本文選題：急性肺損傷　切入點：肺纖維化　出處：《南華大學》2012年碩士論文　論文類型：學位論文

【摘要】：目的通過檢測油酸致大鼠急性肺損傷早期肺纖維化不同時間點肺組織程序性死亡受體-1(Programmed death-1，PD-1)、PD-1基因、細胞凋亡及硫氫化鈉干預的大鼠肺組織PD-1的變化，探討硫氫化鈉對油酸致大鼠急性肺損傷早期肺纖維化的保護作用。 方法應用尾靜脈注射OA復制大鼠ALI模型。清潔級健康、Sprague Dawley（SD）大鼠54只，適應性飼養(yǎng)一周后，隨機分為對照組；OA致傷模型組；干預組。模型組按3天、7天、14天、28天4個時間點分為4個亞組；干預組按3天、7天、14天、28天4個時間點分為4個亞組。每個亞組及對照組均為6只大鼠。模型組和干預組尾靜脈注射OA (0.12mg/kg)誘導急性肺損傷，對照組在相同條件下給予生理鹽水。第二天起干預組大鼠每天經(jīng)尾靜脈注射硫氫化鈉(3.12mg/kg)，其余兩組相同條件下給予助溶劑羧甲基纖維素鈉。相應時間點處死動物取出肺組織，進行病理學觀察；測定肺組織中羥脯氨酸含量；用免疫組化和RT-PCR觀察各組鼠肺組織PD-1蛋白及mRNA表達的水平；TUNEL法檢測28天各組大鼠肺組織細胞凋亡。 結果 1、肺組織HE染色、Masson染色：對照組大鼠觀察未見明顯病變。模型組大鼠第3天表現(xiàn)為明顯的急性炎癥期，肺泡間隔增寬、肺間質內有中性粒細胞滲出，伴有水腫和輕度出血。7天肺泡壁水腫、增厚比較明顯，主要以巨噬細胞和中性粒細胞為主的炎性細胞浸潤，伴有毛細血管增生，成纖維細胞增多，肺間隔仍增寬，有膠原沉積及斑片狀的纖維化改變；14天組，肺泡炎達高峰，，肺泡結構破壞，大量膠原沉積在新生的毛細血管周圍，肺泡間隔及部分肺泡腔被膠原和纖維蛋白占據(jù)，間質內炎性細胞逐漸向以淋巴細胞浸潤為主過渡。28天組，肺間質纖維化成分明顯增多，肺纖維化最重，肺泡結構紊亂，部分坍陷和消失，毛細管腔明顯增厚。模型組、干預組肺泡炎各期均明顯比對照組重，而肺纖維化僅第14天和第28天與對照組比較差異顯著。干預組與模型組病理變化有同一規(guī)律，但肺泡炎、肺纖維化程度都輕于模型組。說明硫氫化鈉可以減輕肺泡炎和肺纖維化程度。 2羥脯氨酸（HYP）含量：模型組HYP含量均高于對照組及干預組（P0.01）,模型組HYP含量隨造模時間的延長逐漸增高，28天達到最高值；干預組HYP含量成低水平增高趨勢，HYP含量均高于對照組而低于模型組（P0.01或P0.05）。 3肺組織PD-1蛋白和PD-1mRNA在肺纖維化過程中的表達：免疫組化以及RT-PCR結果顯示模型組大鼠PD-1蛋白和PD-1mRNA表達量高于對照組及干預組，各時間點與兩組比較差異均有統(tǒng)計學意義（P0.01），干預組PD-1和PD-1mRNA表達量高于對照組，與對照組比較差異均有統(tǒng)計學意義（P0.05）。 4肺組織細胞凋亡變化：模型組第28天大鼠肺組織中細胞凋亡指數(shù)為(13.7±3.4)%，對照組為(1.9±0.3)%和干預組(5.34±1.54)%，差異有統(tǒng)計學意義(F=79.90，P0.01)。 結論： 1、急性肺損傷早期肺纖維化肺組織PD-1基因表達及細胞凋亡指數(shù)的上調，可能在急性肺損傷早期肺纖維化發(fā)展中發(fā)揮了作用。 2、硫氫化鈉通過抑制PD-1的過度表達從而減輕油酸誘發(fā)的大鼠急性肺損傷和肺纖維化。PD-1可能是ALI和肺纖維化治療的一個新的靶分子。
[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.

【學位授予單位】：南華大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R563.8

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