【作者】 付清；

【导师】 柳林； 李闻捷；

【作者基本信息】 第二军医大学 ， 眼科学， 2009， 博士

【摘要】 晶状体中的蛋白质主要包括晶体蛋白(crystallins)、膜蛋白(membraneprotein)和细胞骨架蛋白(cytosteletal protein)等。按水溶性与否可分为可溶性蛋白(晶体蛋白)、非可溶性蛋白(膜蛋白、细胞骨架蛋白、聚合的晶体蛋白)。人晶状体内90%的可溶性蛋白是晶体蛋白:分别是α、β和γ晶体蛋白。目前对α-晶体蛋白的功能研究比较广泛,它的分子伴侣作用已得到世界公认。对β-晶体蛋白的研究还比较少,其中βB2晶体蛋白(βB2 crystallin,Crybb2)在β族晶体蛋白中含量最高,而且它的水溶性成分呈年龄依赖性增高,因此,它的正常结构及其水溶性成分水平,对维持晶状体的高折射系数和透明至关重要。这种反常性增高势必包含了它在维持晶状体透光性中的重要作用,因此它的功能值得进一步研究。基因敲除是研究蛋白功能的一个重要手段,已有研究证明α-晶体蛋白基因敲除可以导致白内障,而且α或β-晶体蛋白基因突变也可以导致白内障。文献报道Crybb2基因突变可降低生殖功能。目前国内外尚未有Crybb2基因敲除动物模型的报道,因此我们选择此蛋白作为靶点,利用Crybb2基因敲除小鼠模型,来研究该蛋白在维持晶状体透明性中的作用和其晶状体外生物学功能。本文主要通过观察Crybb2基因敲除对小鼠晶状体结构的影响及其在晶状体内和晶状体外组织的定位,分析该蛋白在维持晶状体透明性中的作用并研究其晶状体外功能。主要方法:1.Crybb2基因敲除小鼠模型的建立及鉴定此部分工作是应第二军医大学第一附属医院中心实验室要求,与美国纽约iGTL实验室合作并最终在美国iGTL实验室完成,最后以馈赠方式供我们研究专用。小鼠品系为C57BL/C小鼠,小鼠模型为10只杂合子,已成功进行繁殖超过30代,子代小鼠生长发育状况良好。取鼠尾末端组织,通过PCR技术对小鼠基因型进行鉴定;取小鼠眼晶状体进行匀浆离心后取上清液,通过Western blot技术对晶体蛋白的表达进行鉴定。鉴定后将小鼠分类饲养繁殖。2.Crybb2基因敲除小鼠晶状体结构特征用暗视野显微镜观察经氧化损伤后晶状体体外培养的混浊程度。HE染色观察晶状体囊膜下皮质的厚度随年龄的变化,并与野生型同品系小鼠对比。免疫组织化学研究其在晶状体内的定位。3.Crybb2基因敲除小鼠晶状体外功能研究图形视网膜电图(pattern electroretinogram,P-ERG)研究Crybb2基因敲除后视觉电生理的改变。Western Blot,PCR和免疫组织化学方法检测Crybb2 mRNA,Crybb2在晶状体外阳性表达(视网膜、大脑、脊髓、睾丸、卵巢),尽管是低水平表达。数小鼠产仔数,精子计数,睾丸称重,大体形态和HE染色观察形态学改变研究Crybb2基因敲除后小鼠生殖功能的改变,并与野生型同品系小鼠对比。实验结果:1.Crybb2基因敲除小鼠模型建立后,经Western blot鉴定,纯合小鼠晶体蛋白未检测到Crybb2的表达,杂合及野生型小鼠晶体蛋白在分子量23KD处检测到该蛋白的表达。2.Crybb2基因敲除导致小鼠白内障的发生,白内障均发生在前或后皮质部,最早发生于出生后8周左右。Crybb2定位于晶状体皮质囊膜下,基因敲除后,晶状体抗氧化能力下降,随年龄增加,皮质变薄。3.P-ERG检查发现Crybb2基因敲除小鼠振幅降低。野生型小鼠视网膜,大脑,脊髓,睾丸,卵巢中可检测到Crybb2 mRNA,Crybb2阳性表达。Crybb2基因敲除后,精子活力下降,计数减少,小鼠睾丸和卵巢外观减小,睾丸重量减轻。小鼠生仔数目轻度减少,窝数减少。结论:1.Crybb2基因敲除可以导致C57BL/C小鼠白内障的发生,为皮质性白内障,皮质混浊程度随年龄增加而增加,皮质厚度随年龄的增加而变薄。2.Crybb2基因敲除可以降低C57BL/C小鼠生殖功能。更多还原

