【作者】 张艳；

【导师】 陈平；

【作者基本信息】 中南大学 ， 内科学， 2011， 博士

【摘要】 目的探讨经腹腔注射香烟提取物(cigarette smoke extract, CSE)建立肺气肿小鼠模型,并评价模型的病理改变,肺功能,细胞凋亡,蛋白酶表达,抗氧化能力。方法21只SPF级BALB/C小鼠随机分为PBS组(n=7)和CSE组(n=14),制备香烟提取物。两组分别在0,11,22天经腹腔注射0.3mlPBS或CSE,第28天处理两组小鼠,收集标本。PLY3211小动物肺功能检测系统进行小鼠肺功能检查；采用静脉留置针行灌洗术留取支气管肺泡灌洗液(BALF),并用终点比色法检测BALF总抗氧化能力(T-AOC);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肺泡平均截距(MLI),肺泡破坏指数(DI)和平均肺泡隔厚度(MAST);末端转移酶标记技术(TUNEL法)测定肺组织细胞凋亡指数(AI)；免疫组织化学法测定肺组织MMP-2和MMP-9表达。结果(1)与PBS组比较,CSE组小鼠动态肺顺应性减低(p<0.05),气道阻力升高(p<0.05)。(2)病理学观察示：CSE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。部分小血管管腔狭窄,甚至闭锁,血管壁增厚,平滑肌增殖。病理形态学定量分析显示：与PBS组比较,CSE组小鼠肺泡平均截距及肺泡破坏指数增高(均p<0.05),、平均肺泡隔厚度减少(p<0.05)。(3)与PBS组比较,CSE组小鼠肺组织细胞凋亡率升高(p<0.05)。(4)与PBS组比较,CSE组小鼠肺组织MMP-2及MMP-9表达增高(p<0.05)。(5)与PBS组比较,CSE组小鼠支气管肺泡灌洗液抗氧化能力减低(p<0.05)。结论经腹腔注射CSE可以建立肺气肿小鼠模型,该模型稳定可靠,与人类慢性阻塞性肺疾病高度相似。目的探讨内皮特异性分子-1(Endothelial cell specific molecule-1, ESM-1)在经腹腔注射香烟提取物所致慢性阻塞性肺疾病小鼠中的变化情况,了解其与肺组织结构细胞凋亡的相关性。方法21只6周雄性SPF级BALB/C小鼠随机分为2组：PBS组,CSE组。PBS组7只,CSE组14只。分别在0,11,22天经腹腔注射PBS/CSE,第28天处死小鼠。留取肺组织,冻存在—86℃冰箱中,提取组织蛋白,采用Western Blotting法检测肺组织ESM-1的表达。肺组织包埋切片,用免疫组织化学法检测肺组织ESM—1原位表达情况。用ELISA法测定小鼠血清ESM-1浓度。结果(1)与PBS组比较,CSE组小鼠肺组织ESM-1表达(Western Blotting)降低(p<0.05)。(2)与PBS组比较,CSE组小鼠肺组织ESM-1表达(免疫组织化学法)降低(p<0.05)。(3)与PBS组比较,CSE组小鼠血清ESM-1浓度降低(p<0.05)。(4)肺组织细胞凋亡指数(AI)与肺组织ESM-1表达(Western Blotting法) (r=-0.998,p<0.001)呈负相关,与血清ESM-1浓度呈负相关(r=-0.774,p<0.05)。结论本研究证明了肺气肿小鼠肺组织ESM-1的表达以及血清浓度降低,且与AI呈负相关。ESM-1可能参与了COPD细胞凋亡/修复失衡机制。目的探讨内皮特异性分子-1(Endothelial cell specific molecule-1, ESM-1)在慢性阻塞性肺疾病(Chronic obstructive pulmonary disease, COPD)患者体内的变化情况,了解其与肺组织结构细胞凋亡及肺功能的相关性。方法收集COPD患者及正常人的肺组织、血清。肺组织分组：A组：非吸烟(?)COPD者；B组：吸烟并COPD组(稳定期轻度)；C组：吸烟并COPD患者(稳定期中度)。血清分组：D组：健康不吸烟者；E组：吸烟并COPD组(稳定期中度)；F组：吸烟并COPD患者(稳定期重度)；G组：吸烟并COPD患者(稳定期极重度)。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肺泡平均截距(MLI),肺泡破坏指数(DI)和平均肺泡隔厚度(MAST);末端转移酶标记技术(TUNEL法)测定肺组织细胞凋亡指数(AI)；免疫组织化学法检测肺组织ESM-1原位表达情况。ELISA法测定人血清ESM-1浓度。结果(1)病理学观察示：B组及C组人肺组织肺泡腔扩大、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。部分小血管管腔狭窄,甚至闭锁,血管壁增厚,平滑肌增殖。病理形态学定量分析显示：B组及C组MLI较A组明显增高,差异有统计学意义(p<0.05)。C组MLI较B组明显增高,差异有统计学意义(p<0.05)。B组及C组DI较A组明显增高,差异有统计学意义(p<0.05)。C组DI较B组明显增高,差异有统计学意义(p<0.05)。B组及C组MAST较A组明显降低,差异有统计学意义(p<0.05)。C组MAST较B组明显降低,差异有统计学意义(p<0.05)。(2)B组及C组凋亡细胞指数(AI)较A组明显增高,差异有统计学意义(p<0.05)。C组AI较B组明显增高,差异有统计学意义(p<0.05)。(3)B组及C组ESM-1蛋白表达较A组明显降低,差异有统计学意义(p<0.05)。C组ESM-1蛋白表达较B组明显降低,差异有统计学意义(p<0.05)(4)E组、F组、G组血清ESM-1浓度较D组明显降低,差异有统计学意义(p<0.05)。F组、G组血清ESM-1浓度较E组明显降低,差异有统计学意义(p<0.05)。G组血清ESM-1浓度较F组明显降低,差异有统计学意义(p<0.05)(5)肺组织细胞凋亡指数(AI)与肺组织ESM-1表达(r--0.886,p<0.001)呈负相关结论本研究证明了COPD患者肺组织ESM-1的表达以及血清浓度降低,且与AI呈负相关,且与肺功能相关。ESM-1可能参与了COPD细胞凋亡/修复失衡机制。更多还原

