内毒素诱导葡萄膜炎中炎症细胞凋亡与TRAIL表达的研究

目的:在内毒素诱导的葡萄膜炎(endotoxin induced uveitis,EIU)模型中,观察葡萄膜炎病变中炎症细胞凋亡的发生,研究大鼠虹膜组织中炎症细胞肿瘤坏死因子相关凋亡诱导配体(TRAIL)的表达与炎症细胞凋亡的关系,探讨TRAIL凋亡途径可能参与炎症细胞的凋亡。方法:以1mg/kg内毒素注射于大鼠后足垫建立EIU模型,分别于注射后不同时间点观察大鼠眼内的炎症反应。吸取房水观察细胞数。摘取眼球,行HE(hematoxylin eosin)染色观察大鼠虹膜组织的病理改变;TUNEL(terminal deoxynucleotidyl transferase mediated dUTP nick-end labeling)法检测炎症细胞凋亡情况;同时运用SABC法检测在内毒素诱导后的不同时间TRAIL在炎症细胞上的表达,并行图像分析。结果:内毒素注射6h开始出现炎症,以后炎症加重,于18～24h达到炎症高峰,48h炎症明显消退。房水中的细胞数也在24h组达到最多。HE染色显示:内毒素注射后6h,即出现炎症细胞,细胞数目在24h组最多,多数为多形核中性粒细胞,少数为单核细胞和淋巴细胞,48h组炎症细胞几乎消失。TUNEL染色显示:在内毒素注射后6h组即有炎症细胞出现阳性着色,24h组凋亡数达到最多。免疫组化显示:TRAIL蛋白在大鼠的虹膜色素上皮层呈弱阳性着色;TRAIL在炎症细胞上呈阳性着色,24h组在炎症细胞中的着色数量及着色强度达到最高。结论:以1mg/kg的内毒素注射于SD大鼠后足垫可诱导出葡萄膜炎反应,炎症反应在24h组最为强烈。炎症细胞凋亡可能是EIU炎症迅速消退的原因之一。TRAIL可能参与了炎症细胞的凋亡。

Expression of TNF-related apoptosis-inducing ligand system and infiltrated cells apoptosis in endotoxin-induced uveitis

AIM:To investigate the expression and function of TNF- related apoptosis-inducing ligand(TRAIL)apoptosis system on infiltrated cells in Sprague-Dawley(SD)rats with endotoxin-induced uveitis(EIU), and to study the role of TRAIL on apoptosis.

METHODS: EIU model was established by injecting a dose of 1mg/kg endotoxin(LPS)into the footpads of SD rats. At different time points(6, 12, 18, 24, 48 hours)after endotoxin injection, clinical symptoms were observed with ophthalmoscope. The cells in the aqueous humor one eye in each rat were counted. Eyes were enucleated for histological examination at different time points. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay(TUNEL)method was used to assess the infiltrated cells apoptosis. The expression of TRAIL was determined by sreptavidin-biotin complex(SABC)immune histochemistry. The positive stain section was analyzed by computer-based image analysis system.

RESULTS: Intraocular inflammation appeared 6 hours after LPS injection, peaked at 18-24 hours, and obviously weakened at 48 hours. The cells of aqueous humor were gradually increased peaked at 24 hours. TUNEL stain indicated: a number of apoptotic infiltrated cells could be seen at the time of 6 hours to 24 hours after endotoxin injection. TRAIL was constitutively expressed on the rat iris weakly. TRAIL was expressed on the infiltrated cells. The intensity was weak in the 6 hours, 12 hours, at a peak in the 18 to 24 hours.

CONCLUSION: Uveitis was induced in the rat eyes with injecting 1mg/kg endotoxin. The short duration of EIU may be associated with apoptosis of infiltrated cells in inflammation. The TRAIL system may be associated with the infiltrated cells apoptosis in EIU.