hCTLA-4Ig转基因巴马香猪内耳发育畸形及听功能异常动物模型建立

发布时间：2018-06-10 03:20

本文选题：CTLA-4Ig + 感音神经性耳聋　；参考：《中国人民解放军医学院》2017年硕士论文

【摘要】：CTLA-4 (cytotoxic T lymphocyte-associated antigen-4, CTLA-4),中文名称细胞毒T淋巴细胞相关抗原4,作为白细胞分化抗原的一种,又名CD152,是T细胞表面上的一种跨膜受体,与CD28共同竞争享有B7分子配体,当CTLA-4与B7分子结合后,可以导致T细胞表达无反应性,进而表达免疫反应的负调节。CTLA-4Ig主要是由CTLA-4分子细胞外功能区域和人自身免疫球蛋白IgG1的恒定区进行结合的一种融合蛋白,是一种经典的、研究功能明确的免疫抑制分子,CTLA-4Ig可以导致T细胞正常免疫反应受限,从而导致免疫功能降低。为了研究CTLA-4Ig在内耳发育及表达情况,我们与第三军医大学合作,以体细胞核移植方法构建人源性hCTLA-4Ig转基因巴马香猪,角蛋白(K14)作为启动子,靶向表达,从而避免了因全身性表达CTLA-4lg导致的动物在胚胎发育期死亡。本次研究通过对hCTLA-4lg转基因巴马香猪种群中个体的转基因鉴定、听力学功能表型的分析和多种内耳形态学方法的研究,对hCTLA-4Ig转基因巴马香猪耳聋做了初步研究。本次研究包括如下三个部分。第一部分hCTLA-4Ig转基因巴马香猪耳聋动物模型基因型鉴定及蛋白分布表达首先,为了明确本研究的科学性,我们应首先确保我们通过体细胞移植获得的封闭家系种群后代的基因型,确保其基因稳定遗传,可靠。此外还还需明确CLTA-4基因是否在正常巴马香猪发育过程中表达,及CTLA-4Ig是否在内耳表达。我们采用了 PCR (Polymerase Chain Reaction,聚合酶链式反应)方法进行阳性巴马香猪的鉴定以及用于种群繁衍的筛选。其中,在PCR结果中,因巴马香猪转基因猪个体表达了 326bp的目的条带,而正常野生型巴马香猪未见明显目的条带表达,说明转基因克隆猪家系稳定。第二部分hCTLA-4Ig转基因巴马香猪听力学功能检测为了研究CTLA-4Ig在内耳表达中的作用,我们首先通过对hCTLA-4Ig转基因巴马香猪的听力学功能情况进行研究,在本次研究中,我们对hCTLA-4Ig转基因巴马香猪封闭种群20只转基因克隆猪进行了听力学筛查,采用了听觉脑干反应(auditory brainstem response , ABR)阈值检测,并在相同检测条件下测试了20只正常野生型巴马香猪ABR阈值,进行统计学配对分析,发现hCTLA-4Ig转基因巴马香猪组ABR阈值与野生型巴马香猪组ABR阈值之间具有明显的统计学差异(P0.05)。通过听力学功能的检测,我们发现hCTLA-4Ig转基因巴马香猪出现了不同程度的感音神经性耳聋,从而说明hCTLA-4Ig转基因巴马香猪家系出现了听力损失。第三部分hCTLA-4Ig转基因巴马香猪内耳形态学观察在明确了解hCTLA-4lg转基因巴马香猪听力学表型特征后,我们通过对内耳进行形态学变化研究,进一步从形态学和病理学角度判断hCTLA-4Ig转基因巴马香猪异常特征。首先我们通过取材后直接在显微镜下比较观察,hCTLA-4Ig转基因巴马香猪耳蜗大小体积、耳蜗回数、听小骨等发育未见与野生型巴马香猪无明显差异。耳蜗标本经过火棉胶包埋后,采用连续切片,经过HE染色后观察,发现hCTLA-4Ig转基因巴马香猪耳蜗形态存在异常,出现corti' s器结构异常,前庭膜塌陷,中阶狭窄等。其中耳蜗标本制备后进行扫描电镜观察,纤毛多数成病理改变,毛细胞脱落。在免疫荧光结果中,我们可以发现,hCTLA-4Ig在转基因猪的耳蜗内有表达,多集中在血管纹、螺旋神经节部位,而在正常野生型巴马香猪耳蜗表达情况较低。通过对如上方法的形态学分析,我们可以看出,hCTLA-4Ig转基因巴马香猪的耳蜗具有病理改变,而这些病理改变是由于基因异常引起的,从而说明CTLA-4Ig突变与耳聋相关。
[Abstract]:CTLA-4 (cytotoxic T lymphocyte-associated antigen-4, CTLA-4), the Chinese name cytotoxic T lymphocyte associated antigen 4, as a type of leukocyte differentiation antigen, also known as CD152, is a transmembrane receptor on the surface of T cells, and shares a B7 ligand with CD28. The negative regulatory.CTLA-4Ig, which expresses the immune response, is a fusion protein composed mainly of the CTLA-4 molecule extracellular function region and the constant region of human autoimmune globulin IgG1. It is a classic, functional immune suppressor molecule, and CTLA-4Ig can lead to the limited immune response of T cells, thus leading to the regulation of the normal immune response of T cells. In order to study the development and expression of CTLA-4Ig in the inner ear, we work with Third Military Medical University to construct a human hCTLA-4Ig transgenic Bama pig by somatic cell nuclear transplantation. The keratin (K14) is used as the promoter to target the expression, thus avoiding the development of the animal in the embryo due to the systemic expression of CTLA-4lg. This study has made a preliminary study on the deafness of the hCTLA-4Ig transgenic Bama pig by the identification of the individuals in the hCTLA-4lg transgenic Bama pig population, the analysis of the auditory functional phenotype and the study of various internal ear morphology methods. This study includes the following three parts. The first part is the hCTLA-4Ig transgenic Bama pig. In order to clarify the scientific nature of this study, we should first ensure that the gene type of the descendants of the closed family population obtained by somatic cell transplantation