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张召才，严静，蔡国龙，虞意华，胡才宝

浙江医院ICU,杭州市310013，中国

Expression of integrin genes in heart of septic rat

ZHANG Zhao-cai*, YAN Jing, CAI Guo-long, YU Yi-hua, HU Cai-bao.

*Intensive Care Unit (ICU), <?xml:namespace prefix = st1 ns = "urn:schemas-microsoft-com:office:smarttags" />Zhejiang Hospital, Hangzhou310013, China.

Corresponding author: Zhang Zhao-cai, email:zhaocai@gmail.com

Objective: To investigate the expression profile of integrin genes in heart of septic rat and relevant mechanisms responsible for sepsis-induced heart injury.Methods: Twelve 3-month-old male Wistar rats were randomized to 2 equal groups, they were treated with caecal-ligation and perforation (CLP: n=6) to establish sepsis model or sham (Sham: n=6) procedure to serve as controls. The hearts of rats were rapidly excised 24 h later. After determination of hemodynamic parameters by using Langendorff apparatus, the isolated hearts were examined histopathologically. Thereafter, expression of intergrin genes in hearts of both groups of rats were analysed by oligonucleotide microarrays. Results: In comparison with sham controls, no overt pathological changes were detected in hearts of septic rats, but decreased cardiac output (CO)(57.8±2.4 vs 29.4±3.3 ml.min-1), Stroke volume (SV) (0.18±0.01 vs 0.12±0.01 ml), heart rate (HR) (302±12 vs 256±6 bpm), Left ventricular developed pressure (LVDP) ( 90.0±2.7 vs 75.6±4.9 mmHg), LV end-diastolic pressure (LVEDP) (8.0±0.3 vs 7.5±0.3 mmHg) and Maximum rate of LV pressure rise (dP/dtmax) (2601±34 vs 2167±159 mmHg. s-1) were determined in CLP rats (P<0.01 for all), indicating that the myocardium in septic rat was depressed. Microarray analysis showed that 20 out of 24 integrin genes were up-regulated in septic rat heart by over 2 folds, among which the integrin αV and β2 gene were upregulated and integrin β1 gene was of relatively insufficient expression. Conclusion: Maladjustment in expression of integrin gene was present in septic rat heart, up-regulation of integrin αV and β2 gene might accelerate the heart injury mediated by inflammatory mediators, the relatively insufficient expression of integrin β1 gene may contribute to cardiac dysfunction in septic rat.

Key words: Integrin，Sepsis，Oligonucleotide microarray，Heart injury

脓毒症是全身性炎症反应综合征，是ICU患者主要死亡原因。脓毒症患者中大约有40％～50％会合并心脏功能不全，其中出现严重心衰者约7％，这种现象已经发现多年，但其病理生理基础仍然不清楚^[1，2]。近年来，整合素与心脏疾病的关系受到关注，整合素参与了正常心脏发育和某些心脏疾病如心脏肥厚、扩张性心肌病和心肌梗死等的发生过程^[3]，整合素β1的表达减少或受到抑制可以诱发心功能不全甚至心衰^[4]。脓毒症时，整合素β1和β2在中性粒细胞中表达增多，参与了脓毒症导致的肺损伤^[5]，它们是否参与脓毒症相关的心脏损伤尚不清楚。本研究应用寡核苷酸基因芯片检测整合素家族成员在脓毒症大鼠心肌组织中的基因表达情况，了解整合素与脓毒症导致的心功能不全之间的关系，探讨脓毒症导致心脏损伤的相关机制。

材料与方法

1.脓毒症动物模型的建立和鉴定

雄性、12周龄、纯种近系Wistar大鼠12只,体重300g-370g，购自中科院上海实验动物中心。在标准条件适应3天后，以苯巴比妥腹腔注射麻醉(30 mg/kg)、无菌条件下经股静脉置入PE50导管（Intermedic, Sparks, MD, USA)供输液和采集血样之用，然后将大鼠随机均分为脓毒症模型组和假手术对照组（每组6只）。模型组腹腔正中切开、钝性分离腹肌、打开腹膜，结扎盲肠远端、保持肠道畅通，以18号针头针刺盲肠3次后关腹，术后以生理盐水（3mL/h）经股静脉导管输注24小时以维持血压和血流动力学稳定；假手术对照组仅开腹手术而不结扎盲肠，术后处理同模型组。

24小时后，再次以苯巴比妥麻醉大鼠，静脉注射肝素(300 IU/kg)后，其后快速摘除心脏并固定于Langendorff 灌流装置；然后从肺静脉置入左心导管，参照文献^[6]方法分别测定两组大鼠的血流动力学指标：左室舒张末压（LVEDP）、左室发展压 (LVDP)，左室压最大上升速率 (dP/dt max), 心脏输出量(CO)和每搏输出量、心率(HR)。

2.心脏组织留取与处理

完成以上各项检测后，将两组大鼠心脏纵向剪成2半，一半以福尔马林固定，用于组织病理学检测；余下部分液氮冻存，用于基因芯片检测。

3.基因芯片检测

大鼠粘连分子和细胞外基质基因的寡核苷酸芯片（Oligo GEArray®，ORN013）购自美国SuperArray公司，用于同步检测每个心脏标本中的整合素基因表达变化，所有操作严格参照厂商提供的实验方法进行。主要步骤如下：

3.1 总RNA提取：Trizol试剂盒（Invitrogen）抽提各冻存的心脏组织样品总RNA，紫外吸收测定法和变性琼脂糖凝胶电泳法确认RNA纯度及完整性；

3.2合成生物素标记的cRNA探针：采用TrueLabeling-AMP?线性RNA扩增试剂盒（SuperArray），首先将样品RNA逆转录合成cDNA，然后与扩增混合液（由RNA 扩增缓冲液、生物素标记的UTP和扩增酶类混合液组成）混和，线性扩增生成生物素标记的cRNA探针；

3.3 cRNA 纯化和标记效率测定：使用ArrayGrade cRNA纯化试剂盒（SuperArray）将cRNA 纯化，紫外吸收测定法测定cRNA其浓度并评估cRNA纯度；以化学发光检测法进行封闭和检测，确认cRNA标记效率。

3.4 芯片预杂交和杂交：在杂交管中，将Oligo GEArray®芯片与GEAhyb杂交液预杂交，然后与预先制备的生物素标记的cRNA探针混合，完成芯片杂交。

3.5 膜封闭和化学发光检测：应用SuperArray化学发光检测试剂盒进行，探针与芯片杂交后，封闭杂交膜，再加链亲和素偶联的碱性磷酸酶（AP），其后进行化学发光底物反应，X射线胶片曝光在胶片上显示检测结果。

3.6图像采集和处理：将胶片上的图像用扫描仪扫描并转换为灰度TIFF格式的图片文件保存，运行ScanAlyze软件，将灰度TIFF格式图片的点阵转化为数字型数据，将此原始数据储存为Microsoft Excel文件。 

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表1.两组大鼠血流动力学参数比较（Х±s）

表2. 模型组表达上调的整合素基因列表