| CTLA-4(cytotoxic T lymphocyte-associated antigen-4, CTLA-4), which is also named as CD 152 as a kind of leukocyte antigen, is a transmembrane receptor in T cell surface. It can compete with CD28 in combination of B7 molecular ligand. When CTLA-4 And B7 molecules combined, it can decrease T cell activity. CTLA-4Ig is a fusion protein that binds the extracellular domain of the CTLA-4 molecule to the human immunoglobulin IgG1 constant region. It's a classic immune-suppressive molecule, it can limit T cell immune response, resulting in immune function decreased. In order to study the development and expression of CTLA-4Ig in the inner ear, we constructed the hCTLA-4Ig genotype Bama pig with somatic cell nuclear, thus avoiding the expression of CTLA-4Ig of animals died during embryonic development. In this study, CTLA-4Ig genotype Bama pig was studied in genotype identification, auditory functional phenotype analysis and a variety of inner ear morphological methods for the expression of hCTLA-4Ig genotype Bama pig Deafness made a preliminary study. This study includes the following three parts.Part ? The expression of hCTLA-4Ig Bama mini pig hearing loss animal model genotype identification and protein expressionIn order to ensure the scientific characterization of the hCTL-4Ig genotype Bama pig's audiometry and the inner ear morphological study, we ensure the genotype of the offspring population obtained by somatic cell transplantation is stably hereditary and reliable. We use PCR (Polymerase Chain Reaction) to identify positive Bama pig and screening for population multiplication. Finally, 326bp of the target bands were expressed in the genotype pigs of the Bama pig, while the wild type Bama pigs did not show the obvious bands.Part ? Hearing level of hCTLA-4Ig Transgenic Bama PigsIn order to study the role of CTLA-4Ig in the expression of the inner ear, we should first understand the auditory function of the hCTLA-4Ig transgenic Bama pig, which in this study we targeted the expression of hCTLA-4Ig transgenic The auditory brainstem response (ABR) was used to detect the auditory brainstem response (ABR), and under normal conditions, 10 normal wild-type Bama pigs were tested for ABR, and the statistics were performed. Statistical analysis showed that there was a statistically significant difference between the ABR threshold of hCTLA-4Ig transgenic Bama pig group and the ABR threshold of wild type Bama pig group (P <0.05). Through the detection of auditory function, we found that hCTLA-4Ig transgenic Bama pigs showed varying degrees of sensory nerve deafness, indicating that hCTLA-4Ig resulted in Bama pig The hearing loss.Part ? The CTLA-4Ig transgenic Bama pig internal ear morphological observationAfter a clear understanding of the hCTLA-4Ig transgenic Bama hogs, we observed morphological studies of the inner ear, and further visualized the expression of hCTLA-4Ig transgenic Bama from morphological and pathologically Abnormal characteristics of pig. First, we observed the expression of hCTLA-4Ig transgenic Bama husk cochlea, the number of cochlea, and the development of small bones were not significantly different from those of wild-type Bama pigs.The cochlear specimens were embedded in the cotton shoots, and the sections were observed by HE staining. The expression of hCTLA-4Ig transgenic Bama pig was abnormal and the hair cells were obviously absent. The cochlear specimens were prepared by scanning electron microscopy, ciliated majority of pathological changes. In addition, we also immunized with frozen sections of the cochlea and found that CTLA-4Ig was extensively inside the cochlea.Through the morphological analysis of the above method,we can see that the hCTLA-4Ig transgenic Bama pig has a pathological change in the cochlea,and these pathological changes lead to transgenic Bama pig Organic changes,indicating that CTLA-4Ig mutation associated with deafness. |
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