【Abstract】 Objective To set and evaluate a mice model of emphysema induced by cigarette smoke extract.Methods BALB/c mice were injected with PBS/CSE(cigarette smoke extract) at day 0,11,22, and were sacrificed at day 28. Pulmonary function, pathology of lung tissue(Mean linear intercept, Destructive index, Mean alveolar septal thickness), antioxidant activity of bronchoalveolar lavage fluid, pulmonary parenchymal apoptosis index, expression of MMP-2, MMP-9 were measured. Results(1)Compared to PBS-group, airway resistance were higher in CSE-group (p<0.05), and airway dynamic compliance were lower in CSE-group(p<0.05).(2)Compared to PBS-group, destructive index and mean linear intercept were higher in CSE-group (p<0.05), and mean alveolar septum thickness were lower in CSE-group (p<0.05). (3)Compared to PBS-group, apoptosis index (AI) were higher in CSE-group (p<0.05).(4)Compared to PBS-group, antioxidant activity were lower in CSE-group (p<0.05).(5)Compared to PBS-group, expression of MMP-2 and MMP-9 increased in CSE-group (p<0.05).Conclusions Intraperitoneal injection of CSE causes emphysema and increase of alveolar septal cell apoptosis in mice. Objective A characteristic pathogenesis of COPD is pulmonary vascular endothelial apoptosis. Endothelial cell specific molecule-1 (ESM-1) is a proteoglycan mainly secreted by pulmonary endothelium. It playes an important role in angiogenesis and proliferation of endothelium. We hypothesized that ESM-1 might be involved in pathogenesis of COPD. To investigate expression of ESM-1 and apoptosis of alveolar septal cells.Methods BALB/c mice were injected with PBS/CSE(cigarette smoke extract) at day 0,11,22, and were sacrificed at day 28. ESM-1 in lung tissue, and concentration of ESM-1 in serum were measured. Results(1)Expression of ESM-1 in lung tissue and concentration of ESM-1 in serum were lower in CSE-group compared with PBS-group (p<0.05).(2)Expression of ESM-1 in lung tissue was inversely correlated with AI (r =-0.998, p<0.001). Concentration of ESM-1 in serum was inversely correlated with AI (r=-0.774, p<0.05). Conclusions ESM-1 decreases in emphysema mice and relates to pulmonary parenchymal apoptosis. It suggestes that ESM-1 may be involved in pathogenesis of COPD. Objective A characteristic pathogenesis of COPD is pulmonary vascular endothelial apoptosis. Endothelial cell specific molecule-1 (ESM-1) is a proteoglycan mainly secreted by pulmonary endothelium. It playes an important role in angiogenesis and proliferation of endothelium. We hypothesized that ESM-1 might be involved in pathogenesis of COPD.This research investigated expression of ESM-1 and apoptosis of alveolar septal cells in COPD patients.Methods Collecting lung tissue and serum of COPD patients and normal people. Lung group:A group:non-smoking non-COPD; B Group: Smoking and COPD group (stable period I); C group:smoking and COPD patients (stable period II). Serum group:D:non-smokers health; E Group:Smoking and COPD group (stable period II); F Group: Smoking and COPD patients (stable period III); G Group:Smoking and COPD patients (stable period IV). Pulmonary function, pathology of lung tissue, ESM-1 in lung tissue, and concentration of ESM-1 in serum were measured. Results (1)Compared to A Group, the MLI and DI were significantly increased and the MAST was decreased in B Group and C Froup(All p<0.05).(2)Compared to A Group, the AI were significantly increased in B Group and C Froup(All p<0.05).(3)Compared to A Group, the expression of ESM-1 in lung tissue was significantly decreased in B Group and C Froup(All p<0.05).(4)Compared to A Group, the concentration of ESM-1 in serum was significantly decreased in B Group and C Froup(All p<0.05).(5)Expression of ESM-1 in lung tissue was inversely correlated with AI (r =-0.886, p<0.05).Conclusions ESM-1 decreases in COPD patients and relates to pulmonary parenchymal apoptosis. It suggestes that ESM-1 may be involved in pathogenesis of COPD.更多还原