【作者】 曹君；

【导师】 陈平；

【作者基本信息】 中南大学 ， 内科学， 2010， 博士

【摘要】 目的探讨内皮祖细胞(Endothelial progenitor cells, EPCs)的培养及鉴定,比较密度梯度离心和全骨髓培养法分离培养EPCs的差异。方法取4周雄性近交系C57BL／6J小鼠骨髓,分别采用密度梯度离心及全骨髓培养法进行培养。相差倒置显微镜观察各组细胞贴壁情况和细胞形态；采用免疫荧光检测细胞摄取Dil-acLDL及结合FITC-UEA-I能力；采用免疫荧光检测细胞血管性血友病因子(vWF)表达,免疫细胞化学检测内皮型一氧化氮合酶(eNOS)表达及流式细胞术检测细胞表面标志CD34、CD133和Flk-1的表达。结果(1)全骨髓培养法培养贴壁细胞多数呈长梭形铺展生长,部分细胞呈类圆形及纺锤形；密度梯度离心培养细胞多呈类圆形、纺缍形、梭形或多边形,部分细胞呈线状排列,培养过程中可见典型铺路石样外观及形成血管样结构。(2)密度梯度离心培养摄取Dil-acLDL和结合FITC-UEA-I双阳性率较全骨髓培养法高[(95.3±1.8)%Vs.(59.7±8.6)%,P<0.05]。(3)密度梯度离心培养细胞vWF及eNOS表达阳性率均高于全骨髓培养法[(92.9±1.5)%vs.(51.6±3.5)%,(90.3±1.5)%vs.(45.8±1.6)%,均P<0.05]。(4)密度梯度离心培养细胞CD34+Flk-1+、CD34+CD133+Flk-1+细胞百分比均高于全骨髓培养法[(26.09±1.62)%vs.(7.58±0.70)%,(18.41±0.97)%vs.(5.00±0.19)％,均P<0.05],CD34+CD133+细胞百分比低于全骨髓培养法[(49.51±1.79)%vs.(72.68±5.30)%,P<0.05]。(5)随着培养时间延长,密度梯度离心CD34+CD133+、CD34+Flk-1+、CD34+CD133+Flk-1+细胞百分比逐渐增高,至第14 d已分别达(45.34±2.12)%、(11.65±1.01)%及(9.513±0.87)%。结论(1)密度梯度离心及全骨髓培养法在EGM-2MV培养体系下均可培养出EPCs。(2)密度梯度离心较全骨髓培养法培养的EPCs纯度高。目的探讨单纯烟雾暴露所致慢性阻塞性肺疾病(Chronic obstructive pulmonary disease, COPD)小鼠模型建立及病理学、气道炎症、肺功能评价及全身表现。方法16只C57BL／6J小鼠随机分为正常对照组及COPD组,每组8只,采用自制烟雾暴露箱进行香烟烟雾暴露,6支／次,4次／d,连续90 d,定期检测体重。烟雾暴露90 d并观察30 d后处理两组小鼠,收集标本。PLY3211小动物肺功能检测系统行小鼠肺功能检查；采用静脉留置针行灌洗术并留取支气管肺泡灌洗液(BALF)行细胞计数及分类计数；肺组织病理切片苏木素-伊红(HE)染色后观察形态学改变并定量测定平均肺泡隔厚度(MAST)、平均内衬间隔(MLI)及肺泡破坏指数(DI)；终点比色法检测血清总抗氧化能力(T-AOC);骨骼肌组织病理切片苏木素-伊红(HE)染色后观察形态学改变并采用TUNEL法检测骨骼肌凋亡率。结果(1)与正常对照组比较,COPD组小鼠气道阻力(Raw)增高[(1.91±0.58) cmH2O·mL-1·min-1 vs. (0.22±0.12) cmH2O·mL-1·min-1,P<0.05],动态肺顺应性(Cdyn)降低[(1.019±0.004) mL/cmH2O vs. (3.100±1.367) mL/cmH2O, P<0.05]。(2)与正常对照组相比,COPD组小鼠BALF中细胞总数[(5.85±0.67)×108/Lvs.(1.47±0.24)×108/L]、巨噬细胞数(AM)[(4.45±0.63)×108／L vs.(1.34±0.14)×108／L]、中性粒细胞数(N)[(7.76±0.92)×107／Lvs.(0.77±0.09)×107/L]、中性粒细胞所占比例(N%)[(13.7±2.4)%vs.(8.7±1.0)%]均增高(均P<0.05)。(3)病理学观察示COPD组小鼠肺组织肺泡腔扩大、部分肺泡间隔断裂、肺气肿形成,气道上皮排列紊乱、部分气道上皮增生、周围炎症细胞浸润并伴有平滑肌增生。(4)病理形态学定量分析显示：与正常对照组比较,COPD组MLI及DI增高[(51.8±7.3)μm vs.(33.3±3.2)μm,(38.9±4.3)%vs.(12.9±3.2)%,均P<0.05],MAST减低[(4.3±0.6)μm vs.(8.1±1.0)μm, P<0.05]。(5)外周骨骼肌病理显示：COPD组小鼠股四头肌纤维则排列松散,胞浆染色深浅不一,可见胞浆空泡,胞核相对减少伴核内移。与正常对照组比较,COPD组小鼠体重及血清抗氧化能力均降低[(23.1±1.5)g vs.(26.9±0.7)g,(11.2±0.4)U／mLvs.(18.5±0.4)U／mL,均P<0.05],骨骼肌凋亡率增高[(20.6±4.5)%vs.(2.9±0.6)%,均P<0.05]。(7)小鼠体重与血清总抗氧化能力呈正相关(r=0.815,P<0.05),与骨骼肌细胞凋亡呈负相关(r=-0.852,P<0.05)；骨骼肌细胞凋亡与血清总抗氧化能力呈负相关(r=-0.941,P<0.05)。结论单纯烟雾暴露可以成功建立小鼠COPD模型且稳定可靠,与人类COPD相似性高。目的探讨内皮祖细胞(EPCs)对烟雾暴露所致慢性阻塞性肺疾病(COPD)小鼠肺功能、气道炎症、肺病理、肺上皮及内皮细胞凋亡、抗氧化能力及基质金属蛋白酶(MMPs)的影响,以了解其对COPD是否有保护作用及机制。方法(1)密度梯度离心分离4周雄性C57BL／6J小鼠骨髓单个核细胞于EGM-2MV中诱导培养成EPCs。(2) 40只6周雄性C57BL／6J小鼠随机分为5组：正常对照组、PBS早期干预组、PBS晚期干预组、EPCs早期干预组和EPCs晚期干预组,每组8只。除正常对照组外,其余各组均予香烟烟雾暴露,6支烟／次,4次／d,连续90 d。EPCs早期干预和EPCs晚期干预组分别于烟雾暴露第30 d、第90 d经气道注入30μL 1×105个CM-DiI标记的EPCs进行干预,而PBS早期干预组及PBS晚期干预组在分别于烟雾暴露第30 d、第90 d经气道注入30μL PBS。