同种异体骨髓间充质干细胞对脓毒症大鼠腹腔巨噬细胞极化改变的影响  

Effects of allogeneic bone marrow mesenchymal stem cells on polarization of peritoneal macrophages in rats with sepsis

作　　者：郑远华 熊兵[2] 邓医宇[3] 赖文[2] 郑少逸[2] 卞徽宁[2] 刘族安[2] 黄志锋[2] 孙传伟[2] 李汉华[2] 罗红敏[2] 马亮华[2] 陈瀚熙[2] 

机构地区：[1]广东汕头大学医学院,515041 [2]广东省人民医院(广东省医学科学院)烧伤与创面修复外科,510080 [3]广东省人民医院(广东省医学科学院)急危重症医学部,510080

出　　处：《中华烧伤杂志》2017年第4期217-223,共7页Chinese Journal of Burns

基　　金：国家自然科学基金（81471237、81271329）;广东省医学科学技术研究基金（A2016108）

摘　　要：目的 探讨同种异体骨髓间充质干细胞（BMSC）对LPS诱导的脓毒症大鼠腹腔巨噬细胞极化改变的影响。方法 （1）取5只SD大鼠，采用全骨髓贴壁法分离纯化培养BMSC，取第3代细胞行形态学观察，流式细胞仪检测干细胞表面标志物CD29、CD44、CD45、CD90的表达，并进行成骨细胞、成脂肪细胞诱导分化鉴定。（2）取45只SD大鼠，按随机数字表法分为假伤组5只、LPS对照组20只、BMSC治疗组20只。对LPS对照组、BMSC治疗组大鼠予尾静脉按5 mg/kg注射LPS致脓毒症，假伤组大鼠注射等量的生理盐水模拟致伤。伤后1 h，BMSC治疗组大鼠另予尾静脉注射1 mL BMSC（2×106个/mL），另2组大鼠注射等量PBS。假伤组大鼠于伤后24 h，LPS对照组、BMSC治疗组于伤后6、12、24、48 h各取5只大鼠处死后取肺组织行HE染色观察病理变化。假伤组伤后24 h的大鼠与LPS对照组、BMSC治疗组伤后24、48 h的大鼠另同时通过腹腔注射低糖DMEM培养液后取腹腔液培养巨噬细胞。流式细胞仪检测假伤组伤后24 h，LPS对照组、BMSC治疗组伤后24、48 h大鼠腹腔巨噬细胞标志物CD68（用于设门）、CD11c、CD206阳性表达情况。对数据行单因素方差分析、LSD检验。结果 （1）分离培养的第3代贴壁细胞形态呈均一纤维样，与Fb相似；CD29、CD44、CD90呈阳性表达，CD45呈弱阳性表达；诱导后细胞可分化为成骨细胞和成脂肪细胞，鉴定为BMSC。（2）伤后24 h，假伤组大鼠肺泡结构清晰、完整，无充血或炎性细胞浸润。伤后6 h，LPS对照组、BMSC治疗组大鼠肺泡结构清晰，少量炎性细胞浸润，肺组织轻微充血，肺间质稍增厚。伤后12 h，LPS对照组大鼠肺组织较BMSC治疗组炎症反应明显，可见大量炎性细胞浸润，肺组织充血，肺间质明显增厚；而BMSC治疗组大鼠与伤后6 h情况基本一致。伤后24 h，LPS对照组、BMSC治疗组大鼠肺组织病理改变较伤后1Objective To explore the effects of allogeneic bone marrow mesenchymal stem cells （BMSCs） on polarization of peritoneal macrophages isolated from rats with sepsis induced by endotoxin/lipopolysaccharide （LPS）.Methods （1） BMSCs were isolated, cultured and purified from 5 SD rats with whole bone marrow adherent method. The third passage of cells were collected for morphologic observation, detection of expressions of stem cell surface markers CD29, CD44, CD45, and CD90 with flow cytometer, and identification of osteogenic and adipogenic differentiation. （2） Another 45 SD rats were divided into sham injury group （SI, n=5）, LPS control group （LC, n=20）, and BMSCs-treated group （BT, n=20） according to the random number table. Rats in groups LC and BT were injected with LPS （5 mg/kg） via tail vein to induce sepsis; rats in group SI were injected with the same amount of normal saline to simulate the damage. At post injury hour （PIH） 1, rats in group BT were given 1 mL BMSCs （2×106/mL） via tail vein injection; rats in another two groups were injected with equal volume of phosphate buffer saline. Five rats in group SI at PIH 24 and in groups LC and BT at PIH 6, 12, 24, and 48 were sacrificed to harvest lung tissue for pathological observation with HE staining. In addition, rats in group SI at PIH 24 and in groups LC and BT at PIH 24 and 48 were simultaneously performed with intraperitoneal injection of low-glucose DMEM. Then peritoneal fluid was harvested to culture peritoneal macrophages. Flow cytometer was used to assess the positive expression of cell makers of macrophages including CD68 （making gate）, CD11c, and CD206 in group SI at PIH 24 and in groups LC and BT at PIH 24 and 48. Data were processed with one-way analysis of variance and LSD test.Results （1） The third passage of cells showed uniform fiber-like shape similar to fibroblasts. These cells showed positive expressions of CD29, CD44, CD90 and weak positive expression of CD45. They were able to differentiate int

关 键 词：脓毒症 巨噬细胞 骨髓间充质干细胞 

分 类 号：R459.7[医药卫生—急诊医学]