摘要
目的：观察慢性阻塞性肺疾病(COPD)患者外周血内皮祖细胞(EPCs)数量和功能的变化。
方法：共收集2008年9月-2009年4月随访的稳定期COPD患者20例,诊断标准符合2007年《慢性阻塞性肺疾病(COPD)诊治指南》,同时选择20例非COPD者为对照,均无长期吸烟及被动吸烟史,近2月无呼吸系统疾病史,经病史询问、体格检查、胸部X线摄片、肺通气功能测定(包括乙酰甲胆碱激发试验)均未发现肺部异常。密度梯度法分离外周血的单个核细胞,流式细胞仪计数EPCs (CD34+/CD133+/VEGFR-2+细胞为EPCs),并用添加了生长因子的特异性内皮细胞培养基诱导单个核细胞向内皮细胞分化、增殖,以细胞呈“铺路石”样形态,结合FITC-UEA-I并摄取DiI-acLDL双染色阳性,内皮型一氧化氮合酶(eNOS)、血管性血友病因子(vWF)表达阳性,鉴定为EPCs。通过计数贴壁细胞数目测定EPCs的黏附能力,噻唑蓝比色法(MTT法)测定EPCs的增殖能力,硝酸还原法测定EPCs分泌NO的能力,免疫印迹法测定EPCs的内皮型一氧化氮合酶(eNOS)及磷酸化内皮型一氧化氮合酶(P-eNOS)蛋白表达。
结果：[1].外周血EPCs量少,COPD组外周血分离的单个核细胞中EPCs比例低于对照组[(0.54±0.16)%vs(1.15±0.57)%,P＜0.05]。[2].COPD病人外周血单个核细胞培养、诱导分化后EPCs数量增加,培养第7天EPCs比例为(3.18±0.80)%,第10天为(9.41±2.25)%。[3].COPD组EPCs黏附数低于对照组[(18.7±4.8)个／视野vs(45.0±5.9)个／视野,P<0.05]；COPD组EPCs增殖能力(A490nm值)低于对照组[(0.135±0.038)vs(0.224±0.042), P<0.05]; COPD组EPCs培养液NO浓度低于对照组[(25.11±5.27)μmol/L vs(37.72±7.10)μmol/L, P<0.05]。[4]. COPD组EPCs eNOS、P-eNOS蛋白表达OD值均低于对照组[(112.06±10.00)vs(135.41±5.38), (88.89±4.98)vs(117.98±16.49),P均<0.05]。
结论：[1].外周血来源的EPCs可以成功体外扩增。[2]. COPD病人外周血EPCs数量减少,黏附、增殖及分泌NO功能下降。[3]. COPD病人eNOS表达下降。
目的：经气管移植同种异体小鼠骨髓来源的EPCs,观察EPCs在小鼠体内的定位及移植的EPCs对COPD小鼠肺功能、气道炎症、肺泡和肺血管结构改变及肺组织eNOS、P-eNOS蛋白表达的影响。
方法：分离C57BL／6J小鼠骨髓来源的单个核细胞,予特异性内皮培养基和细胞因子诱导分化、培养,经细胞“铺路石”样形态及结合FITC-UEA-I并摄取DiI-acLDL双染色阳性、vWF表达阳性鉴定为EPCs。烟雾暴露法建立COPD小鼠模型,小鼠随机数字表法分为5组：对照组、COPD组、早期干预组、晚期干预组、PBS假干预组,每组8只,假干预组又随机分为早期假干预组、晚期假干预组,每组4只。COPD组小鼠放入自制熏烟箱中,每次予6支烟燃烧,15分钟,每天4次烟雾暴露,共90天。对照组在箱中同样时间,呼吸空气。在烟雾暴露1个月、3个月时分别进行早期、晚期干预,即CM-DiI标记EPCs后,经气管途径进行同种异体EPCs移植。早期假干预组、晚期假干预组在烟雾暴露1个月、3个月时分别气管内注入PBS。停止烟雾暴露1月后收集标本,小动物肺功能仪测肺功能；肺病理切片苏木素-伊红(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)评估肺气肿程度；LOGENE-I病理图像分析仪进行肺血管形态学定量指标观察：测量与呼吸性细支气管伴行的肺小动脉外径、管壁厚度、管腔面积和血管总面积,然后分别计算血管壁厚度与血管外径比值(WT%)和管腔面积与血管总面积比值(VA%)；支气管肺泡灌洗液(BALF)细胞总数计数,Wright染色后细胞分类计数并计算比例；硝酸还原法测定BALF中NO含量；免疫印迹法测定肺组织内皮型一氧化氮合酶(eNOS)及磷酸化内皮型一氧化氮合酶(P-eNOS)的蛋白表达。
