【作者】 何平平；

【导师】 张平；

【作者基本信息】 南华大学 ， 内科学， 2007， 硕士

【摘要】 目的局部肾素-血管紧张素-醛固酮系统(RAAS)被发现参与一些组织器官的纤维化过程。本实验于不同位点阻断RAAS,观察阻断RAAS的不同位点对大鼠肺间质纤维化的影响并探讨相关的作用机制,为肺间质纤维化的的防治提供有效的治疗方法和理论依据。方法将75只SD大鼠随机分成:正常对照组(N组)、肺纤维化模型组(B组)、卡托普利干预组(C组)、螺内酯干预组(S组)和氯沙坦干预组(L组),每组15只大鼠。模型组及各干预组大鼠气管内注入博莱霉素(BLM,5mg/kg)诱导肺纤维化,对照组气管内注入等量生理盐水。动物清醒后各组分别每日给予1.5ml等量液体灌胃1次,N组和B组均以生理盐水灌注,C组予卡托普利60 mg/kg、L组予氯沙坦10 mg/kg、S组予螺内酯100mg/kg灌胃。用药期间观察大鼠一般情况、体重增长速度及死亡情况;每组于给药后第7、14、28天分别随机麻醉5只,取肺泡灌洗液(BALF)进行细胞计数和细胞分类;腹腔放血处死后取肺组织行HE染色、Masson胶原染色及测定肺组织羟脯氨酸(HYP)含量;免疫组化法检测单核细胞趋化蛋白-1(monocyte chemoattractant protein-1,MCP-1)和肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)蛋白表达;RT-PCR观察各组大鼠肺组织MCP-1mRNA表达的水平;TUNEL法检测各组大鼠肺组织细胞凋亡情况。结果1.一般情况:实验前各组大鼠一般情况均好,体重无显著性差异。实验期间B组大鼠一般情况差,体重增长缓慢,有3只大鼠死亡,C、S、L各干预组一般情况较B组好,体重增长速度较B组快,实验期间各有1只大鼠发生死亡。2. BALF细胞计数与分类:B组BALF中细胞总数明显增加,各时间点显著高于N组(P﹤0.01),以后呈逐渐下降趋势。C、S、L各干预组细胞总数高于N组(P﹤0.01),但明显低于B组(P﹤0.01)。B组巨噬细胞比例相对下降,各时间点均明显低于N组(P﹤0.01),以后呈逐渐上升趋势,中性粒细胞比例明显高于N组(P﹤0.01),以后呈逐渐下降趋势,淋巴细胞比例显著低于N组(P﹤0.01),以后呈逐渐下降趋势。C、S、L各组巨噬细胞比例明显高于N组(P﹤0.01)和B组(P﹤0.01),以后呈逐渐上升趋势,中性粒细胞比例明显低于B组(P﹤0.01),以后呈逐渐下降趋势,第14、28天恢复至N组水平(P﹥0.05),淋巴细胞比例低于N组(P﹤0.01),但明显高于B组(P﹤0.01),以后呈逐渐下降趋势。C、S、L各组之间比较无统计学差异(P﹥0.05)。3.肺泡炎及肺纤维化程度:B组肺泡炎和肺纤维化的程度一直高于N组(P﹤0.01)。C、S、L各组和B组病理变化有相似规律,但各时间点肺泡炎和肺纤维化的程度明显低于B组(P﹤0.01)。C、S、L各干预组肺泡炎和肺纤维化程度无统计学差异(P﹥0.05)。4. MCP-1蛋白表达: B组表达水平在各时间点均明显高于N组(P﹤0.01)。C、S、L各干预组表达与B组有同一规律,但表达均较弱,显著低于B组(P﹤0.01)。C、S、L各组之间比较无统计学差异(P﹥0.05)。5. MCP-1mRNA表达: B组MCP-1mRNA表达量于各时间点均明显高于N组(P﹤0.01)。C、S、L各干预组MCP-1mRNA表达量于各时间点均明显低于B组(P﹤0.01或﹤0.05)。C、S、L各组之间比较无统计学差异(P﹥0.05)。6. TNF-α蛋白表达:B组表达水平在各时间点均明显高于N组(P﹤0.01)。C、S、L各干预组表达与B组有同一规律,但表达均较弱,显著低于B组(P﹤0.01)。C、S、L各组之间比较无统计学差异(P﹥0.05)。7.凋亡指数: B组细胞凋亡指数各时间点均明显高于N组(P﹤0.01), C、S、L各干预组凋亡指数明显低B组(P﹤0.01),干预组之间差异无显著性(P﹥0.05)。8. HYP测定:B组HYP含量一直显著高于N组(P﹤0.01),呈逐渐上升趋势。C、S、L各干预组成低水平增高趋势,各时间点比B组明显降低(P﹤0.01)。C、S、L各组之间比较无统计学差异(P﹥0.05)。结论1.通过气管内注入博莱霉素的方法成功地复制了大鼠肺纤维化动物模型,结果显示本实验动物模型与临床肺纤维化表现相似。2.局部RAAS均参与了肺纤维化的形成,阻断RAAS可减轻肺纤维化。3. RAAS不同环节阻断剂可能通过抑制炎性反应,下调MCP-1和TNF-α表达,减轻HYP的合成,抑制肺泡上皮细胞凋亡减轻肺纤维化的形成。更多还原

