大鼠同种异体角膜移植排斥反应动物模型的建立及NK细胞在角膜排斥反应中作用的研究

发布时间：2018-03-28 14:33

本文选题：角膜移植　切入点：动物模型　出处：《第二军医大学》2011年硕士论文

【摘要】：角膜疾病是眼科临床工作中最常见的疾病之一,其致盲率在我国盲目流行病学调查中排名第二,仅次于白内障。且角膜损伤的瘢痕修复及角膜内皮细胞的不可再生性都严重影响角膜病的治疗和预后,因此,对于治疗角膜盲,角膜移植是临床上唯一可靠、有效的复明手段,与其他脏器移植比较.角膜移植有明显的免疫学特点即免疫特惠,尽管如此,随着现代显微手术和角膜保存技术的发展,移植术后的免疫排斥反应已成为角膜移植失败的主要原因。所以,建立合适的角膜移植动物模型,对进一步模拟人的角膜移植排斥反应,进行更加深入的基础研究具有非常重要的意义。角膜移植排斥反应的机制是多种免疫细胞和免疫分子参与的复杂的免疫反应过程,有效地防治角膜移植术后免疫排斥反应的发生是降低角膜移植排斥率的实际问题,大量研究依据辅助T淋巴细胞(helper t lymphocyte, Thl/Th2)学说探讨各类免疫细胞及细胞因子参与角膜移植排斥反应的调控机制,现今的研究和临床药物大多数针对T淋巴细胞系统,而近来实验证明剔除T淋巴细胞的动物角膜移植模型,仍然会发生排斥反应。自然杀伤细胞(Nature Killed Cell,NK细胞)是一类独立的淋巴细胞群,有报道提出NK细胞具有对异体抗原的识别,然后释放细胞因子来激活不同的效应细胞的作用,而国内关于NK细胞在角膜移植排斥反应中作用的研究尚未有相关报道。研究目的：建立大鼠同种异体角膜移植模型,观察角膜排斥反应的临床表现及进行排斥反应的病理学研究,分析手术中注意事项及操作技巧、术后并发症的原因及对策,提高大鼠角膜移植模型的成功率。初步研究NK细胞在大鼠同种异体角膜移植排斥反应中的作用。主要方法： 1.大鼠同种异体穿透性角膜移植动物模型的建立 1.1实验组：Wistar雌性大鼠40只,SD(Sprague-Dawley)雌性大鼠40只,用直径3.5mm的环钻钻取SD大鼠右眼角膜,作为同种异体角膜移植植片,用直径3.0mm的环钻钻取Wistar大鼠右眼角膜作为植床,用10-0尼龙线将角膜植片间断缝合于角膜植床上,为同种异体角膜移植组。 1.2对照组：(?)(?)istar雌性大鼠40只,用3.0mm环钻钻取右眼角膜,于12：00位做标记,旋转180°后,用10-0尼龙线原位间断缝合于植床上,为同体角膜移植对照组。 1.3术后观察：观察术后角膜移植排斥反应发生的进程,于术后不同时间对实验组及对照组大鼠角膜植片情况进行排斥反应评分。 2.大鼠同种异体角膜移植排斥反应发生的病理学研究及并发症的分析实验组及对照组分别于PO6、PO8、PO12、PO24、PO45各取4只术眼眼球,10%甲醛固定,石蜡包埋,HE染色。观察随着排斥反应的发生发展,角膜植片可出现基质细胞浸润,植片厚度,植片新生血管,植片内皮细胞数目等方面变化。观察记录和总结术中术后并发症的发生和预防方法。 3.自然杀伤细胞在大鼠同种异体角膜移植排斥反应中作用的研究分别于PO2、PO4、PO6、PO8、PO10、PO12等不同时间点,取实验组及对照组大鼠外周血,应用CD3-CD161+的NK细胞特异性抗体标记外周血内NK细胞,行流式细胞仪计数,分析NK细胞术后不同时间在淋巴细胞中所占比例的变化。实验结果 1.SD大鼠与Wistar大鼠间同种异体角膜移植组与Wistar大鼠自体角膜移植组之间角膜排斥反应指数(Rejection Value RV值)存在明显差异,实验组排斥反应发生率为100%,异体移植片存活时间为(9.65+0.77)天。 2.同种异体角膜移植组与自体角膜移植组大鼠植片病理切片中,在角膜植片基质浸润、植片厚度、新生血管等方面存在显著差异,两组共80例动物模型中术后出现虹膜前粘连6例,白内障4例,眼内感染3例,虹膜脱出3例。 3.同种异体角膜移植组术后早期大鼠外周血中NK细胞占淋巴细胞比例明显高于对照组,结果有显著差异,NK细胞计数峰值出现在术后第(4.4±0.82)天。结论 1.大鼠角膜主要组织相容性抗原的表达与人类角膜相似,且排斥反应发生率高,作为较理想的角膜移植动物模型,为模拟人角膜移植排斥反应,进行深入实验研究提供基础条件。选用远交系大鼠、偏中心植片及保留缝线等方法可增加角膜排斥反应的发生率 2.熟练的显微技术、锐利的手术器械、充分的扩瞳及术后前房的形成是减少术后并发症的关键。 3.NK细胞在大鼠同种异体角膜移植早期排斥反应起到一定作用。
[Abstract]:Corneal transplantation is one of the most common diseases in ophthalmology clinical work . Its blindness rate is second in blind epidemiological investigation in our country . It is secondary to cataract . Corneal transplantation is the only reliable and effective method for corneal transplantation .

