急性肺损伤早期肺纤维化大鼠肺组织的PD-1基因表达及硫氢化钠的干预作用

发布时间：2018-03-09 20:10

本文选题：急性肺损伤　切入点：肺纤维化　出处：《南华大学》2012年硕士论文　论文类型：学位论文

【摘要】：目的通过检测油酸致大鼠急性肺损伤早期肺纤维化不同时间点肺组织程序性死亡受体-1(Programmed death-1，PD-1)、PD-1基因、细胞凋亡及硫氢化钠干预的大鼠肺组织PD-1的变化，探讨硫氢化钠对油酸致大鼠急性肺损伤早期肺纤维化的保护作用。方法应用尾静脉注射OA复制大鼠ALI模型。清洁级健康、Sprague Dawley（SD）大鼠54只，适应性饲养一周后，随机分为对照组；OA致伤模型组；干预组。模型组按3天、7天、14天、28天4个时间点分为4个亚组；干预组按3天、7天、14天、28天4个时间点分为4个亚组。每个亚组及对照组均为6只大鼠。模型组和干预组尾静脉注射OA (0.12mg/kg)诱导急性肺损伤，对照组在相同条件下给予生理盐水。第二天起干预组大鼠每天经尾静脉注射硫氢化钠(3.12mg/kg)，其余两组相同条件下给予助溶剂羧甲基纤维素钠。相应时间点处死动物取出肺组织，进行病理学观察；测定肺组织中羟脯氨酸含量；用免疫组化和RT-PCR观察各组鼠肺组织PD-1蛋白及mRNA表达的水平；TUNEL法检测28天各组大鼠肺组织细胞凋亡。结果 1、肺组织HE染色、Masson染色：对照组大鼠观察未见明显病变。模型组大鼠第3天表现为明显的急性炎症期，肺泡间隔增宽、肺间质内有中性粒细胞渗出，伴有水肿和轻度出血。7天肺泡壁水肿、增厚比较明显，主要以巨噬细胞和中性粒细胞为主的炎性细胞浸润，伴有毛细血管增生，成纤维细胞增多，肺间隔仍增宽，有胶原沉积及斑片状的纤维化改变；14天组，肺泡炎达高峰，，肺泡结构破坏，大量胶原沉积在新生的毛细血管周围，肺泡间隔及部分肺泡腔被胶原和纤维蛋白占据，间质内炎性细胞逐渐向以淋巴细胞浸润为主过渡。28天组，肺间质纤维化成分明显增多，肺纤维化最重，肺泡结构紊乱，部分坍陷和消失，毛细管腔明显增厚。模型组、干预组肺泡炎各期均明显比对照组重，而肺纤维化仅第14天和第28天与对照组比较差异显著。干预组与模型组病理变化有同一规律，但肺泡炎、肺纤维化程度都轻于模型组。说明硫氢化钠可以减轻肺泡炎和肺纤维化程度。 2羟脯氨酸（HYP）含量：模型组HYP含量均高于对照组及干预组（P0.01）,模型组HYP含量随造模时间的延长逐渐增高，28天达到最高值；干预组HYP含量成低水平增高趋势，HYP含量均高于对照组而低于模型组（P0.01或P0.05）。 3肺组织PD-1蛋白和PD-1mRNA在肺纤维化过程中的表达：免疫组化以及RT-PCR结果显示模型组大鼠PD-1蛋白和PD-1mRNA表达量高于对照组及干预组，各时间点与两组比较差异均有统计学意义（P0.01），干预组PD-1和PD-1mRNA表达量高于对照组，与对照组比较差异均有统计学意义（P0.05）。 4肺组织细胞凋亡变化：模型组第28天大鼠肺组织中细胞凋亡指数为(13.7±3.4)%，对照组为(1.9±0.3)%和干预组(5.34±1.54)%，差异有统计学意义(F=79.90，P0.01)。结论： 1、急性肺损伤早期肺纤维化肺组织PD-1基因表达及细胞凋亡指数的上调，可能在急性肺损伤早期肺纤维化发展中发挥了作用。 2、硫氢化钠通过抑制PD-1的过度表达从而减轻油酸诱发的大鼠急性肺损伤和肺纤维化。PD-1可能是ALI和肺纤维化治疗的一个新的靶分子。
[Abstract]:The purpose of the early damage of lung fibrosis at different time points of programmed death receptor -1 detection of oleic acid induced acute lung in rats by (Programmed death-1, PD-1), PD-1 gene, cell apoptosis and sodium hydrosulfide intervention in lung tissue of PD-1 rats, to investigate the protective effect of sodium hydrosulfide on oleic acid induced acute lung injury and pulmonary fibrosis rat.
Methods intravenous injection of OA replication in ALI model rats. Clean healthy, Sprague Dawley (SD) 54 rats afteradaptivebreedingforoneweek, were randomly divided into control group; model group induced OA injury; intervention group. The model group on 3 days, 7 days, 14 days, 28 days and 4 hours points are divided into 4 subgroups; the intervention group on 3 days, 7 days, 14 days, 28 days 4 time points are divided into 4 sub groups. Each sub group and control group were 6 rats. Model group and intervention group were injected with OA (0.12mg/kg) - induced acute lung injury, control group were given normal saline in the same condition. The second day daily intervention group rats by intravenous injection of sodium hydrosulfide (3.12mg/kg), the other two groups under the same conditions to help solvent sodium carboxymethyl cellulose. The corresponding time after animal lung tissue and pathological observation; Determination of hydroxyproline content in lung tissue with; immunohistochemistry and RT-PCR were observed in rats The expression level of PD-1 protein and mRNA in lung tissue; TUNEL assay was used to detect the apoptosis of lung tissue in every group of rats in 28 days.
Result
