川崎病模型小鼠内皮祖细胞的状态

Status of endothelial progenitor cell in murine model of Kawasaki disease

目的探讨川崎病模型小鼠冠状动脉损伤的发生与内皮祖细胞（EPC）数量及功能改变的相关性。方法制备酪乳杆菌细胞壁提取物（LCWE），取0．5ml经腹腔单次注射制备C57BL／6小鼠的川崎病模型。将24只小鼠以随机数字表法分为3组：LCWE注射14d组；LCWE注射56d组及对照组（磷酸盐缓冲液腹腔注射），每组8只。分别检测其外周血CD34^＋Flk-1^＋CD45^-的EPC含量，同时提取各组小鼠的骨髓单个核细胞在体外进行EPC培养，培养至第7天，通过噻唑蓝（MTT）比色法、黏附实验和迁移实验分别测定EPC的增殖、黏附和迁移能力。结果LCWE注射14d组小鼠冠状动脉主干及其各级分支周围可以见到以淋巴细胞为主的大量炎细胞浸润，其外周血中循环EPC的含量（0．017％±0．008％）明显低于对照组（0．028％±0．007％），LCWE注射56d组冠状动脉弹力层的破坏清晰可见，而其外周血循环EPC的含量（0．016％±0．007％）也明显低于对照组（均P〈0．01）。通过骨髓单个核细胞的体外培养显示，LCWE注射14d组小鼠骨髓EPC的集落形成能力明显低于对照组，其体外增殖能力检测的MTr实验中吸光度（A值）为0．39±0．11，低于对照组（0．61±0．14，P〈0．01）；其黏附能力和迁移能力[（3．1±0．6）、（3．2±0．6）个／高倍视野）]也均低于对照组[（6．4±1．2）、（6．2±0．5）个／高倍视野，均P〈0．01）]。LCWE注射56d组小鼠骨髓EPC集落形成能力仍明显低于对照组，其增殖能力、黏附能力和迁移能力[0．38±0．09、（3．1±0．6）个／高倍视野和（3．3±0．6）个／高倍视野]也显著低于对照组（均P〈0．01）。结论川崎病模型小鼠冠状动脉损伤的发生可能与EPC数量及功能的下调存在一定的相关性。

Objective 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）. Methods 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. Results 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 lymphoeytes. 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-formingability 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 MTY 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, allP〈0.01）.Conelusion The occurrence of coronary artery lesion may be correlated with the down-regulation of EPC number and function in murine model of KD.