粒细胞-巨噬细胞集落刺激因子联合骨髓间充质干细胞对高氧暴露新生大鼠肺损伤的干预作用  

Effects of recombinate mouse granulocyte-macrophage-colony-stimulating factor along with marrow-derived mesenchymal stem cells on lung injury induced by hyperoxia exposure in newborn rats

作　　者：田兆方[1] 李玉红[1] 付雪梅[2] 封志纯[2] 

机构地区：[1]南京医科大学附属淮安第一医院新生儿科,南京223300 [2]北京军区总医院八一儿童医院新生儿中心

出　　处：《中华急诊医学杂志》2010年第1期32-36,共5页Chinese Journal of Emergency Medicine

基　　金：国家自然基金面上资助项目（30772036）;南京医科大学科技发展基金重点项目（07NMUZ038）

摘　　要：目的探讨重组鼠粒细胞-巨噬细胞集落刺激因子（granulocyte macrophage—colony stimulating factor，GM—CSF）联合大鼠骨髓来源的间充质干细胞（mesenchymal stem cells，MSCs）对高氧暴露新生大鼠肺损伤的影响。方法采用贴壁选择法分离、培养、扩增大鼠骨髓来源MSCs，并以4’，6二脒基-2-苯吲哚（4’，6-diamidino-2-phenylindole，DAPI）进行标记，注射前以PBS按要求剂量予以稀释；3日龄清洁级SD新生大鼠32只，置于95％氧环境下7d后，随机（随机数字法）分为4组，每组8只；高氧＋GM—CSF组＋MSCs组（A），高氧＋GM—CSF组（B），高氧＋MSCs组（c），高氧对照组（D）；A，B组大鼠于模型结束后皮下注射GM—CSF9μg/kg，A组同时还予腹腔注射5X104MSCs；C组予模型后腹腔注射5X104MSCs，D组仅注射相同容积的PBS，于注射后72h（13f3龄）处死全部动物，肺组织病理学检测辐射状肺泡计数（radical alveolar counts，RAC）；ELASA法检测肺组织匀浆TNF-α，IL-1β含量；免疫组化检测肺组织端粒酶逆转录酶（telomerase reverse transcrip tase，TERT）积分；荧光显微镜观察DAPI阳性细胞分布情况。结果（1）四组在RAC（P〈0．01）、肺组织匀浆TNFG（P〈0．01），IL-1β（P〈0．01）水平等方面差异均有统计学意义；与D组相比，A，B，C三组RAC值增加（P〈0．05），TNF-α,IL-1β水平下降（P〈0．05）；A组与B、A组与C组差异也均有统计学意义（P〈0．05）。（2）四组在TERT积分差异也有统计学意义（P〈0．01）；A，B组明显增高（P〈0．05）；而C，D两组 间差异均无统计学意义（P〉O．05）。（3）免疫荧光显微镜下A，C组可见DAPI阳性细胞，分别为（6．23±1．88），（5．10±0．91）（P〈0．05）。结论GM—CSF、MSCs对高氧暴露可引致新生大鼠肺损伤均具有保护作用，涉及多种作用机制，联合治疗具有协同作用。Objective To investigate the effects of reeombinate mouse granulocyte-macrophage-colony-stimulating factor （GM-CSF） alone with mesenchymal stem cells （MSCs） on injured lung of rats after exposure to hyperoxia. Method Mouse MSCs were separated, cultured, amplificated, identified and labeled with 4＇, 6-diamidino-2-phenylindole（DAPI）. Thirty-two 3-day-old Sprague Dawley （SD） rats from four litters were randomly divided （random number） into four groups, namely hyperoxia exposured ＋ GM-CSF ＋ MSCs group（ group A）, hyperoxia exposured ＋ GM-CSF group（ group B）, hyperoxia exposured ＋ MSCs group （group C） and hyperoxia exposured group（group D）. All rats were placed in a closed Plexiglas chamber with a minimal flow in and out, providing six to seven exchanges of the chamber vohune per hour and maintaining O2 levels above 95 %. Seven days later, all of them were taken out of the chamber. Rats in group A were treated with 5×10^4 MSCs intraperitoneally alone with 9 μg/kg GM-CSF subcutaneously, rats in group B received 9 μg/kg GM-CSF subcutaneously, rats of group C were treated with 5×10^4 MSCs intraperitoneally and rats of group D were administrated with phosphatebuffered solution （PBS）. Three days latter, all animals were sacrificed by an injection of 120 mg/kg sodium pen- tobarbital, perfused with cold 0.9 % NaCl, and the left lungs were removed. The upper lobe of them were grinded to make tissue homogenates used for ELASA, and the lower lobes of them were made into frozen sections for fluo rescence microscope. The right lungs were fixed in situ for 2 hours by intratracheal instillation of 10% neutral formatin and then were still fixed for additional 24 hours. Sagittal sections of paraffin-embedded middle lobe and upper lobe of left lung tissue were used for Immunohistochemistry and histological study respectively.Immunohistochemistry was used to detect the expression of telomerase reverse transcrip tase （TERT） grade. Results Among 4 groups, there were significantly d

关 键 词：粒细胞-巨噬细胞集落刺激因子 高氧 新生 骨髓间充质干细胞 

分 类 号：R722.1[医药卫生—儿科]