(3)烟雾暴露停止后第30 d,麻醉各组小鼠,采用PLY3211小动物肺功能检测系统测定肺功能,心脏取血处死小鼠,收集血清、BALF及肺组织标本。肺病理切片HE染色后测定平均肺泡隔厚度(MAST)、平均内衬间隔(MLI)和肺泡破坏指数(DI)；免疫荧光法定位肺组织细胞角蛋白(Pan-cytokeratin); BALF行细胞计数及分类计数；比色法测定BALF及血清总抗氧化能力(T-AOC); TUNEL法检测肺内细胞凋亡水平并计算凋亡指数(apoptotic index,博士学位论文中文摘要AI)；ELISA法检测BALF中MMP-2、MMP-9及TIMP-1水平；免疫组化检测肺组织MMP-2及MMP-9表达；明胶酶谱法测定肺组织MMP-2及MMP-9活性；RT-PCR法检测肺组织MMP-2、MMP-9及TIMP-1mRNA表达水平。结果(1)小鼠骨髓来源的单个核细胞在EGM-2MV培养体系中可形成典型“铺路石”样外观及血管样结构并鉴定为EPCs。(2)CM-DiI标记EPCs经气道注入,在小鼠气道及血管可见荧光表达。(3)与正常对照组相比,PBS早期干预组、PBS晚期干预组、EPCs早期干预组及EPCs晚期干预组Raw均增高,Cdyn均降低(均P<0.05)。EPCs早期干预组Raw较PBS早期干预组低,EPCs晚期干预组Raw较PBS晚期干预组低,EPCs早期干预组Raw较EPCs晚期干预组低(均P<0.05)。EPCs早期干预组Cdyn较PBS早期干预组高,EPCs晚期干预组Cdyn较PBS晚期干预组高,EPCs早期干预组Cdyn较EPCs晚期干预组高(均P<0.05)。Raw及Cdyn在PBS早期干预组及PBS晚期干预组间差异无统计学意义(P>0.05)。PEF在各组之间比较差异无统计学意义(均P>0.05)。(4)PBS早期干预组及PBS晚期干预组肺组织可见明显肺泡腔变大、肺泡间隔断裂、肺气肿形成,EPCs早期干预组及EPCs晚期干预组病变程度较轻。与正常对照组相比,PBS早期干预组、PBS晚期干预组、EPCs早期干预组及EPCs晚期干预组MAST均降低,ⅦMLI及DI均增高(均P<0.05)。EPCs早期干预组MAST大于PBS早期干预组,而EPCs晚期干预组MAST大于PBS晚期干预组,且EPCs早期干预组MAST大于EPCs晚期干预组(均P<0.05)。EPCs早期干预组MLI及DI均较PBS早期干预组低,EPCs晚期干预组MLI及DI均较PBS晚期干预组低,EPCs早期干预组MLI及DI均较EPCs晚期干预组低(均P<0.05)。MAST、MLI及DI在PBS早期干预组及PBS晚期干预组间差异无统计学意义(均P>0.05)。(5)免疫荧光显示上皮特异标志物细胞角蛋白(Pan-cytokeratin)阳性部位可见CM-DiI红色荧光表达,双阳部位呈黄色或橙色荧光。(6)PBS早期干预组、PBS晚期干预组、EPCs早期干预组及EPCs晚期干预组BALF中细胞总数、巨噬细胞数(AM)、中性粒细胞数(N)及中性粒细胞所占比例(N%)均高于正常对照组(均P<0.05)。EPCs早期干预组BALF中细胞总数、AM及N均较PBS早期干预组及EPCs晚期干预组低(均P<0.05)。EPCs晚期干预组BALF细胞总数、AM、AM%、N及N%较PBS晚期干预组稍低,但差异无统计学意义(均P>0.05)。BALF细胞总数、AM、AM%、N及N%在PBS早期干预组和PBS晚期干预组中差异亦无统计学意义(均P>0.05)。(7) BALF及血清总抗氧化能力在PBS早期干预组、PBS晚期干预组、EPCs早期干预组及EPCs晚期干预组均低于正常对照组(均P>0.05)。EPCs早期干预组BALF及血清总抗氧化能力高于PBS早期干预组,EPCs晚期干预组BALF及血清总抗氧化能力高于PBS晚期干预组均P<0.05)。EPCs早期干预组BALF及血清总抗氧化能力高于EPCs晚期干预组(均P<0.05)。BALF及血清总抗氧化能力在PBS早期干预组和PBS晚期干预组中差异无统计学意义。(8) BALF中MMP-2及MMP-9水平在PBS早期干预组、PBS晚期干预组、EPCs早期干预组及EPCs晚期干预组均高于正常对照组(均P>0.05)。EPCs早期干预组BALF中MMP-2及MMP-9水平较PBS早期干预组低,而EPCs晚期干预组BALF中MMP-2及MMP-9水平亦较PBS晚期干预组低,差异均有统计学意义(均P<0.05)。EPCs早期干预组BALF中MMP-2及MMP-9水平亦较EPCs晚期干预组低(均P<0.05)。与正常对照组相比,PBS早期干预组、PBS晚期干预组、EPCs早期干预组及EPCs晚期干预组BALF中TIMP-1的水平均减低(均P<0.05)。EPCs早期干预组BALF中TIMP-1的水平较PBS早期干预组高,EPCs晚期干预组BALF中TIMP-1的水平较PBS晚期干预组高(均P<0.05)；EPCs早期干预组BALF中TIMP-1水平亦高于EPCs晚期干预组(均P<0.05)。BALF中MMP-2、MMP-9及TIMP-1水平在PBS早期及晚期干预组间差异无统计学差异(均P>0.05)。(9)各组均可见凋亡细胞。PBS早期干预组、PBS晚期干预组、EPCs早期干预组及EPCs晚期干预组肺泡隔及肺血管内皮AI均较正常对照组高(均P<0.05)。EPCs早期干预组肺泡隔及肺血管内皮AI均较PBS早期干预组低,EPCs晚期干预组肺泡隔及肺血管内皮AI均较PBS晚期干预组低(均P<0.05)。EPCs早期干预组肺泡隔及肺血管内皮AI亦低于EPCs晚期干预组(均P<0.05)。肺泡隔及肺血管内皮AI在PBS早期干预组和PBS晚期干预组中差异无统计学意义。(10)免疫组化显示：与正常对照组相比,PBS早期干预组、PBS晚期干预组、EPCs早期干预组及EPCs晚期干预组肺组织MMP-2及MMP-9表达均增强(均P<0.05)。EPCs早期干预组肺组织MMP-2及MMP-9表达均较PBS早期干预组低,EPCs晚期干预组肺组织MMP-2及MMP-9表达均较PBS晚期干预组低(均P<0.05)。EPCs早期干预组肺组织MMP-2及MMP-9表达亦低于EPCs晚期干预组(均P<0.05)。