本文選題：CTLA-4Ig + 感音神經性耳聾　； 參考：《中國人民解放軍醫(yī)學院》2017年碩士論文

【摘要】：CTLA-4 (cytotoxic T lymphocyte-associated antigen-4, CTLA-4),中文名稱細胞毒T淋巴細胞相關抗原4,作為白細胞分化抗原的一種,又名CD152,是T細胞表面上的一種跨膜受體,與CD28共同競爭享有B7分子配體,當CTLA-4與B7分子結合后,可以導致T細胞表達無反應性,進而表達免疫反應的負調節(jié)。CTLA-4Ig主要是由CTLA-4分子細胞外功能區(qū)域和人自身免疫球蛋白IgG1的恒定區(qū)進行結合的一種融合蛋白,是一種經典的、研究功能明確的免疫抑制分子,CTLA-4Ig可以導致T細胞正常免疫反應受限,從而導致免疫功能降低。為了研究CTLA-4Ig在內耳發(fā)育及表達情況,我們與第三軍醫(yī)大學合作,以體細胞核移植方法構建人源性hCTLA-4Ig轉基因巴馬香豬,角蛋白(K14)作為啟動子,靶向表達,從而避免了因全身性表達CTLA-4lg導致的動物在胚胎發(fā)育期死亡。本次研究通過對hCTLA-4lg轉基因巴馬香豬種群中個體的轉基因鑒定、聽力學功能表型的分析和多種內耳形態(tài)學方法的研究,對hCTLA-4Ig轉基因巴馬香豬耳聾做了初步研究。本次研究包括如下三個部分。第一部分hCTLA-4Ig轉基因巴馬香豬耳聾動物模型基因型鑒定及蛋白分布表達首先,為了明確本研究的科學性,我們應首先確保我們通過體細胞移植獲得的封閉家系種群后代的基因型,確保其基因穩(wěn)定遺傳,可靠。此外還還需明確CLTA-4基因是否在正常巴馬香豬發(fā)育過程中表達,及CTLA-4Ig是否在內耳表達。我們采用了 PCR (Polymerase Chain Reaction,聚合酶鏈式反應)方法進行陽性巴馬香豬的鑒定以及用于種群繁衍的篩選。其中,在PCR結果中,因巴馬香豬轉基因豬個體表達了 326bp的目的條帶,而正常野生型巴馬香豬未見明顯目的條帶表達,說明轉基因克隆豬家系穩(wěn)定。第二部分hCTLA-4Ig轉基因巴馬香豬聽力學功能檢測為了研究CTLA-4Ig在內耳表達中的作用,我們首先通過對hCTLA-4Ig轉基因巴馬香豬的聽力學功能情況進行研究,在本次研究中,我們對hCTLA-4Ig轉基因巴馬香豬封閉種群20只轉基因克隆豬進行了聽力學篩查,采用了聽覺腦干反應(auditory brainstem response , ABR)閾值檢測,并在相同檢測條件下測試了20只正常野生型巴馬香豬ABR閾值,進行統(tǒng)計學配對分析,發(fā)現(xiàn)hCTLA-4Ig轉基因巴馬香豬組ABR閾值與野生型巴馬香豬組ABR閾值之間具有明顯的統(tǒng)計學差異(P0.05)。通過聽力學功能的檢測,我們發(fā)現(xiàn)hCTLA-4Ig轉基因巴馬香豬出現(xiàn)了不同程度的感音神經性耳聾,從而說明hCTLA-4Ig轉基因巴馬香豬家系出現(xiàn)了聽力損失。第三部分hCTLA-4Ig轉基因巴馬香豬內耳形態(tài)學觀察在明確了解hCTLA-4lg轉基因巴馬香豬聽力學表型特征后,我們通過對內耳進行形態(tài)學變化研究,進一步從形態(tài)學和病理學角度判斷hCTLA-4Ig轉基因巴馬香豬異常特征。首先我們通過取材后直接在顯微鏡下比較觀察,hCTLA-4Ig轉基因巴馬香豬耳蝸大小體積、耳蝸回數(shù)、聽小骨等發(fā)育未見與野生型巴馬香豬無明顯差異。耳蝸標本經過火棉膠包埋后,采用連續(xù)切片,經過HE染色后觀察,發(fā)現(xiàn)hCTLA-4Ig轉基因巴馬香豬耳蝸形態(tài)存在異常,出現(xiàn)corti' s器結構異常,前庭膜塌陷,中階狹窄等。其中耳蝸標本制備后進行掃描電鏡觀察,纖毛多數(shù)成病理改變,毛細胞脫落。在免疫熒光結果中,我們可以發(fā)現(xiàn),hCTLA-4Ig在轉基因豬的耳蝸內有表達,多集中在血管紋、螺旋神經節(jié)部位,而在正常野生型巴馬香豬耳蝸表達情況較低。通過對如上方法的形態(tài)學分析,我們可以看出,hCTLA-4Ig轉基因巴馬香豬的耳蝸具有病理改變,而這些病理改變是由于基因異常引起的,從而說明CTLA-4Ig突變與耳聾相關。
[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.
【學位授予單位】：中國人民解放軍醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R764;R-332

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相關期刊論文 前2條

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2 胡博華;;噪聲性耳聾:基礎研究進展和展望[J];中華耳科學雜志;2016年06期

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本文編號：2001800