TLR2/MyD88信号系统在大鼠角膜移植术后排斥反应中的作用

Role of TLR2/MyD88 signaling system in immune rejection after corneal transplantation in rat

目的探讨Toll样受体2(Toll like receptor,TLR2)/MyD 88信号系统在大鼠角膜移植术后排斥反应中的作用。方法取14只SD大鼠为供体,28只Wistar大鼠为受体,建立28个同种异体穿透性角膜移植模型。建模后分为同种异体角膜移植组14只(6只用于术后观察排斥情况),同种异体角膜移植TLR2单克隆抗体组14只(6只用于术后观察排斥情况)。另选16只Wistar大鼠,分为同种自体角膜移植组8只,正常对照组8只。3个手术组大鼠分别行穿透性角膜移植术。术后TLR2单克隆抗体组给予0.5 g·L-1TLR2单克隆抗体,分别于术后0 d、2 d、4 d、6 d、8 d分5次经球结膜下注射给药。正常对照组、同种自体角膜移植组及同种异体角膜移植组给等量生理盐水。术后每天于裂隙灯下观察角膜透明度、新生血管,并用排斥反应指数评分。第9天取材,行HE、免疫组织化学染色和实时荧光定量PCR检测。结果术后随时间变化各手术组角膜植片均出现不同程度的水肿、混浊和新生血管生长,以同种异体角膜移植组最为明显。HE染色结果显示在同种异体角膜移植组,角膜基质层出现水肿、大量炎症细胞浸润和新生血管,而在同种自体角膜移植组和TLR2单克隆抗体组中炎症反应较轻。免疫组织化学检测结果:TLR2和MyD 88分子在正常对照组、同种自体角膜移植组及TLR2单克隆抗体组的角膜上皮中均有微量表达,同种异体角膜移植组角膜上皮、基质细胞中TLR2和MyD 88分子的表达明显增多,尤以基质层明显。实时荧光定量PCR结果显示同种异体角膜移植组角膜组织TLR2 mR NA、MyD 88 mR NA的表达较同种自体角膜移植组(P=0.000、0.004)和正常对照组(P=0.000、0.000)显著增加,两者的表达亦较TLR2单克隆抗体组显著增加(P=0.000、0.003)。结论 TLR2单克隆抗体抑制TLR2及其下游信号分子MyD 88的表达;TLR2/MyD 88信号系统参与了角膜移植术后排斥反应,影响了角膜植片的转归。

Objeetive To explore the role of Toll like receptor （TLR2）/MyD88 signaling system in immtme rejection after penetrating keratoplasty （PKP） in rats. Methods Fourteen SD rats were chosen as the donators, and 28 Wistar rats as recep- tor,28 allograft corneal transplantation models were established. The models were divid- ed into 14 allograft corneal transplantation group （6 cases were used to observe the postoperative rejection） and 14 allograft corneal transplantation treated with TLR2 mon- oclonal antibody group （6 cases were used to observe the postoperative rejection）. An- other 16 Wistar rats were chosen and divided into isograft corneal transplantation group （8 cases） and normal control group （ 8 cases）. PKP was performed in three corneal transplantation groups. The isograft and allograft groups were treated with saline, while TLR2 monoclonal antibody group with equal numbers of 0.5 g ~ L-1 TLR2 monoclonal antibody,they were injected at 0 day,2 days,4 days,6 days and 8 days from the bulbar conjunetiva,respectively. The corneal opacity and neovascularization were observed by slit-lamp microscope and scored according to the rejection index, with normal cornea serving as the control. The corneal tissues were sampled at 9 days after the transplanta- tion for HE and immunohistochemistry staining. The expression of TLR2 and MyD88 detected by qPCR. Results With the passage of time, edema, opacities and neovascularization of the corneal graft occurred after the operation in three corneal transplantation groups, especially in aliograft group. HE staining showed that severe cor- neal edema,a lots of inflammatory cells infiltration and new vessels in stroma were seen in allograft group, while mild inflammatory response was found in isograft group and TLR2 monoclonal antibody group. Immunohistochemistry staining result display that TLR2 and MyD88 were weakly detected in normal group ,isograft group and TLR2 mono- clonal antibody group, they were increased＊ in epithelium and stroma in allograft group, particularly in stroma. The expression of TLR2 mRNA and MyD88 mRNA in aUograft ~roup were significantly higher than those in isograft group （P = 0.000,0. 004 ） and nor- mal control group（P =0. 000,0. 000 ） ,in addition,the expression in TLR2 monoclonal antibody ： roup were significantly decreased in comparison with those in allograft group （P = 0. 000,0. 003 ）. Coneluslon TLR2 monoclonal antibody inhibits the expression of TLR2 and its downstream signal MyD88 ;TLR2/MyD88 signaling system is involved in corneal transplantation immune re- jection, and effect the outcome of corneal graft.