结果：[1].小鼠骨髓来源的单个核细胞在添加了生长因子的特异性内皮培养基中呈梭形、多边形、铺路石样形态,结合FITC-UEA-I并摄取DiI-acLDL双染色阳性,vWF表达阳性,鉴定为EPCs。[2]. CM-DiI标记的EPCs经气管移植后在小鼠肺血管、气道可见荧光表达。[3].经烟雾暴露3个月,COPD组小鼠肺病理切片示肺气肿改变。[4].在对照组、COPD组、早期干预组、晚期干预组、早期假干预组、晚期假干预组平均肺泡隔厚度(MAST)分别为(8.09±1.02)μm、(4.32±0.62)μm、(6.29±1.01)μm、(5.48±0.65)μm、(4.51±0.61)μm、(4.44±0.63)μm；平均内衬间隔(MLI)分别为(33.26±3.22)μm、(51.79±7.25)μm、(40.25±2.65)μm、(45.21±2.25)μm、(50.98±7.68)μm、(51.05±7.43)μm；破坏指数(DI)分别为(12.85±3.22)%、(38.86±4.32)%、(20.37±3.43)%、(28.17±3.56)%、(37.47±4.76)%、(38.46±4.50)%。CODP组MAST小于对照组,差异有统计学意义(P<0.05)；MAST在CODP组、早期假干预组、晚期假干预组之间差异无统计学意义(P均>0.05)；早期干预组MAST小于对照组,大于COPD组和早期假干预组,差异均有统计学意义(P均<0.05)；晚期干预组MAST小于对照组,大于COPD组和晚期假干预组,差异均有统计学意义(P均<0.05)；早期干预组和晚期干预组MAST数值差异无统计学意义(P>0.05)。CODP组MLI、DI大于对照组,差异均有统计学意义(P均<0.05)；MLI、DI在CODP组、早期假干预组、晚期假干预组之间比较差异均无统计学意义(P均>0.05)；早期干预组MLI、DI均大于对照组,小于COPD组和早期假干预组,差异均有统计学意义(P均<0.05)；晚期干预组MLI、DI均大于对照组,也均大于早期干预组,但小于COPD组和晚期假干预组,差异均有统计学意义(P均<0.05)。[5].在对照组、COPD组、早期干预组、晚期干预组、早期假干预组、晚期假干预组血管壁／血管外径(WT%)分别为(7.96±2.66)%、(26.66±2.57)%、(13.10±2.07)%、(17.67±1.44)%、(26.45±2.14)%、(26.33±2.26)%；管腔面积／血管面积(VA%)分别为(78.44±9.77)%、(54.88±4.80)%、(67.88±4.91)%、(63.66±7.89)%、(52.34±5.98)%、(53.46±5.04)%。COPD组WT%大于对照组,差异有统计学意义(P<0.05)；WT%在COPD组、早期假干预组、晚期假干预组之间比较差异无统计学意义(P均>0.05)；早期干预组WT%大于对照组,但小于COPD组和早期假干预组,差异均有统计学意义(P均<0.05)；晚期干预组WT%大于对照组,也大于早期干预组,但小于COPD组和晚期假干预组,差异均有统计学意义(P均<0.05)。COPD组VA%小于对照组,差异有统计学意义(P<0.05)；VA%在COPD组、早期假干预组、晚期假干预组之间比较差异无统计学意义(P均>0.05)。