肺组织MMP-2及MMP-9表达在PBS早期干预组和PBS晚期干预组中差异无统计学意义。(11)明胶酶谱示PBS早期干预组、PBS晚期干预组、EPCs早期干预组及EPCs晚期干预组肺组织MMP-2、MMP-9活性均较正常对照组增高(均P<0.05)。EPCs干预能抑制增高的MMP-2和MMP-9活性,且EPCs早期干预组肺组织MMP-2、MMP-9活性均低于EPCs晚期干预组(均P<0.05)。PBS早期及晚期干预组间肺组织MMP-2及MMP-9活性差别无统计学意义。(12)与正常对照组相比,PBS早期干预组、PBS晚期干预组、EPCs早期干预组及EPCs晚期干预组肺组织MMP-2、MMP-9mRNA表达均增高,TIMP-1mRNA表达减低(均P<0.05)。EPCs早期干预组肺组织MMP-2、MMP-9 mRNA表达均低于PBS早期干预组,EPCs晚期干预组肺组织MMP-2、MMP-9 mRNA表达均低于PBS晚期干预组,且EPCs早期干预组肺组织MMP-2、MMP-9 mRNA表达均低于EPCs晚期干预组(均P<0.05)。EPCs早期干预组肺组织TIMP-1 mRNA表达较PBS早期干预组高,EPCs晚期干预组肺组织TIMP-1 mRNA表达较PBS晚期干预组高,且EPCs早期干预组肺组织TIMP-1mRNA表达高于EPCs晚期干预组(均P<0.05)。结论(1) EPCs经气管注入可部分抑制烟雾暴露所致慢性阻塞性肺疾病小鼠气道炎症浸润、肺内细胞凋亡、MMPs表达及活性,提高局部及全身抗氧化能力,改善肺功能,促进肺组织修复,减轻肺气肿程度。(2)EPCs早期干预较晚期干预效果好。更多还原

【Abstract】 Objective To discuss the cultivation and identification methods of endothelial progenitor cells (EPCs) and observe the differences between whole bone marrow culture method and density gradient centrifugation method in isolating and cultivating EPCs.Methods Bone marrow obtained from healthy C57BL/6J mice were cultivated by whole bone marrow culture method and density gradient centrifugation method respectively in ECM-2MV medium. The morphologic features of cells were observed under inverted microscope, and the abilities of DiI-acLDL uptaking and FITC-UEA-I binding were detected through fluorescence stain. Immunofluorescence was used to investigate the expression of von Willebrand factor (vWF). Immunocytochemistry was used to investigate the expression of endothelial nitric oxide synthase (eNOS). The cell surface markers were analysed by flow cytometry.Results(1) Primary cells culture with density gradient centrifugation method showed the characteristics of forming capillary structure and "cobblestone" morphology. Under the method of whole bone marrow culture, most adhere cells were long fusiform-shaped and grown in spreading style.(2) The double-positive rate in uptaking DiI-acLDL and binding FITC-UEA-I of adhere cells in density gradient centrifugation method [(95.3±1.8)%] was higher than that in whole bone marrow culture method [(59.7±8.6)%] (P<0.05).(3) The expression rate of vWF and eNOS of adhere cells in density gradient centrifugation method [(92.9±1.5)%,(92.9±1.5)%] were higher than that in whole bone marrow culture method [(51.6±3.5)%, (45.8±1.6)%](all P<0.05).(4) More CD34+Flk-1+and CD34+CD133+Flk-1+cells were detected in density gradient centrifugation method than whole bone marrow culture method [(26.09±1.62)% vs. (7.58±0.70)%, (18.41±0.97)%vs. (5.00±0.19)%, all P<0.05], but the ratio of CD34+CD133+cells were higher in whole bone marrow culture method than in density gradient centrifugation method [(49.51±1.79)% vs. (72.68±5.30)%, P<0.05].