医学论文发表：分泌型糖基化磷蛋白
医学论文发表：
摘要：
Changes of expressions of osteopontin and integrin β3 mRNA in right ventricle of pulmonary hypertensive rats
[Abstract] Objective:To investigate the changes of expressions of osteopontin (OPN)and integrin messenger RNA in right heart remodeling associated with pulmonary
hypertensive rats ．Methods: 60 SD were randomly divided into 5 groups. 12 in each group. A control group and 4 experimental pulmonary hypertension (PAH). Samples have
been produced at 4 time points (MCT injection after the first week 1,2,3,4). MCT will be injected 1,2,3,4 weeks after experiment in 4 experimental pulmonary
hypertension (PAH), which were referred to as PAH1, PAH2, PAH3 and PAH4 experimental group. Determination of mean pulmonary arterial pressure (mPAP) and right
ventricular hypertrophy index. The right ventricle myocardial osteopontin (OPN) and integrin β3 receptor mRNA expression levels were detected by immunohistochemistry
,western blot and RT-PCR method at different time points in rats in each group. Results: ① In control group, OPN of and integrin β3receptor mRNA expression level in
the right ventricle myocardial of rats is very low, and pulmonary hypertension in each group (PHA1-4), OPN and integrin β3 expression in the right ventricle
myocardial of rats were significantly increased. Compared with the control group,there was a significant difference in each group (PHA1-4) (all,P<0.O1). ②OPN and
integrinβ3 receptor protein expression levels increased in left and right ventricular myocardial of pulmonary hypertension group, their expression levels have
increased along with the extension of the time with pulmonary hypertension. Compared with the control group, differences were significant in pulmonary hypertension
group (P<0.01).Correlation analysis showed that between the right ventricle myocardial 0PN mRNA expression and right ventricular hypertrophy index was positively
correlated (r = 0.828, P<0.01). Between the right ventricle myocardial integrin β3 mRNA expression and right ventricular hypertrophy index was positively correlated
(r = 0.822, P<0.01). Conclusion: In the process of pulmonary hypertension and right ventricular hypertrophy formation in rats, OPN and integrin β3 receptor gene
expression increased in the right ventricle myocardial, their expression probably plays an important role in the right ventricle myocardial remodeling.
Key words:osteopontin； integrin β3； pulmonary hypertension；western blot; reverse transcription polymerse chain reaction
骨桥蛋白(osteopontin，OPN)是一种分泌型糖基化磷蛋白，因其分子结构上含有RGD(Arg-Gly-Asp)三肽序列，具有细胞黏附功能，既是一种细胞外基质又是一种细胞信号转导因子。整合素
(integrin)是一类重要的黏附分子，由α和β亚基组成，介导细胞和细胞外基质(ECM)及细胞之间的黏附和信号转导。其中αvβ3整合素通过识别并结合OPN，经跨膜信号转导至胞内，与细胞
的迁移、增殖和分化有关，在心血管病理、生理机制中发挥重要的作用[1] 。正常状态下，心脏心肌细胞低表达0PN，但在多种病理状态下心肌高表达的OPN在心肌肥大和心室重塑中起着重要
的作用[2]。本研究在建立肺动脉高压大鼠模型上，观察OPN及其整合素β3受体基因在右心室心肌中表达变化。
1 材料和方法
1.1 试验动物 二级雄性Sprague-Dawley(SD)大鼠，昆明医学院实验动物中心提供，体重250～300 g。
1.2 主要试剂 Trizol～reagent RNA提取试剂盒、随机引物和M-MLV和RNase inhibitor(Invitrogen，USA)，Taq DNA polymerase(Promega公司生产)。
1．3 肺动脉高压和右心室重塑动物模型的制备 健康雄性SD大鼠60只,体质量250～300 g,随机等分为5组，每组12只:1个对照组和4个肺动脉高压实验组(PAH组),分别于4个时间点（MCT注射后
第1,2,3,4周）取材。将MCT注射后第1,2,3,4周的4个肺动脉高压实验组(PAH组)分别简称PAH1、PAH2、PAH3和PAH4实验组。大鼠饲养适应1周后按60 mg/kg皮下注射野百合碱(MCT,美国Sigma
Chemical Co.)。MCT注射后第1,2,3,4周测量血流动力学指标后留取标本,每个时间点取10只大鼠［3］。对照组行饲养适应1周后皮下注射生理盐水适应期及注射后4周重复测压。放血处死动物
，迅速取出心肺，沿室间隔剪下右心室，分别称取右心室(RV)，左心室加室间隔(LV+S)的重量，计算右心室肥厚指数[Rv／(LV+S)]。心肺组织置于液氦内冷冻待测。
1.4 骨桥蛋白和整合素β3 mRNA表达的测定 根据Trizol一步法从冻存右心室中提取总RNA。采用20μl反应体系，逆转录合成cDNA。根据Genebank资料，以osteopontin mRNA、integrinβ3
mRNA和β-actin mRNA为模板，经primer premier5.0计算机软件设计引物(引物委托北京赛百盛生物有限公司合成，引物序列见表1)。优化RT-PCR条件，分别进行OPN、integrin β3与β-
actin共扩增。扩增产物经1.5％琼脂糖凝胶电泳后用凝胶成像仪观察，用Image Master Total lab v1.Ol软件半定量分析条带峰面积，分别计算OPN、integrinβ3与β-actin光密度的比值。
表l 大鼠OPN，integrin β3及β-actin引物设计
Gene Primer Annealing
Temperature(℃) PCR products
(bp)
OPN 5’-CTGTGTCCTAAGAAACGG-3’
5’-ATCTGTGGCATCGGGATACTG-3’ 61.0 335
Integrin β3 5’-GTTTACCACTGATGCCAAGA-3’
5’-CATCAGACAGGACTACCACT-3’ 58.0 266
β-actin 5’-GCCCATCTATGAGGGTTAC-3’
5’-CTGGAAGGTGGACAGTGAG-3’ 60.0 571
来源：医学论文发表
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