早期干预组VA%小于对照组,但大于COPD组和早期假干预组,差异均有统计学意义(P均<0.05)；晚期干预组VA%小于对照组,差异有统计学意义(P<0.05),VA%在晚期干预组与COPD组之间及晚期干预组和晚期假干预组之间比较差异无统计学意义(P均>0.05)。[6].在对照组、COPD组、早期干预组、晚期干预组、早期假干预组、晚期假干预组气道阻力(Raw)分别为(0.0365±0.0196)Kps/L/s、(0.3179±0.0969)Kpa/L/s、(0.1029±0.0460)Kpa/L/s、(0.1999±0.0113) Kpa/L/s、(0.2893±0.0624)Kpa/L/s、(0.3188±0.0889)Kpa/L/s；肺顺应性(CL)分别为(0.3100±0.1067)L/Kpa、(0.1019±0.0004)L/Kpa、(0.2942±0.0228)L/Kpa、(0.2078±0.0823)L/Kpa、(0.1208±0.0186) L/Kpa、(0.1213±0.0216)L/Kpa；呼气峰流速(PEF)分别为(8.38±1.08)L／S、(7.20±0.97)L／S、(7.95±0.95)L／S、(8.10±0.96)L／S、(7.45±1.05)L／S、(7.75±0.83)L／S。COPD组Raw高于对照组,差异有统计学意义(P<0.05)；Raw在COPD组、早期假干预组、晚期假干预组之间比较差异无统计学意义(P均>0.05)；早期干预组Raw低于COPD组和早期假干预组,差异均有统计学意义(P均<0.05),与对照组比较差异无统计学意义(P>0.05)；晚期干预组Raw高于对照组,也高于早期干预组,但低于COPD组和晚期假干预组,差异均有统计学意义(P均<0.05)。COPD组CL低于对照组,差异有统计学意义(P<0.05)；CL在COPD组、早期假干预组、晚期假干预组之间比较差异无统计学意义(P均>0.05)；早期干预组CL高于COPD组和早期假干预组,差异均有统计学意义(P均<0.05),与对照组比较差异无统计学意义(P>0.05)；晚期干预组CL低于对照组,也低于早期干预组,但高于COPD组和晚期假干预组,差异均有统计学意义(P均<0.05)。PEF在各组之间比较差异无统计学意义(P>0.05)。[7].在对照组、COPD组、早期干预组、晚期干预组、早期假干预组、晚期假干预组BALF中NO浓度分别为(15.55±8.74)μmol/L、(52.65±12.16)μmol/L、(36.10±10.95)μmol/L、(43.76±13.19)μmol/L.(50.56±10.64)μmol/L、(53.37±12.83)μmol/L。COPD组BALF中NO浓度高于对照组,差异有统计学意义(P<0.05);BALF中NO浓度在COPD组、早期假干预组、晚期假干预组之间比较差异无统计学意义(P均>0.05)；早期干预组BALF中NO浓度高于对照组,但低于COPD组和早期假干预组,差异均有统计学意义(P均<0.05)；晚期干预组BALF中NO浓度高于对照组,差异有统计学意义(P<0.05),BALF中NO浓度在晚期干预组与COPD组之间及晚期干预组与晚期假干预组之间比较差异无统计学意义(P均>0.05)。[8].