【Abstract】 Crystallins,membrane protein and cytosteletal protein are the main compositon proteins of the len.Crystallins is water-soluble,but cytosteletal protein and membrane protein and polymerized crystallins are urea-soluble.90%of the water-soluble proteins in the heman eye lens are the crystallins:α、βandγcrystallin.The study of the function ofα-crystallin is widespread and the molecular chaperone of it was generally accepted.But the study toβ-crystallin is ralatively less. The concentration ofβB2-crystallin is not only higher than otherβ-crystallin but also increase with age.So the function of it is deserved to research.Crystallin gene knockout is an important method to study the function of crystallin.Some research have find that theα-crystallin gene knockout can lead to cataract,so do theαorβ-crystallin gene mutation.It has not been reported the animal model ofβB2-crystallin gene knock out all over the world.So we chose the crystallin as the target and set up the animal model so as to research its function on maintaining the transparency of lens and its extralenticular functions.In our study,pattern electroretinogram was used to study the function of retinal ganglion cell after the gene knock out.The lens ofβB2-crystallin gene knockout mice was studied in this article.We examined the in vitro construction of len and analysis the function ofβB2-crystallin in keeping transparency of the lens and observed influence on reproduction of the mouse with targeted knockout of the mouseβB2-crystallin gene compared with the wild tipe.Methods:1.Generation and verification ofβB2-crystallin gene knockout mice.βB2-crystallin gene knockout mice were produced by the in Genious Targeting Laboratory.The model of mice were 10 heterozygote which have been breed in the central lab and have reproduced over 30 generations.Gene expression were characterized by PCR and western blot. 2.The structure and character of lens inβB2-crystallin gene knockout miceFresh lens from 8-week-old gene knockout and wild type mice were removed from the eyeballs and cultured in TC-199 medium for 2 hours in a CO₂ incubator,and then the gene knockout mice were screened for the degree of opacity with dark field biomicroscopy.Microstructure of lens was analyzed by hematoxylin and eosin stain and immunohistochemistry.By studying cortex of different ageβB2-crystallin gene knockout mice lens,we observed the influence to development of cataract.3.Fertility were studied using knock out ofβB2-crystallin mice and the wild typePattern electroretinogram was used to study the function of retinal ganglion cell after the gene knock out.We used Western Blot,PCR and immunohistochemistry to detect mRNA ofβB2-crystallin gene,βB2-crystallin outside the len（retina,brain, spinal cord,ovary and testis） although it was expressed at lower levels in several extralenticular locations.Through the babies of the mouse cipher,Sperm count,testis weigh,using morphshape and hematoxylin and eosin stain observation method,we studied the reproduction function of the mice of targeted knockout of the mouseβB2-crystallin gene compared with the wild tipe.RESULTS:1.Western blot failed to detect anyβB2-crystallin in the lens ofβB2-crystallin gene knockout mice.2.The gene knockout mice were screened more severe opacity with dark field biomicroscopy.Hematoxylin and eosin stain and immunohistochemistry found that theβB2-crystallin was located in subcapsular of the cortex and the cortex was thiner in the gene knockout mice with ageing.3.The vibration of the Pattern electroretinogram was lower after gene knock out. Through the mice the baby cipher,Sperm count,testis weigh,we found that the productivity were lower in the gene knock mice.Using morphshape and hematoxylin and eosin stain,we observed smller shape and structure chaos in the mice with targeted knockout of theβB2-crystallin gene compared with the wild tipe.Conclusions:1.Targeted knockout of the mouseβB2-crystallin gene can induce cataract which is located in the subcapsular,and then the cortex of the lens grow thinner with age.2.Targeted knockout of the mouseβB2-crystallin gene induces subfertility更多还原