(5) Under the method of density gradient centrifugaion, the ratio of CD34+CD133+, CD34+Flk-1+and CD34+CD133+Flk-1+were increased during the culture time (all P<0.05)., and reached to (45.34±2.12)%, (11.65±1.01)% and (9.513±0.87)% in day 14 respectively.Conclusion (1) EPCs could be obtained from mice bone marrow under density gradient centrifugation method and whole bone marrow method under the culture system of EGM-2MV. (2) Compared with whole bone marrow culture method, density gradient centrifugation method was better for higher purity of EPCs. Objective To set and evaluate a mouse model of COPD induced by cigarette smoke (CS) and observe the systemic manifest.Methods Adult (n=8,18-20g body wt) male C57BL/6J mice (COPD group) were exposed 4 times per day, whole body, to CS from six cigarettes for continuous 90 days and then to observe for another 30 days. The control group (n=8) was sham-smoked. BAL with venous indwelling catheter was used to collected bronchoalveolar lavage fluid (BALF). Pulmonary function was measured by PLY 3211 small animal pulmonary function testing system. The total cell count and classification of BALF were measured, and morphology manifests of lung tissues were evaluated by H&E stain. Systemic manifests of mice were detected, including body weight, morphology manifests in H&E stain and apoptosis of skeletal muscles by TUNEL, and total antioxidant capacity (T-AOC) in serum measured by colorimetric technique.Results(1) Compared with the normal control group, higher Raw[(1.91±0.58) cmH2O·mL-1·min-1 vs. (0.22±0.12) cmH2O·mL-1·min-1, P<0.05], lower Cdyn [(1.019±0.004) mL/cmH20 vs. (3.100±1.367) mL/cmH2O, P <0.05] were found in COPD group. (2) Compared with the normal control group, COPD group owned more total cell count [(5.85±0.67)×108/L vs. (1.47±0.24)×108/L], AM [(4.45±0.63)×108/L vs. 1.34±0.14)×108/L], N [(7.76±0.92)×107/L vs. (0.77±0.09)×107/L] and higher N%[(13.7±2.4)% vs. (8.7±1.0)%] in BALF. (3) Morphology detection of lung tissues showed significant enlarged airspace, disruption of alveolar septum and formation of emphysema in COPD group. (4) Quantitative morphological analysis showed:compared with the normal control group, MLI and DI [(51.8±7.3)μm vs.(33.3±3.2)μm, (38.9±4.3)% vs. (12.9±3.2)% respectively, all P<0.05] were higher in COPD group, while MAST [(4.3±0.6)μm vs.(8.1±1.0)μm, P<0.05] was lower in COPD group. (5) Morphology detection of peripheral skeletal muscle showed random loose arrangement of fibers, inhomogenous stain or vacuole forming in endochylema, lessen of nuclei or even "nuclei ingression" in quadriceps femoris of COPD group. Compared with the normal control group, COPD group showed higher apoptosis index [(20.6±4.5)% vs.