在对照组、COPD组、早期干预组、晚期干预组、早期假干预组、晚期假干预组BALF细胞总数分别为(1.47±0.24)×108／L、(5.85±0.67)×108／L、(3.61±0.38)×108／L、(5.47±0.71)×108／L、(5.67±0.75)×108／L、(5.87±0.72)×108／L；巨噬细胞(AM)分别为(1.34±0.14)×108／L、(4.45±0.63)×108／L、(2.86±0.34)×108／L、(4.11±0.69)×108／L、(4.32±0.73)×108／L、(4.54±0.65)×108／L；中性粒细胞(N)分别为(0.77±0.09)×107/L、(7.76±0.92)×107／L、(5.01±0.78)×107／L、(7.58±0.96)×107／L、(7.62±0.89)×107／L、(7.87±0.81)×107／L；巨噬细胞比例(AM%)分别为(86.55±8.90)%、(76.28±8.25)%、(78.81±7.96)%、(75.42±7.68)%、(76.36±8.03)%、(76.11±8.42)%；中性粒细胞比例(N%)分别为(8.68±0.97)%、(13.71±2.42)%、(12.31±1.98)%、(13.51±2.09)%、(13.01±2.13)%、(13.81±2.39)%。COPD组BALF细胞总数、AM、N、N%高于对照组,差异均有统计学意义(P均<0.05)；BALF细胞总数、AM、N、AM%、N%在COPD组、早期假干预组、晚期假干预组之间比较差异无统计学意义(P均>0.05)；早期干预组BALF细胞总数、AM、N高于对照组,但低于COPD组和早期假干预组,差异均有统计学意义(P均<0.05),N%高于对照组(P<0.05),与COPD组和早期假干预组比较差异均无统计学意义(P均>0.05)；晚期干预组BALF细胞总数、AM、N高于对照组,也高于早期干预组,差异均有统计学意义(P均<0.05),BALF细胞总数、AM、N、AM%、N%在晚期干预组与COPD组之间及晚期干预组与晚期假干预组之间比较差异无统计学意义(P均>0.05)。[9].在对照组、COPD组、早期干预组、晚期干预组、早期假干预组、晚期假干预组肺组织eNOS、P-eNOS的蛋白表达OD值分别为[(253.89±18.69),(193.98±17.56)]、[(142.53±10.27),(140.08±8.43)]、[(162.66±13.61),(156.20±10.09)]、[(168.28±13.65),(152.81±10.43)]、[(150.43±10.71),(141.88±10.54)]、[(148.57±11.16),(140.12±8.92)]。COPD组肺组织eNOS. P-eNOS的蛋白表达均低于对照组,差异均有统计学意义(P均<0.05)；肺组织eNOS、P-eNOS的蛋白表达在COPD组、早期假干预组、晚期假干预组之间比较差异无统计学意义(P均>0.05)；早期干预组肺组织eNOS、P-eNOS的蛋白表达均低于对照组,但高于COPD组和早期假干预组,差异均有统计学意义(P均<0.05)；晚期干预组肺组织eNOS、P-eNOS的蛋白表达均低于对照组,但高于COPD组和晚期假干预组,差异均有统计学意义(P均<0.05)；肺组织eNOS、P-eNOS的蛋白表达在早期干预组和晚期干预组之间差异无统计学意义(P>0.05)。
结论：[1].小鼠骨髓来源的EPCs可在体外成功扩增。[2]. EPCs移植可减轻COPD小鼠肺组织破坏和血管增生,早期移植较晚期移植作用明显。[3].EPCs移植可使COPD小鼠气道阻力下降,肺顺应性改善,早期移植较晚期移植作用明显。[4].早期EPCs移植可减轻COPD小鼠气道炎症。
Objective:To observe the number and function of circulating endothelial progenitor cells(EPCs) in the patients with chronic obstructive pulmonary disease (COPD).