The mechanism of corneal transplantation rejection is a complex immune response process involving many kinds of immune cells and immune molecules . The mechanism of immune rejection after corneal transplantation can be effectively controlled . The mechanism of corneal transplantation rejection is discussed based on helper T lymphocyte ( Thl / Th2 ) theory . The present research and clinical drugs are a kind of independent lymphocyte population . It is reported that NK cells have the recognition of alloantigens , then release cytokines to activate different effector cells , while the domestic study on NK cells ' s role in corneal transplantation rejection has not been reported .

Purpose of study :

To establish a rat model of allografting corneal transplantation , to observe the clinical manifestation of corneal rejection and to study the pathological study of rejection , to analyze the reasons and countermeasures of the complications after operation and to improve the success rate of the rat corneal transplantation model .

Main methods :

1 . Establishment of rat model of allograft penetrating corneal transplantation

1.1 The experimental group : 40 male Wistar female rats , 40 SD ( Sprague - Dawley ) female rats were drilled with a circular drill with a diameter of 3.5mm to obtain the right eye cornea of the SD rat , and the right eye cornea of the Wistar rat was drilled with a circular drill with a diameter of 3.0 mm as the implantation bed .

1.2 Control group : ( ? ) ( ? ) ( ? ) istar female rat 40 , right eye cornea was drilled with 3.0mm ring drill , mark was done at 12 : 00 , and after 180 掳 rotation , 10 - 0 nylon thread was used to suture in situ on the implant bed , and the control group was transplanted into the cornea of the same body .

1.3 Post - operative observation : To observe the course of corneal transplantation rejection after operation , and to evaluate the rejection rate of corneal graft in experimental group and control group at different time after operation .

2 . Pathological study and complications of allograft rejection in rats

In the experimental group and control group , PO6 , PO8 , PO12 , PO24 , PO45 were collected 4 eyes , 10 % formaldehyde fixed , paraffin embedded and HE stained .

3 . Effect of natural killer cells on allograft rejection in rats

The NK cells were labeled with CD3 - CD161 + NK cell - specific antibody in peripheral blood of the experimental group and the control group at different time points , such as PO2 , PO4 , PO6 , PO8 , PO10 and PO12 , respectively .

experimental results

1 . There was a significant difference in corneal rejection index ( RV ) between SD rats and Wistar rats . The incidence of rejection in experimental group was 100 % , and the survival time of allograft was ( 9.65 + 0.77 ) days .

2 . There was a significant difference in corneal graft matrix infiltration , graft thickness , neovascularization and so on . There were 6 cases of anterior iris adhesion , 4 cataract cases , 3 cases of intraocular infection and 3 cases of iris dislocation .

3 . The percentage of NK cells in the peripheral blood of the early rats was significantly higher than that in the control group . The results showed that the peak of NK cell count appeared on the 4th postoperative day ( 4.4 卤 0.82 ) days .

Conclusion

1 . The expression of compatible antigen in cornea of rat cornea is similar to that of human cornea , and the rate of rejection is high . As an ideal animal model for corneal transplantation , it provides basic condition for simulating human corneal transplantation rejection , and it can increase the incidence of corneal rejection .

2 . skilled micro - technology , sharp surgical instruments , adequate dilation and post - operative anterior chamber formation are key to reducing postoperative complications .

3 . NK cells play a role in the early rejection of allografting in rats .

【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R779.65

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