本文選題：角膜移植　切入點：動物模型　出處：《第二軍醫(yī)大學(xué)》2011年碩士論文

【摘要】：角膜疾病是眼科臨床工作中最常見的疾病之一,其致盲率在我國盲目流行病學(xué)調(diào)查中排名第二,僅次于白內(nèi)障。且角膜損傷的瘢痕修復(fù)及角膜內(nèi)皮細(xì)胞的不可再生性都嚴(yán)重影響角膜病的治療和預(yù)后,因此,對于治療角膜盲,角膜移植是臨床上唯一可靠、有效的復(fù)明手段,與其他臟器移植比較.角膜移植有明顯的免疫學(xué)特點即免疫特惠,盡管如此,隨著現(xiàn)代顯微手術(shù)和角膜保存技術(shù)的發(fā)展,移植術(shù)后的免疫排斥反應(yīng)已成為角膜移植失敗的主要原因。所以,建立合適的角膜移植動物模型,對進(jìn)一步模擬人的角膜移植排斥反應(yīng),進(jìn)行更加深入的基礎(chǔ)研究具有非常重要的意義。 角膜移植排斥反應(yīng)的機(jī)制是多種免疫細(xì)胞和免疫分子參與的復(fù)雜的免疫反應(yīng)過程,有效地防治角膜移植術(shù)后免疫排斥反應(yīng)的發(fā)生是降低角膜移植排斥率的實際問題,大量研究依據(jù)輔助T淋巴細(xì)胞(helper t lymphocyte, Thl/Th2)學(xué)說探討各類免疫細(xì)胞及細(xì)胞因子參與角膜移植排斥反應(yīng)的調(diào)控機(jī)制,現(xiàn)今的研究和臨床藥物大多數(shù)針對T淋巴細(xì)胞系統(tǒng),而近來實驗證明剔除T淋巴細(xì)胞的動物角膜移植模型,仍然會發(fā)生排斥反應(yīng)。自然殺傷細(xì)胞(Nature Killed Cell,NK細(xì)胞)是一類獨立的淋巴細(xì)胞群,有報道提出NK細(xì)胞具有對異體抗原的識別,然后釋放細(xì)胞因子來激活不同的效應(yīng)細(xì)胞的作用,而國內(nèi)關(guān)于NK細(xì)胞在角膜移植排斥反應(yīng)中作用的研究尚未有相關(guān)報道。 研究目的： 建立大鼠同種異體角膜移植模型,觀察角膜排斥反應(yīng)的臨床表現(xiàn)及進(jìn)行排斥反應(yīng)的病理學(xué)研究,分析手術(shù)中注意事項及操作技巧、術(shù)后并發(fā)癥的原因及對策,提高大鼠角膜移植模型的成功率。初步研究NK細(xì)胞在大鼠同種異體角膜移植排斥反應(yīng)中的作用。 主要方法： 1.大鼠同種異體穿透性角膜移植動物模型的建立 1.1實驗組：Wistar雌性大鼠40只,SD(Sprague-Dawley)雌性大鼠40只,用直徑3.5mm的環(huán)鉆鉆取SD大鼠右眼角膜,作為同種異體角膜移植植片,用直徑3.0mm的環(huán)鉆鉆取Wistar大鼠右眼角膜作為植床,用10-0尼龍線將角膜植片間斷縫合于角膜植床上,為同種異體角膜移植組。 1.2對照組：(?)(?)istar雌性大鼠40只,用3.0mm環(huán)鉆鉆取右眼角膜,于12：00位做標(biāo)記,旋轉(zhuǎn)180°后,用10-0尼龍線原位間斷縫合于植床上,為同體角膜移植對照組。 1.3術(shù)后觀察：觀察術(shù)后角膜移植排斥反應(yīng)發(fā)生的進(jìn)程,于術(shù)后不同時間對實驗組及對照組大鼠角膜植片情況進(jìn)行排斥反應(yīng)評分。 2.大鼠同種異體角膜移植排斥反應(yīng)發(fā)生的病理學(xué)研究及并發(fā)癥的分析 實驗組及對照組分別于PO6、PO8、PO12、PO24、PO45各取4只術(shù)眼眼球,10%甲醛固定,石蠟包埋,HE染色。觀察隨著排斥反應(yīng)的發(fā)生發(fā)展,角膜植片可出現(xiàn)基質(zhì)細(xì)胞浸潤,植片厚度,植片新生血管,植片內(nèi)皮細(xì)胞數(shù)目等方面變化。觀察記錄和總結(jié)術(shù)中術(shù)后并發(fā)癥的發(fā)生和預(yù)防方法。 3.自然殺傷細(xì)胞在大鼠同種異體角膜移植排斥反應(yīng)中作用的研究 分別于PO2、PO4、PO6、PO8、PO10、PO12等不同時間點,取實驗組及對照組大鼠外周血,應(yīng)用CD3-CD161+的NK細(xì)胞特異性抗體標(biāo)記外周血內(nèi)NK細(xì)胞,行流式細(xì)胞儀計數(shù),分析NK細(xì)胞術(shù)后不同時間在淋巴細(xì)胞中所占比例的變化。 實驗結(jié)果 1.SD大鼠與Wistar大鼠間同種異體角膜移植組與Wistar大鼠自體角膜移植組之間角膜排斥反應(yīng)指數(shù)(Rejection Value RV值)存在明顯差異,實驗組排斥反應(yīng)發(fā)生率為100%,異體移植片存活時間為(9.65+0.77)天。 2.同種異體角膜移植組與自體角膜移植組大鼠植片病理切片中,在角膜植片基質(zhì)浸潤、植片厚度、新生血管等方面存在顯著差異,兩組共80例動物模型中術(shù)后出現(xiàn)虹膜前粘連6例,白內(nèi)障4例,眼內(nèi)感染3例,虹膜脫出3例。 3.同種異體角膜移植組術(shù)后早期大鼠外周血中NK細(xì)胞占淋巴細(xì)胞比例明顯高于對照組,結(jié)果有顯著差異,NK細(xì)胞計數(shù)峰值出現(xiàn)在術(shù)后第(4.4±0.82)天。 結(jié)論 1.大鼠角膜主要組織相容性抗原的表達(dá)與人類角膜相似,且排斥反應(yīng)發(fā)生率高,作為較理想的角膜移植動物模型,為模擬人角膜移植排斥反應(yīng),進(jìn)行深入實驗研究提供基礎(chǔ)條件。選用遠(yuǎn)交系大鼠、偏中心植片及保留縫線等方法可增加角膜排斥反應(yīng)的發(fā)生率 2.熟練的顯微技術(shù)、銳利的手術(shù)器械、充分的擴(kuò)瞳及術(shù)后前房的形成是減少術(shù)后并發(fā)癥的關(guān)鍵。 3.NK細(xì)胞在大鼠同種異體角膜移植早期排斥反應(yīng)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 擄 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 鹵 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 .

【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R779.65

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

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