【Abstract】 Objective The local rennin-angiotensin-aldosterone system (RAAS) has been found to take part in the process of pulmonary fibrosis in some tissue and organs. The experiment is designed to study the effect on pulmonary fibrosis in rats after the blockage of the rennin– angiotensin -aldosterone system at different sites and explore its mechanism;so as to offer some effective theropy method and theory support for the prevention and cure of pulmonary fibrosis.Method Seventy-five SD rats were randomly divided into five groups: normal control group(group N),pulmonary fibrosis model group(group B), captopril group(group C),spironolactone group(S group), losartan group (group L),with 15 rats in each. Group B and every treatment groups received intratracheal instillation of bleomycin (BLM,5mg/kg) to induce pulmonary fibrosis, while group N received normal saline by intratracheal instillation. After the rats wake up, the rats of every groups were perfused into stomach with the same volume fluid (1.5ml) once everyday. Group N and group B were perfused normal saline, group C received captopril 60mg/kg, group L received losartan 10 mg/kg, group S received spironolactone 100mg/kg. In the period of treatments, we observed the general data, increase of weight, death data.Five rats in each group were narcosised on 7, 14, 28 days respectively after intratracheal instillation. Total cell counts and differential analysis of branchoalveolar lavage fluid(BALF) were performed under microscope. After the rats were sacrificed by bleeding, we received lung tissue. Histological changes of the lungs were evaluated by HE stain, Masson′s trichrome stain and hydroxyproline concentration of the lung tissue. The expression of monocyte chemoattractant protein-1 ( MCP-1 ) and tumor necrosis factor-α(TNF-α) was measured by immunhistochemical method with image analyse system. The mRNA expression of MCP-1 was evaluated by RT-PCR quantitative analysis. TUNEL was used to detect the change of cell apoptosis.Results1. General data: Before use the medicine, all the rats of every groups show on significance on general data and weight. In the experiment,for the group of B ,the general data were bad , the increase of weight was slow, three rats had dead during the process. For the group of C,S,L, the general data were better than the group of B significantly, the increase of weight were fast than the group of B, only one rat had dead during the process in each group.2.Analysis in BALF from rats: The total number of cells in the group of B increased,which were significantly higher than that in the group of N(P﹤0.01),there were a degressive trend anon. In the group of C、S、L,the total number of cells were significantly higher than that in the group of N(P﹤0.01),but were significantly lower than that in the group of B(P﹤0.01). The concentrations(%) of macrophages in the group of B descend relatively and were significantly lower than that in the group of N(P﹤0.01)at every time, there were a ascensive trend anon. The concentrations(%)of neutrophils in the group of B were significantly higher than that in the group of N(P﹤0.01)at every time, there were a degressive trend anon. The concentrations(%)of lymphocytes in the group of B were significantly lower than that in the group of N(P﹤0.01)at every time, there were a degressive trend anon. In the group of C、S、L,the concentrations(%) of macrophages were significantly higher than that in the group of N(P﹤0.01)and B(P﹤0.01),and there were a ascensive trend anon. The concentrations(%)of neutrophils were significantly lower than that in the group of B(P﹤0.01),and there were a degressive trend anon, came back the level of the group of N at the 14th,28th day. The concentrations(%)of lymphocytes were significantly lower than that in the group of N(P﹤0.01),but were significantly higher than that in the group of B(P﹤0.01), and there were a degressive trend anon.There were no statistic significant differences in total cell counts and differential analysis in the group of B、C、S、L(P﹥0.05).3. The degree of pulmonary alveolitis and fibrosis :The degree of alveolitis and pulmonary fibrosis of the group of B were in every phase was markedly severe compared with normal contro(lP﹤0.01).The group of C,S,L have the same tendency as the group of B;but the degree of alveolitis and pulmonary fibrosis decreased, compared with the group of B(P﹤0.01).Between C,S,L,there were no significant differences(P>0.05).4.The protein expression of MCP-1: The result of immunohistochemistry suggested that the expression of MCP-1 in lung tissue of the group of B was much higher than that of the group of N at every time(P<0.01). The group of C,S,L have the same tendency as the group of B ,but the MCP-1 concentration were lower than that of the group of B(P<0.01),but there were no significant difference between them(P>0.05).5. The mRNA expression of MCP-1: The result of RT-PCR suggested that the expression of MCP-1mRNA in lung tissue of the group of B was much higher than that of the group of N at every time(P<0.01). The group of C,S,L have the same tendency as the group of B ,but the MCP-1 mRNA were lower than that of the group of B at every time (P<0.01 or<0.05 ),but there were no significant difference between them(P>0.05).6.The protein expression of TNF-α:The result of immunohistochemistry suggested that the expression of TNF-αin lung tissue of the group of B was much higher than that of the group of N at every time(P<0.01). The group of C,S,L have the same tendency as the group of B ,but the TNF-αconcentration were lower than that of the group of B(P<0.01),and there were no significant difference between them(P>0.05).7. Apoptisis index: The apoptosis index of lung cells at every time in the group of B were higher than that in the group of N(P<0.01). The apoptosis index of lung cells in the group of C,S,L were significantly lower than that in the group of B(P<0.01).There were on significant difference between the three treatment groups (P>0.05).8. HYP concentration: HYP concentrations of the group of B were markedly higher then the group of N(P<0.05,P<0.01)and that of B group increased gradually.That of the group of C,S,L took on low level,but increased too,HYP concentration were lower than that of the group of B at every time(P﹤0.01).There were no significant difference in treatment groups(P>0.05).Conclusion1.The rats had received intratracheal instillation with bleomycin (BLM) to induce pulmonary fibrosis models successfully.The results suggested that pulmonary fibrosis of the models were exactly mimicked the clinical procedure of pulmonary fibrosis.2.The local rennin-angiotensin-aldosterone system (RAAS)might take part in the formation of pulmonary fibrosis, the blokage of RAAS can delieve the degree of pulmonary fibrosis.3.The blokage of different sites of RAAS might delieve the degree of pulmonary fibrosis through depressing inflammatory reaction, down-regulate the expression of MCP-1 and TNF-α,delieveing the concentration of HYP and suppressing cell apoptosis.更多还原