(2.9±0.6)%, P<0.05] in peripheral skeletal muscle. Lower total antioxidant capacity (T-AOC) in serum was detected in COPD group than that in normal control. (6) Body weight was positively correlated with T-AOC (r=0.815, P<0.05), and was negatively correlated with the apoptosis of skeletal muscle (r=-0.852, P<0.05). The apoptosis of skeletal muscle was negatively correlated with T-AOC (r=-0.941, P<0.05).Conclusions Cigarette Smoke could establish COPD model stably in mice, this mouse model owned high similarity to human COPD. Objective This study was designed to determine the effect of intratracheal allogenic EPCs transplantation on cigarette smoke-induced COPD model of mice and its mechanism.Methods (1) Isolated mononuclear cells from C57BL/6J mice bone marrow were cultured in EGM-2MV medium for 10-12 days, yielding EPCs. (2) Forty male C57BL/6J mice were randomly divided into five groups:normal control group, PBS early treated group, PBS late treated group, EPCs early treated group and EPCs late treated group. All groups except the normal control group which was sham-smoked were exposed passively to cigarette smoke (CS) (6 cigarettes for 15 minutes,4 times per day for continuous 90 days). At the 30th-day and 90th-day after cigarette smoke exposure, allogenic EPCs (105 cells in 30μL PBS) were administered into the trachea of mice in EPCs early treated group and EPCs late treated group respectively. Correspondingly, the PBS early treated group and PBS late treated group were treated with 30μL PBS at the 30th-day and 90th-day respectively. (3) 30 days after the final cigarette smoke exposure, mice were anesthetized to measure lung function by PLY 3211 small animal pulmonary function testing system, then sacrificed by intracardiac bloodletting, and the samples of serum, BALF and lung tissues were collected. Lung paraffin sections were stained with hematoxylin and eosin (HE), and measured mean alveolar septal thickness (MAST), mean linear intercept (MLI) and destructive index (DI) to evaluate the degree of emphysema. Frozen sections of lung were stained with immunofluorescence to located Pan-cytokeratin. The total cell and classification were counted in BALF. The total antioxidant capacity (T-AOC) in serum and BALF were measured by colorimetric technique, while the levels of MMP-2, MMP-9 and TIMP-1 in BALF were detected by ELISA. TUNEL was performed to observe the DNA damaged cells in lung and the expressions of MMP-2 and MMP-9 were determined by immunohistochemisty. MMP-2 and MMP-9 activities were investigated by gelatin zymography. And mRNA levels of MMP-2, MMP-9 and TIMP-1 were measured by reverse transcriptase PCR (RT-PCR).Results(1) Mononuclear cells from C57BL/6J mice bone marrow cultured in EGM-2MV medium had EC-liked shape and were identified as EPCs.