多器官功能障碍综合征大鼠肾组织细胞外基质损伤

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中华急诊医学杂志2012年8月第21卷第8期Chin J Emerg Med,August 2012,Vol.21,No.8

P819-824

【摘要】目的 检测Ⅳ型胶原及基质金属蛋白酶-9在多器官功能障碍综合征（MODS）大鼠肾组织及血清中变化，探讨细胞外基质的破坏与MODS肾功能衰竭的关系。方法 实验在三峡大学医学院实验中心完成。将40只成年雄性SD大鼠（随机数字表法）分为健康对照组（8只）、MODS模型组（32只），模型组又分为造模后6、12、24、48 h不同时点，每组8只。 采用“二次打击”即眼球失血加腹腔注射脂多糖（LPS）制备MODS模型：大鼠左眼球取血2 ml/100g,在放血4 h后予无菌腹腔注射LPS 5 mg/kg。对照组予以腹腔注射等量的生理盐水，造模完成后在不同的时相点搜集标本。血生化检测各组血清肌酐（Cr）及尿素氮（BUN）的变化；光镜及电镜观察肾组织的形态及超微结构变化；酶联免疫吸附试验（ELISA）法检测血清中Ⅳ型胶原及MMP-9的浓度变化；western印迹技术测定肾组织中Ⅳ型胶原及MMP-9的蛋白水平。组间比较采用单因素方差分析（SNK法）。结果 （1）与对照组相比，MODS组各个时点肾功能损害较为明显，Cr及BUN均明显增高(P＜0.05)。（2）光镜下对照组肾组织结构无改变；MODS组肾组织损伤较重。电镜下对照组肾组织基底膜胶原纤维连续完整，MODS组肾组织基底膜水肿、胶原纤维分布缺损明显。（3）与对照组比较，造模后6～48 h，MODS组大鼠血清中MMP-9水平均明显增高，于造模后12 h达到高峰(P＜0.05)。造模后血清Ⅳ型胶原的质量浓度各组均高于对照组，6～12 h组升高不明显（P＞0.05），24～48 h显著高于对照组(P＜0.01)。(4) 造模后肾组织MMP-9的浓度6～12 h比对照组明显增加（P＜0.01），24 h达到高峰（P＜0.01），随后48 h下降。造模后各组肾组织Ⅳ型胶原的质量浓度均较对照组明显降低 (P＜0.05)。结论 细胞外基质的破坏是引起MODS肾损伤的一个重要因素，可能成为MODS 早期肾功能损害的诊疗和干预靶点。

【关键词】多器官功能障碍综合征；基质金属蛋白酶-9 ；内皮细胞；细胞外基质；Ⅳ胶原；脂多糖；全身炎症反应综合征；二次打击；大鼠

Observation of extracellular matrix damage in the kidney of rats with multiple organ dysfunction syndrome YUAN Yu-rong， TENG Lin，YU Min， LIU Xian-zhe. The People's Hospital of Three Gorges University, The first People's Hospital of Yichang , Yichang 443000, China

Corresponding author: LIU Xian-zhe, Email:

【Abstract】Objective To determine the levels of type Ⅳ collagen and matrix metalloproteinase-9（MMP-9）in the serum and kidney of rats with the multiple organ dysfunction syndrome （MODS）and investigate the mechanism of extracellular matrix damage in renal failure of MODS. Methods Fortymale Sprague-Dawley（SD）rats were randomly (ramdom number) divided into two groups, namely the normal control group (n=8) and the MODS model group (n=32). The rats of model group were further divided into four sub-groups as per different intervals, 6 h, 12 h, 24 h and 48 h, after modeling (n=8 in each). The animal models of MODS were established by two hits, the left eyeball of each model rat was removed to bleed to 2 ml blood/100 g body weight and lipopolysaccharide (LPS, 5 mg/kg) was injected into intraperitoneal cavity of model rats four hours later. The same volume of saline instead was injected intraperitoneally into rats of control group. All rats were sacrificed at different intervals. Creatinine （Cr）and blood urea nitrogen (BUN) were determined by using a Hitachi Automatic Biochemical Analyzer. The histological changes in renal tissue were observed under light microscope and transmission electronic microscope. The levels of serum type Ⅳ collagen and MMP-9 protein were measured by using enzyme linked immunosorbent assay (ELISA). Type Ⅳ collagen and MMP-9 protein levels in renal tissue were detected by western blot. One-way ANOVA was used for comparison among multiple groups. Results Compared with the control group, Cr and BUN were significantly higher in MODS group (P