骨髓间充质干细胞来源exosome对大鼠肾缺血再灌注损伤的保护作用  

Bone marrow mesenchymal stem cell-derived exosome protects kidney against ischemia reperfusion injury in rats

作　　者：王汝霖[1] 林淼[1] 黎力平[1] 李龙[1] 戚贵生[1] 戎瑞明[1] 许明[1] 朱同玉[1] 

机构地区：[1]复旦大学附属中山医院泌尿外科上海市器官移植重点实验室,上海200032

出　　处：《中华医学杂志》2014年第42期3298-3303,共6页National Medical Journal of China

基　　金：国家自然科学基金（81070595）;上海市科委科研计划（09DZ2260300、09411952000）

摘　　要：目的 探讨骨髓间充质干细胞（BM-MSC）来源外泌体（exosome）对大鼠肾缺血再灌注损伤（IRI）的保护作用及机制.方法 原代分离、培养、鉴定大鼠BM-MSC.提取BM-MSC来源exosome,使用电镜观察exosome,并检测其表面CD63表达.构建雄性SD大鼠IRI模型,随机数字表法分为假手术组（Sham组）、缺血再灌注组（IR组）、MSC处理组（IR＋MSC组）、MSC来源exosome处理组（IR＋MSC-ex组）、成纤维细胞来源exosome处理组（IR＋F-ex组）,每组6只大鼠.检测各组大鼠血清肌酐和尿素氮水平,HE染色观察肾脏组织形态学改变,TUNEL染色评价细胞凋亡,RT-PCR检测炎症因子白细胞介素1β（IL-1β）、肿瘤坏死因子α（TNF-α）表达水平,Western印迹法检测肾组织半胱氨酸天冬氨酸蛋白酶3（caspases-3）表达水平.结果 （1）成功分离培养大鼠BM-MSC,其表面高表达CD29、CD44,低表达CD34,可成功诱导分化为成骨细胞和脂肪细胞;（2）透射电镜下观察鉴定exosome,其表面标志CD63表达阳性;（3）与IR组相比,IR＋MSC组、IR＋MSC-ex组血肌酐、尿素氮均显著降低,病理损伤明显减轻,凋亡细胞数减少,部分炎症因子表达降低,促凋亡蛋白caspase-3表达减少（IR组：4 310±616,IR＋ MSC组：2569±530,IR＋MSC-ex组：3144±343,均P＜0.05）.结论 大鼠BM-MSC来源exosome对大鼠肾脏IRI具有保护作用,可降低炎症和凋亡水平.Objective To explore the protective effects and mechanism of exosomes derived from bone marrow mesenchymal stem cells （BM-MSC） on ischemia reperfusion injury （IRI） in rats.Methods Rat-MSC were isolated,cultured and identified.Exosome was extracted from BM-MSC and observed under transmission electron microscope （TEM）.The expression of surface molecular marker CD63 was tested by llow cytometry.Male Sprague-Dawley rats were randomly grouped into sham-operated （Sham）,ischemia reperfusion （IR）,MSC-treated （IR ＋ MSC）,MSC-derived exosome-treated （IR ＋ MSC-ex） and fibroblastderived exosome-treated （IR ＋ F-ex） groups.The model for ischemia reperfusion injury was constructed.The serum levels of creatinine and blood urea nitrogen （BUN） were tested in each group.The histomorphological changes in of renal tissue samples were examined by hematoxylin and eosin-stained tissue samples.Cellular apoptosis was examined by terminal deoxynucleotidyl transferase-mediated nick-end labeling （TUNEL） staining.The expression levels of proinflammatory cytokines interleukin 1 β and tumor necrosis factor α were examined by reverse transcription （RT）-PCR And the expression level of caspase-3 was examined by Western blot.Results Rat BM-MSCs were successfully isolated and cultured.Dectection of surface markers revealed high expression levels of CD29 and CD44 and a low expression level of CD34.MSC differentiated successfully into osteoblasts and lipocytes after growing in osteoblast-and lipocyte-inducing media respectively.Typical appearances of exosome were observed under transmission electron microscope.CD63 was positive on flow cytometry.Compared with the IR group,the IR ＋ MSC and IR ＋ MSC-ex groups showed low levels of serum creatinine and BUN,mild pathological injury,decreased number of apoptotic cells and low expression of inflammatory factors and caspase-3（IR group,4 310 ±616;IR ＋ MSC group,2 569 ±530; IR ＋ MSC-ex group,3 144 ± 343,both P ＜ 0.05）.Conclusion Rat BM-MSC-deri

关 键 词：再灌注损伤 肾 骨髓间充质干细胞 外泌体 炎症 

分 类 号：R692[医药卫生—泌尿科学]