(2) EPCs could immigrate to the airway, alveolar septum and pulmonary blood vessels after intratracheal transplantation.(3) The lung function showed higher airway resistance (Raw) and lower dynamic lung compliance (Cdyn) were detected in PBS or EPCs early and late treated groups than those of the control group (All P<0.05). And EPCs early and late treated groups owned lower Raw and higher Cdyn than corresponding PBS treated groups respectively (All P<0.05). Also, EPCs early treated group owned lower Raw and higher Cdyn than EPCs late treated group (All P<0.05). The differences among groups in PEF were of no significance (All P>0.05).(4) Morphology detection (HE stain) showed significant enlarged airspace, disruption of alveolar septum and formation of emphysema in PBS early and late treated groups, and the degree of alveolar destruction in EPCs early and late treated groups were relieved compared with those of corresponding PBS treated groups respectively. The MLI and DI were significantly increased and the MAST was decreased in the PBS early and late treated groups compared with the control group (All P<0.05). However, The MLI and DI were markedly decreased while the MAST was increased in EPCs treated groups (especially in EPCs early treated group) compared with corresponding PBS treated groups (All P<0.05).(5) The frozen sections stained with immunofluorescence manifest the positive areas in green fluorescence of FITC. Together with the red fluorescence of CM-DiI, some areas that were double stained by FITC and CM-DiI showed yellow or orange fluorenscence.(6) The number of total cells, macrophages, neutrophils and ratio of neutrophils in BALF were increased markedly in PBS early and late treated groups in comparison to the control group, and EPCs early treated group showed fewer total cells, macrophages and neutrophils in BALF than corresponding PBS treated group (All P<0.05). But there were no statistical difference between EPCs late treated group and PBS late treated group (All P>0.05).(7) The total antioxidant capacities (T-AOC) in serum and BALF were significantly decreased in PBS early and late treated groups in comparison with the control group (All P<0.05). After EPCs treatments, the decrease of T-AOC in serum and BALF was inhibited partly compared with corresponding PBS treated group (All P<0.05). And the EPCs early treated group owned higher level of T-AOC in serum and BALF compared with EPCs late treated group (All P<0.05).