Methods:The total study population included 20 COPD patients and 20 control subjects. Mononuclear cells were isolated from human peripheral blood by density gradient centrifugation, the number of EPCs (CD34+/CD133+/VEGFR-2+cells) was calculated by flow cytometer. Mononuclear cells were cultured in endothelial growth medium-2 (EGM-2), cells with following characteristic were identified as EPCs: cells with "slabstone"-like appearance, expression of von Willebrand factor(vWF) and endothelial nitric oxide synthase (eNOS), taking up DiI-acLDL and combining with FITC-UEA-I. The adherent activity was assessed by the number of EPCs adhering to the culture bottle, the proliferative activity was detected by MTT method, the concentration of NO in EPCs culture solution was determined indirectly by measuring the concentration of total nitrite and nitrate to reflect EPCs'secretory activity, protein expression of eNOS and phosphorated eNOS (P-eNOS) were evaluated by western blotting.
Results:[1]. There were few EPCs in circulation. Circulating EPCs in COPD groop was lower than the control with the percentage of (0.54±0.16)% versus (1.15±0.57)% (P<0.05). [2]. EPCs from COPD patients'peripheral blood were increased after induction and culture.The percentage of EPCs was (3.18±0.80)% after 7 days'culture, and (9.41±2.25%) after 10 days'culture. [3]. The number of adhering EPCs in COPD groop was lower than the control[(18.7±4.8)/field vs (45.0±5.9)/field, P<0.05]. EPCs'proliferative activity in COPD groop was poorer than the control, A490nm in COPD groop was lower than the control[(0.135±0.038) vs (0.224±0.042), P<0.05]. The concentration of NO in the EPCs culture solution in COPD groop was lower than the control[(25.11±5.27)μmol/L vs (37.72±7.10)μmol/L, P<0.05]. [4]. ENOS and P-eNOS protein expression in EPCs in COPD groop were lower than the control[(112.06±10.00) vs (135.41±5.38), (88.89±4.98) vs (117.98±16.49), all P<0.05].
Conclusion:[1]. Human peripheral blood-derived EPCs can increase successfully in vitro. [2]. The number and adherent, proliferative, secretory activity of circulating EPCs in COPD patients are decreased when compared with the control. [3]. ENOS protein expression is decreased in COPD patients.
Objective:To observe the EPCs'location, the change of lung function, pulmonary alveoli and pulmonary blood vessels, airway inflammation, eNOS and P-eNOS protein expression in lung tissue in COPD model mice after EPCs'transplantation.
Methods:Bone marrow-derived mononuclear cells (BMMNCs) were gained by density gradient centrifugation from male C57BL/6J mice cavitas medullaris washing solution, and then cultured in EGM-2. Cells with following characteristic were identified as EPCs:cells with "slabstone"-like appearance, expression of von Willebrand factor(vWF) and endothelial nitric oxide synthase (eNOS), taking up DiI-acLDL and combining with FITC-UEA-I. The COPD mice model was established by exposure the mice to the tobacco smoke (6 cigarettes for 15 minutes,4 times a day, for 90 days). There were six groups in this study:the control group:mice exposed to air, COPD group:mice exposed to smoke for 90 days, early-intervention group and late-intervention group:mice were transplanted with CM-DiI labeled EPCs through trachea after 1 month's and 3 months'smoke exposure respectively, early-pseudo-intervention group and late-pseudo-intervention group:in which mice were transplanted with PBS after 1 month's and 3 months'smoke exposure respectively. One month after the last smoke exposure, mice were anesthetized to measure lung function, the right lung were washed with PBS and BALF was collected, total cell number was countered and the differential cell number were calculated after hematoxylin and eosin(HE) staining. The concentration of NO in BALF was determined indirectly by measuring total nitrite and nitrate. After fixed with 4% paraformaldehyde through the tracheal cannula, the left lower lung was removed and stained with HE. The degree of emphysema was evaluated by mean alveolar septal thickness (MAST), mean linear intercept (MLI) and destructive index (DI). The pulmonary vascular characteristic was evaluated by external diameter of pulmonary arteries, thickness of vascular wall, lumen area and vessel area, the ratio of thickness of vascular wall to external diameter of vascular(WT%) and the ratio of lumen area to vessel area(VA%) by LOGENE-Ⅰimage analysis software.The protein expression of endothelial NOS (eNOS) and phosphorated eNOS (P-eNOS) in left upper lungs were evaluated by western blotting.
