL/6 mice received a single intraperitoneal injection of LCWE for inducing KD. Twenty-four mice were categorized randomly into 3 groups: KD model group at Day 14 post-injection, KD model group at Day 56 post-injection and control group with an intraperitoneal injection of phosphate buffered solution (n = 8 each). The number of circulating EPC was defined as CD34(+)Flk-1(+)CD45(-) from mice. Meanwhile, bone marrow mononuclear cells were cultured in vitro to expand EPC for functional analysis. After 7 days of culturing, EPC were inoculated onto culture plate and thiazolyl blue assay was used to measure the absorbance value by enzyme labeling instrument to evaluate the proliferation. The adhesion of EPC was performed by replating cells on fibronectin coated dishes and then counting the number of adherent cells. The migration of EPC was assayed by Transwell.
Focal inflammatory infiltrate was evident in coronary artery trunk and a series of branches at Day 14 post-injection. The inflammatory cell infiltrate consisted of mononuclear lymphocytes. The number of circulating EPC were significantly lower in the Day 14 LCWE-treating murine model versus the controls (0.017% ± 0.008% vs 0.028% ± 0.007%, P < 0.01). Disruption of elastin was consistently observed at Day 56 post-injection. And there was no apparent recovery in number of EPC (0.016% ± 0.007%, P < 0.01). When bone marrow mononuclear cells were cultured in vitro, the colony-forming ability of EPC decreased in the KD model group at Day 14 post-injection versus the controls. Test of proliferating ability showed that the absorbance was 0.39 ± 0.11 in MTT experiment and decreased than the controls (0.61 ± 0.14, P < 0.01). Adhesion and migration were also down-regulated versus the controls ((3.1 ± 0.6) and (3.2 ± 0.6) vs (6.4 ± 1.2) and (6.2 ± 0.5) cells/HPF, both P < 0.01). In the KD model group at Day 56 post-injection, the colony-forming ability of EPC was not recovered significantly. Proliferation ability, adhesion and migration were still decreased compared to the controls (0.38 ± 0.09, (3.12 ± 0.56) cells/HPF and (3.29 ± 0.63) cells/HPF, all P < 0.01).
The occurrence of coronary artery lesion may be correlated with the down-regulation of EPC number and function in murine model of KD.
评估在川崎病（KD）小鼠模型中，冠状动脉病变的发生是否与内皮祖细胞（EPC）数量及功能的变化相关。
制备干酪乳杆菌细胞壁提取物（LCWE），然后给C57BL/6小鼠腹腔注射一次LCWE以诱导KD。24只小鼠随机分为3组：注射后第14天的KD模型组、注射后第56天的KD模型组和腹腔注射磷酸盐缓冲溶液的对照组（每组n = 8）。将小鼠循环EPC的数量定义为CD34(+)Flk-1(+)CD45(-)。同时，体外培养骨髓单个核细胞以扩增EPC用于功能分析。培养7天后，将EPC接种到培养板上，用噻唑蓝法通过酶标仪测量吸光度值以评估增殖情况。通过将细胞重新接种到纤连蛋白包被的培养皿上并计数贴壁细胞数量来检测EPC的黏附情况。通过Transwell检测EPC的迁移情况。
注射后第14天，冠状动脉主干及一系列分支可见局灶性炎性浸润。炎性细胞浸润由单核淋巴细胞组成。与对照组相比，注射后第14天LCWE处理的小鼠模型中循环EPC数量显著降低（0.017% ± 0.008%对0.028% ± 0.007%，P < 0.01）。注射后第56天持续观察到弹性蛋白破坏。并且EPC数量无明显恢复（0.016% ± 0.007%，P < 0.01）。当体外培养骨髓单个核细胞时，注射后第14天KD模型组中EPC的集落形成能力相对于对照组降低。增殖能力检测显示，MTT实验中吸光度为0.39 ± 0.11，低于对照组（0.61 ± 0.14，P < 0.01）。与对照组相比，黏附及迁移能力也下调（(3.1 ± 0.6)和(3.2 ± 0.6)对(6.4 ± 1.2)和(6.2 ± 0.5)个细胞/高倍视野，P均< 0.01）。注射后第56天的KD模型组中，EPC的集落形成能力未显著恢复。与对照组相比，增殖能力、黏附及迁移能力仍降低（0.38 ± 0.09，(3.12 ± 0.56)个细胞/高倍视野和(3.29 ± 0.63)个细胞/高倍视野，P均< 0.01）。
在KD小鼠模型中，冠状动脉病变的发生可能与EPC数量及功能的下调相关。