(8) Compared with the control group, PBS early and late treated groups showed higher level of MMP-2 and MMP-9, and lower level of TIMP-1 in BALF (All P<0.05). The EPCs treatments inhibited the increased level of MMP-2 and MMP-9 and decreased level of TIMP-1 in mice exposed to CS (All P<0.05). And the effects were more obviously in EPCs early treated group than EPCs late treated group (All P<0.05).(9) The TUNEL-positive cells were markedly distributed in alveolar septum and pulmonary blood vessels of the emphysematous lungs of mice in PBS early and late treated groups. The apoptosis index (AI) in both alveolar septum and pulmonary blood vessels were significantly higher in PBS early and late treated groups than that of the control group (All P<0.05). And the AI was reduced in EPCs treated groups, especially in EPCs early treated group (All P<0.05).(10) More MMP-2 and MMP-9 positive cells were distributed in the emphysematous lungs of PBS early and late treated groups comparing with those in the lung of the control group. The expressions of MMP-2 and MMP-9 were apparently reduced in EPCs early and late treated groups compared with those of PBS early and late treated groups respectively (All P<0.05). And the expressions of MMP-2 and MMP-9 in EPCs early treated group were lower than that of EPCs late treated groups (All P<0.05).(11) Mice in PBS early and late groups showed significantly increased MMP-2 and MMP-9 activities in lung homogenates compared with those of the control group(All P<0.05). Expectedly, MMP-2 and MMP-9 activities were reduced significantly in both EPCs early and late treated groups compared with corresponding PBS treated groups (All P<0.05). Also, EPCs early treatment showed more beneficial in inhibiting MMPs activities than EPCs late treatment (All P<0.05).(12) RT-PCR detected increase in mRNA levels of MMP-2 and MMP-9 and decrease in mRNA level of TIMP-lin lungs of PBS early and late treated groups in comparison to those of the control group, and EPCs treated groups (especially EPCs early treated group) owned lower mRNA levels of MMP-2 and MMP-9, and higher mRNA level of TIMP-lthan those of corresponding PBS treated group (All P<0.05).Conclusions(1) We concluded that intratracheal transplantation of EPCs protected against the development of emphysema and improved lung function probably by alleviating inflammatory infiltration, decelerating apoptosis of endothelial cells and epithelial cells, inhibiting proteolytic enzyme activity and expression, and enhancing local and systemic antioxidant activity. (2) EPCs early treatment was more beneficial than EPCs late treatment. EPCs might represent a new therapeutic option in the treatment of emphysema in humans.更多还原