Results:[1]. Mice BMMNCs could differentiate to endothelial cells and increased in the specific endothelial culture medium. [2]. EPCs could immigrate to the airway and pulmonary blood vessels after being transplanted to trachea. [3]. The COPD mice model could be established after 3 months'smoke exposure and it was confirmed by lung function and pathological change. [4]. Mean alveolar septal thickness(MAST) in the control、COPD group、early-intervention group、late-intervention group、early-pseudo-intervention group and late-pseudo-intervention group were (8.09±1.02)μm、(4.32±0.62)μm、(6.29±1.01)μm、(5.48±0.65)μm、(4.51±0.61)μm、(4.44±0.63)μm, respectively. Mean linear intercept (MLI) in the control、COPD group、early-intervention group、late-intervention group、early-pseudo-intervention group and late-pseudo-intervention group were (33.26±3.22)μm、(51.79±7.25)μm、(40.25±2.65)μm、(45.21±2.25)μm、(50.98±7.68)μm、(51.05±7.43)μm, respectively. Destructive index(DI) in the control、COPD group、early-intervention group、late-intervention group、early-pseudo-intervention group and late-pseudo-intervention group were (12.85±3.22)%、(38.86±4.32)%、(20.37±3.43)%、(28.17±3.56)%、(37.47±4.76)%、(38.46±4.50)%, respectively.MAST in COPD group was thinner than the control(P<0.05), There were no statistical difference in MAST among COPD group、early-pseudo-intervention group and late-pseudo-intervention group(all P>0.05). MAST in early-intervention group was thinner than the control, but thicker than COPD group and early-pseudo-intervention group (all P<0.05). MAST in late-intervention group was thinner than the control, but thicker than COPD group and late-pseudo-intervention group (all P<0.05). There was no statistical difference in MAST between early-intervention group and late-intervention group (P>0.05). MLI and DI in COPD group were higher than the control(all P<0.05). There were no statistical difference in MLI and DI among COPD group. early-pseudo-intervention group and late-pseudo-intervention group(all P>0.05).MLI and DI in early-intervention group were higher than the control, but lower than COPD group and early-pseudo-intervention group (all P<0.05). MLI and DI in late-intervention group were higher than the control, also higher than early-intervention group, but lower than COPD group and late-pseudo-intervention group(all P<0.05). [5]. WT% in the control. COPD group. early-intervention group、late-intervention group、early-pseudo-intervention group and late-pseudo-intervention group were (7.96±2.66)%、(26.66±2.57)%、(13.10±2.07)%、(17.67±1.44)%、(26.45±2.14)%、(26.33±2.26)%, respectively. VA% in the control. COPD group、early-intervention group、late-intervention group. early-pseudo-intervention group and late-pseudo-intervention group were (78.44±9.77)%、(54.88±4.80)%、(67.88±4.91)%、(63.66±7.89)%、(52.34±5.98)、(53.46±5.04)%, respectively. WT% in COPD group was higher than the control(P<0.05). There were no statistical difference in WT% among COPD group. early-pseudo-intervention group and late-pseudo-intervention group(all P>0.05).WT% in early-intervention group was higher than the control, but lower than the COPD group and early-pseudo-intervention group(all P<0.05).WT% in late-intervention group was higher than the control, and also higher than early-intervention group, but lower than the COPD group and late-pseudo-intervention group(all P<0.05).VA% in COPD group was lower than the control(P<0.05). There were no statistical difference in VA% among COPD group. early-pseudo-intervention group and late-pseudo-intervention group(all P>0.05).VA% in early-intervention group was lower than the control, but higher than COPD group and early-pseudo- intervention group (all P<0.05). VA% in late-intervention group was lower than the control(P<0.05), there were no statistical difference in VA% among