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

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、扩增人鼠骨髓来源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按要求剂最予以稀释;3日龄清洁级SD新生大鼠32只,置于95%氧环境下7 d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-GSF 9μg/kg,A组同时还予腹腔注射5×10~4MSCs;C组予模型后腹腔注射5×10~4 MSCs,D组仅注射相同容积的PBS,于注射后72 h(13日龄)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);EIASA法检测肺组织匀浆TNF-α,IL-β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TNRα(P<0.01),IL-β(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-β水平下降(P<0.05);A组与B、A组与C组差异也均有统汁学意义(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>0.05).(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.

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

Objective To investigate the effects of recombinate mouse granulocyte-macrophage-colony-stimulating factor(GM-CSF) alone with mesenchymal stem cells(MSCs) on injured lung of rots 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 hypemxia exposured + GM-CSF + MSCs group(group A), hyperoxia exposured + GM-CSF group( group B), hyperoxia exposured + MSCs group( group C) and hypemxia 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 volume per hour and maintaining O_2 levels above 95%. Seven days lair,all of them were taken out of the chamber. Rats in group A were treated with 5 x 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 x 10~4 MSCs intraperitonealiy 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 pentobarbital, 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 fluorescence microscope. The right lungs were fixed in situ for 2 hours by intratracheal instillation of 10% neutral formalin and then were still fixed for additional 24 hours. Sagittal sections of paraffin-embedded middle lobe and upper lobe of left hmg 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 4groups, there were significantly differences in radical alveolar counts(RAC), TNF-α and IL-β1 in tissue homogenates( P < 0. 01 ). Compared with group D, increase in RAC and the decrease in both TNF-α and IL-β1 were found in other groups, and furthermore there were obvious differences in those changes between group A and group B as well as between group A and group C. There were significant differences in TERT(P<0.01) among four groups. The TERT grade in group A and group B were increased more markedly. DAPI-positive cells were found in group A and group C with significantly differences(6.23 ± 1.88, 5.10 ± 0.91, t = 1.53, P<0.05).Conclusions The protective effects of GM-CSF/MSCs on injuried lungs of new born rats after exposure to hyperoxia may be associated with the increase in the proliferation of stem cells improving the local micro-environment of lung tissue. This protective effects against lung injury induced by hyperoxia exposure may be attributed to the synergism between GM-CSF and MSCs.