腺苷A_(2a)受体在博莱霉素诱导的小鼠肺纤维化中的作用  

Effects of adenosine_(2a) receptor( A_(2a)R) on the pulmonary fibrosis induced by bleomycin in mice

作　　者：陈彦凡[1] 何以成 黄卡特[2] 虞晓明 陈马云 陈相[1] 黄晓颖[1] 王良兴[1] CHEN Yah-fan;HE Yi-cheng;HUANG Ka-te;YU Xiao-ming;CHEN Ma-yun;CHEN Xiang;HUANG Xiao-ying;WANG Liang-xing(Department of Respiratory Medicine;Department of Pathology,the First Affiliated Hospital of Wenzhou Medical University,Wenzhou 325000;Department of Respiratory Medicine,Cangnan County People＇ s Hospital,Cangnan 325805,China)

机构地区：[1]温州医科大学附属第一医院呼吸与危重症医学科,浙江温州325000 [2]温州医科大学附属第一医院病理科,浙江温州325000 [3]苍南县人民医院呼吸内科,浙江苍南325805

出　　处：《中国应用生理学杂志》2018年第3期204-208,233,I0001-I0004,共10页Chinese Journal of Applied Physiology

基　　金：国家自然科学基金资助项目(81270110);浙江省卫计委资助项目(2010KYA136)

摘　　要：目的:观察腺苷A_(2a)受体(A_(2a)R)在小鼠肺纤维化形成中的调控作用。方法:30只雄性SPF级野生型BALB/C小鼠和20只A_(2a)R基因敲除BALB/C小鼠,随机分为以下5组:野生型小鼠对照组(A组)、野生型小鼠纤维化组(B组)、A_(2a)R基因敲除小鼠对照组(C组)、A_(2a)R基因敲除小鼠纤维化组(D组)、野生型小鼠纤维化+A_(2a)R激动剂(CGS21680)组(E组),每组各10只。纤维化组小鼠气管内注入博莱霉素(bleomycin,BLM)溶液50μl(5mg/kg体重),对照组在气管内注入等体积生理盐水,注射后立即将小鼠直立旋转3~5 min,使其均匀分布于两肺。A、B、C、D各组每天腹腔注射生理盐水0.5 ml,E组每天腹腔注射A_(2a)R激动剂(CGS21680)0.5 ml(0.25 mg/kg体重),连续28 d。第29天取血检测血清转化生长因子β1(TGF-β1)含量;检测肺组织中羟脯氨酸(hydroxyproline,Hyp)、TGF-β1和A_(2a)R蛋白质含量,TGF-β1 mRNA、A_(2a)R mRNA的表达,并观察肺组织光镜和超微结构。结果:(1)光镜和超微结构提示,B组肺泡壁增厚破坏,肺泡腔狭窄或部分陷闭,纤维增生,炎细胞浸润,I型、II型肺泡上皮细胞明显空泡化,D组较B组明显,E组表现较B组减轻。Masson染色提示B组、D组小鼠肺组织纤维增生明显,E组明显减少,提示基因缺失加重了小鼠肺纤维化的形成。(2)与A组比较,B组血清TGF-β1含量,肺组织Hyp含量,TGF-β1、A_(2a)R蛋白质含量和TGF-β1 mRNA、A_(2a)R mRNA的表达均明显增高(P<0.01,P<0.05)。与C组比较,D组血清TGF-β1含量、肺组织Hyp、TGF-β1含量和TGF-β1 mRNA表达明显增高(P<0.01,P<0.05),提示肺纤维化小鼠肺组织Hyp含量增高,TGF-β1表达上调。(3)与B组相比,D组血清TGF-β1、肺组织Hyp、TGF-β1含量和TGF-β1 mRNA表达明显增高(P<0.01)。与B组比较,E组A_(2a)R蛋白质含量和A_(2a)R mRNA的表达均增高(P<0.01),而血清TGF-β1、肺组织Hyp和TGF-β1蛋白含量及TGF-β1 mRNA表达较B组明显下降(P<0.01,P<0.05),提示A_(2a)R基因敲除小鼠肺Objective： To observe the effects of A2aR on pulmonary fibrosis induced by bleomycin（ BLM） in wild type and A2aR gene knockout mice. Methods： Thirty male BALB/c wild type（ WT） mice and twenty male A2aR gene knockout（ A2aR KO） mice,were randomly divided into 5 groups（ n = 10 each） ： WT control group（ A）,WT fibrosis group（ B）,A2aR KO control group（ C）,A2aR KO fibrosis group（ D）,WT fibrosis ＋ A2aR agonist（ CGS21680） group（ E）. For the induction of pulmonary fibrosis,mice were intratracheally injected with a single dose of bleomycin（ 5. 0 mg/kg body weight）,while the control groups with an equal volume of NS. After injection,the mice were vertically rotated immediately for 3 or 5 minutes in order to make the liquor evenly distributed in lung. The mice in groups A to D received intraperitoneal injection of 0. 5 ml NS,those in E group received 0. 5 ml CGS-21680（ 0. 25 mg/kg body weight） daily for a period of 4 weeks. On the 29 th day,the blood and tissue samples were taken. The chloramine T method was used to detect the content of hydroxyproline（ Hyp） in lung. ELISA was used to detect transforming growth factor-β1（ TGF-β1）in sera. The immunohistochemical technique and Western blot were used to detect the protein expression of TGF-β1 and A2aR. And the mRNA expression of TGF-β1 and A2aR were measured by in situ hybridization. The microstructure,ultrastructure and fiber staining of lung tissue in mice were observed in each group. Results：（1）Thickened and destroyed alveolar walls,disordered,stenosed or partial collapsed alveolar spaces,fibrous proliferation and infiltration of inflammation cells were observed in lung of mice in group B under microscope. The described performance was even more serious in group D,and that the intervention in group E,could significantly ameliorate the pathological changes. The vacuolation in epithelial cells of type I and II as well as the lamellar body,were observed on thevisualizing of ultrastructure of lung tissue in

关 键 词：肺纤维化 博莱霉素 TGF-Β1 A2aR 小鼠 

分 类 号：R332.2[医药卫生—人体生理学]