late-intervention group、COPD group and late-pseudo-intervention group(all P>0.05). [6]. Airway resistance(Raw) in the control、COPD group、early-intervention group、late-intervention group、early-pseudo-intervention group and late-pseudo-intervention group were (0.0365±0.0196)Kpa/L/s、(0.3179±0.0969) Kpa/L/s、(0.1029±0.0460) Kpa/L/s、(0.1999±0.0113) Kpa/L/s、(0.2893±0.0624) Kpa/L/s、(0.3188±0.0889)Kpa/L/s, respectively. Lung compliance(CL) in the control、COPD group、early-intervention group、late-intervention group、early-pseudo-intervention group and late-pseudo-intervention group were (0.3100±0.1067)L/Kpa、(0.1019±0.0004) L/Kpa、(0.2942±0.0228) L/Kpa、(0.2078±0.0823) L/Kpa、(0.1208±0.0186) L/Kpa, (0.1213±0.0216) L/Kpa,respectively.Peak expiratory flow(PEF) in the control、COPD group、early-intervention group、late-intervention group、early-pseudo-intervention group and late-pseudo-intervention group were (8.38±1.08)L/S、(7.20±0.97) L/S、(7.95±0.95) L/S、(8.10±0.96) L/S、(7.45±1.05) L/S、(7.75±0.83) L/S,respectively. Raw in COPD group was higher than the control. There were no statistical difference in Raw among COPD group、early-pseudo-intervention group and late-pseudo-intervention group (all P>0.05).Raw in early-intervention group was lower than COPD group and early-pseudo-intervention group(all P<0.05), there was no statistical difference in Raw between early-intervention group and the control(P>0.05). Raw in late-intervention group was higher than the control,and also higher than early-intervention group, but lower than COPD group and late-pseudo-intervention group (all P<0.05). CL in COPD group was lower than the control(P<0.05). There were no statistical difference in CL among COPD group、early-pseudo-intervention group and late-pseudo-intervention group(all P>0.05).CL in early-intervention group was higher than COPD group and early-pseudo-intervention group (all P<0.05), there was no statistical difference in CL between early-intervention group and the control(P>0.05). CL in late-intervention group was lower than the control,and also lower than early-intervention group,but higher than COPD group and late-pseudo-intervention group (all P<0.05). There were no statistical difference in PEF among the groups(P>0.05). [7]. The concentration of NO in BALF in the control、COPD group、early-intervention group、late-intervention group、early-pseudo-intervention group and late-pseudo-intervention group were (15.55±8.74)μmol/L、(52.65±12.16)μmol/L、(36.10±10.95)μmol/L、(43.76±13.19)μmol/L、(50.56±10.64)μmol/L、(53.37±12.83)μmol/L, respectively. The concentration of NO in BALF in COPD group was higher than the control(P<0.05). There were no statistical difference in the concentration of NO in BALF among COPD group、early-pseudo-intervention group and late-pseudo-intervention group(all P>0.05).The concentration of NO in BALF in early-intervention group was higher than the control, but lower than COPD group and early-pseudo-intervention group (all P<0.05). The concentration of NO in BALF in late-intervention group was higher than the control(P<0.05), there were no significant difference in the concentration of NO in BALF among late-intervention group、late-pseudo-intervention group and COPD group(P>0.05). [8]. The number of totel cells in BALF in the control、COPD group、early-intervention group、late-intervention group、early-pseudo-intervention group and late-pseudo-intervention group were (1.47±0.24) ×108/L、(5.85±0.67)×108/L、(3.61±0.38)×108/L、(5.47±0.71)×108/L、(5.67±0.75)×108/L、(5.87±0.72)×108/L,respectively.The number of macrophage in BALF in the control.COPD group、early-intervention group、late-intervention group、early-pseudo-intervention group and late-pseudo-intervention group were(1.34±0.14)×108/L、(4.45±0.63)×108/L、(2.86±0.34)×108/L、(4.11±0.69)×108/L、(4.32±0.73)×108/L、(4.54±0.65)×108/L,respectively.The number of neutrophil in BALF in the control.COPD group.early-intervention group、late-intervention group、early-pseudo-intervention group and late-pseudo-intervention group were(0.77±0.09)×107/L、(7.76±0.92)×107/L、(5.01±0.78)×107/L、(7.58±0.96)×107/L、(7.62±0.89)×107/L、(7.87±0.81)×107/L, respectively.The percentage of macrophage in the control. COPD group.early-intervention group.late-intervention group.early-pseudo-intervention group and late-pseudo-intervention group were(86.55±8.90)％.