can be ensured to be stable and reliable. In addition, it is necessary to make clear whether the CLTA-4 gene is in the normal development process of normal Bama pig. Expression, and the expression of CTLA-4Ig in the inner ear. We used the PCR (Polymerase Chain Reaction, PCR) method to identify the positive Bama pig and the screening for the population multiplication. In the PCR result, the target band of 326bp was expressed by the Bama pig transgenic pig, and the normal wild Bama pig was used. There was no obvious target band expression, indicating that the transgenic clone pig family was stable. Second hCTLA-4Ig transgenic Bama Xiang pig audiological function test was used to study the role of CTLA-4Ig in the inner ear expression. We first studied the hearing function status of the hCTLA-4Ig transgenic Bama Xiang Pigs. In this study, we were to hCTL 20 transgenic cloned pigs of A-4Ig transgenic Bama pig were screened by audiological screening. The threshold detection of auditory brainstem response (auditory brainstem response, ABR) was used, and the ABR threshold of 20 normal wild Bama pigs was tested under the same test conditions, and the hCTLA-4Ig transgenic Bama was found by statistical matching analysis. There was a significant statistical difference between the ABR threshold of the pig group and the ABR threshold of the wild Bama Xiang pig group (P0.05). Through the audiological function test, we found that the hCTLA-4Ig transgenic Bama Xiang pigs had different degrees of sensorineural deafness, which indicated that the hearing loss of the hCTLA-4Ig transgenic Bama pig family had appeared. Third parts were found. The morphological observation of the internal ear of hCTLA-4Ig transgenic Bama Xiang pig is a clear understanding of the audiological characteristics of the hCTLA-4lg transgenic Bama pig. The morphological changes of the inner ear are studied and the abnormal characteristics of the hCTLA-4Ig transgenic Bama Xiang Pigs are judged from the morphological and pathological aspects. The size of the cochlea in hCTLA-4Ig transgenic Bama pig's cochlea, the number of cochlear cochlea, and the development of the auditory ossicle were not significantly different from that of the wild Bama pig. The cochlear specimens were embedded by the cotton gum, and the cochlea was stained with continuous slices after HE staining, and the abnormal morphology of the cochlea in the hCTLA-4Ig transgenic Bama pig was found to be CORT. The structure of I's was abnormal, the vestibule membrane collapsed, and the middle order stenosis, among which the cochlear specimens were prepared by scanning electron microscopy, most of the cilia were pathological changes, and the hair cells fell off. In the immunofluorescence results, we could find that hCTLA-4Ig was expressed in the cochlea of transgenic pigs and concentrated in the vascular lines, spiral ganglion sites, and in the normal field. The expression of the cochlea of the born Bama pig is low. Through the morphological analysis of the above methods, we can see that the cochlea of the hCTLA-4Ig transgenic Bama pig has a pathological change, and these pathological changes are caused by genetic abnormalities, indicating that the CTLA-4Ig mutation is associated with the deafness.
【学位授予单位】：中国人民解放军医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R764;R-332

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