1, lung tissue HE staining, Masson staining: the rats in control group were no significant pathological changes. The rats in the model group third days showed acute inflammation obviously, alveolar septum, pulmonary interstitial neutrophilic exudate, accompanied by edema and mild bleeding.7 days of alveolar wall edema and thickening obviously, mainly in macrophages and neutrophils infiltration of inflammatory cells, accompanied by capillary proliferation of fibroblasts increased, lung interval still has widened, collagen deposition and patches of fibrosis; 14 day group, alveolitis reached the peak, the damage of alveolar structure, a large number of collagen deposition around the new capillaries and alveolar septum and part the alveolar cavity occupied by collagen and fibrin, inflammatory cells in the interstitial lymphocytic infiltration in order to gradually transition.28 day group, pulmonary fibrosis component increased, pulmonary fibrosis is the heaviest, the alveolar structure disorder, partial collapse Collapse and disappear, the capillary lumen was thickened. The model group, the intervention group alveolitis each period were significantly better than control group, and pulmonary fibrosis in only fourteenth days and twenty-eighth days compared with the control group. Significant difference between the model group and intervention group pathological changes has the same tendency, but the degree of alveolitis, pulmonary fibrosis is lighter than the model group. Sodium hydrosulfide can reduce alveolar inflammation and pulmonary fibrosis.
2 hydroxyproline (HYP) content: the HYP content of the model group were higher than the control group and the intervention group (P0.01), model group, HYP content increased gradually with prolonging the molding time, 28 days to reach the highest value; the intervention group HYP content low level increased, the content of HYP was higher than that of control group and lower than model group (P0.01 or P0.05).
3 the expression of PD-1 protein in lung tissue and PD-1mRNA in pulmonary fibrosis: immunohistochemistry and RT-PCR results showed that the expression of PD-1 protein in the rats of model group and PD-1mRNA was higher than that of the control group and intervention group, each time point and the significant differences between the two groups (P0.01), the intervention group PD-1 and the expression of PD-1mRNA is higher than that of control group the control group, and the differences were statistically significant (P0.05).
4, apoptosis in lung tissue: the apoptotic index in lung tissue of model group was 13.7 + 3.4% in twenty-eighth days, while that in control group was (1.9 + 0.3)% and intervention group (5.34 + 1.54)%, the difference was statistically significant (F=79.90, P0.01).
Conclusion:
1, up regulation of PD-1 gene expression and apoptotic index in lung tissue in early stage of acute lung injury may play a role in the development of pulmonary fibrosis in early stage of acute lung injury.
2, sodium hydrogen sulfide inhibits oleic acid-induced acute lung injury and pulmonary fibrosis in rats by inhibiting the over expression of PD-1..PD-1 may be a new target for ALI and pulmonary fibrosis therapy.

【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R563.8

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