(76.28±8.25)％.(78.81±7.96)％.(75.42±7.68)％.(76.36±8.03)％.(76.11±8.42)％,respectively.The percentage of neutrophil in the control.COPD group.early-intervention group、late-intervention group、early-pseudo-intervention group and late-pseudo-intervention group were(8.68±0.97)％、(13.71±2.42)％、(12.31±1.98)％、(13.51±2.09)％、(13.01±2.13)％、(13.81±2.39)％,respectively.The number of totel cells and macrophage(M)、neutrophil(N)、the percentage of neutrophil(N%)in BALF in COPD group were all larger than those in the control(all P<0.05).There were no statistical difference in the number of totel cells and M、N、N% in BALF among COPD group、early-pseudo-intervention group and late-pseudo-intervention group(all P>0.05).The number of totel cells and M.N in BALF in early-intervention group were highr than those in the control, but lower than those in COPD group and early-pseudo-intervention group (all P<0.05), N% in early-intervention group was higher than the control(P<0.05), there were not statistical difference in N% among early-intervention group、early-pseudo-intervention group and COPD group(P>0.05). The number of totel cells and M、N in BALF in late-intervention group were highr than those in the control, also highr than those in early-intervention group(all P<0.05), there were no statistical difference in the number of totel cells、M、N in BALF among late-intervention group、late-pseudo-intervention group and COPD group(all P>0.05). [9]. OD value of eNOS and P-eNOS protein expression in lung tissue in the control、COPD group、early-intervention group、late-intervention group、early-pseudo-intervention group and late-pseudo-intervention group were [(253.89±18.69), (193.98±17.56)]、[(142.53±10.27), (140.08±8.43)]、[(162.66±13.61), (156.20±10.09)]、[(168.28±13.65), (152.81±10.43)]、[(150.43±10.71), (141.88±10.54)]、[(148.57±11.16), (140.12±8.92)],respectively. ENOS and P-eNOS protein expression in lung tissue in COPD group were lower than those in the control(all P<0.05). There were no statistical difference in eNOS and P-eNOS protein expression in lung tissue among COPD group、early-pseudo-intervention group and late-pseudo-intervention group(all P>0.05).ENOS and P-eNOS protein expression in lung tissue in early-intervention group were lower than the control, but higher than COPD group and early-pseudo-intervention group(all P<0.05).ENOS and P-eNOS protein expression in lung tissue in late-intervention group were lower than the control, but higher than COPD group and late-pseudo-intervention group(all P<0.05).There were no significant difference in eNOS and P-eNOS protein expression in lung tissue between early-intervention group and late-intervention group(all P>0.05).
Conclusion:[1]. Mice bone marrow-derived mononuclear cells (BMMNCs) can differentiate to endothelial cells and increase successfully in vitro. [2]. EPCs transplantation can alleviate lung impairment and pulmonary blood vessel hyperplasia in COPD model mice,early-transplantation is better than late-transplantation. [3]. EPCs transplantation can alleviate airway resistance and increase lung compliance in COPD model mice, early-transplantation is better than late-transplantation. [4]. Early-transplantation with EPCs can alleviate airway inflammation in COPD model mice.
引文
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