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当前 器官移植 治疗 疾病 面临的 最大问题是 供体器官 严重缺乏,器官衰竭患者 多因 无法 及时得到供体器官 而死亡。异种器官移植 已成为 目前 科学界 和 医学界 公认的 解决 供体器官短缺的 有效途径,猪 已被认为 是最适合的 非灵长类 异种器官移植 供体。猪→人 异种器官移植 的 主要屏障 是 超急性排斥反应(HAR),α1,3半乳糖(α1,3Gal)是 引起 HAR的 主要抗原,临床上 将 清除了α1,3 Gal 的 猪器官 移植给 狒狒,仍会发生 排斥反应,提示 在 猪体内 还存在 其他非 α1,3Gal 异种抗原。为了早日 实现 猪→人异种器官移植的 临床应用,广大医学 和 科研工作者 把目光 转向了非 α1,3Gal 基因的 挖掘 和 功能 研究。本试验以 独特的 被誉为 猪→人异种器官移植 良好供体 的BMI 为 研究材料,以与 猪→人异种器官移植 免疫排斥反应 潜在 相关的 CMAH、OXSR1、CD80、ASGR1和B4GALNT2 等5个 非α1,3Gal基因 为候选基因,从分子、蛋白、细胞水平 同时 入手,验证这 5个基因 与猪→人异种器官移植 免疫排斥 反应的 相关性。
Currently, the most significant problem which is encountered by organ transplantation-based treatment of disease is the serious lack of donor organ, therefore most organ failure patients die from not obtaining donor organs timely. On this basis, xenotransplantation has become a well recognized method in scientific and medical community to solve the lack of donor organs, where pig has been regarded as the most suitable on human primate donor for xenotransplantation. The major technique barrier for pig→human xenotransplantation is the hyperacute rejection(HAR), in which α1,3 galactose(α1,3Gal)is the major antigen that causes HAR. In clinical practice, pig organs in which the α1,3 Gal have been removed, rejection action may still occur, indicating that there are still other heteroantigens other than α1,3Gal. To realize clinical application of pig→human
xenotransplantation, massive medical and scientific researchers turn to the exploration of non-α1,3Gal gene and research of related functions. In this experiment, the unique material BMI, known as an excellent donor for pig→human xenotransplantation, was selected as the researched material, and 5 non-α1,3Gal genes that are related with pig→human xenotransplantation rejection reaction such as CMAH、OXSR1、CD80、ASGR1 and B4GALNT were selected as candidate genes. The correlation between the 5 genes and pig→human xenotransplantation rejection reaction were verifed in the molecular, protein, and cellular level simultaneously.
首先 从mRNA水平分离 并 获得 这些 基因的 完全编码区 序列信息,并对 这些 基因 进行 免疫相关 多组织转录表达水平 的qPCR 检测分析,获得 其 在mRNA水平的 表达情况;然后 采用Western Blot方法 进一步 对 相同组织 进行验证,获得 其在 蛋白水平的 表达情况;构建 目的基因 和 绿色荧光报告基因 GFP 的 融合表达载体,转染示踪 进行 细胞水平的 验证;并对 相关蛋白质 进行 基本信息、特征信息、结构域、功能位点、高级结构 等功能生物信息学 分析。
First,mRNA-level separation was conducted to obtain the serial information of these genes in full coding region, qPCR detection analysis in the level of immuno-related multi-org transcription expression was applied to obtain the expression condition in mRNA level; secondly, Western Blot was adopted to further verify the same organs, so as to obtain the expression condition in protein level; the fusion expression vector for target gene and Green fluorescent protein reporter gene (GFP) was established for transfection tracing, so as to perform cellular level verification; moreover, the functional biological information of related proteins were analyzed including basic information, characteristic information, structural domain, functional site, and higher-order structure.
结果表明 猪CMAH、OXSR1、CD80、ASGR1 和 B4GALNT2 基因的 全长CDS序列 分别为 1734 bp、1590 bp、894 bp、861 bp、1509 bp,并 提交到 GenBank 获得了 认证。qPCR分析 和 Western Blot验证 表明CMAH mRNA在 颌下腺组织中 表达 最高;OXSR1 mRNA在 睾丸中 表达最高;CD80 mRNA在 脾组织中 表达最高;ASGR1 mRNA在 肝脏中 特异表达,在其它组织中 不表达;B4GALNT2 mRNA在 扁桃体中 表达最高;揭示 同一基因 在不同组织 和 不同基因 在 相同组织中 差异表达 的现象,也说明 基因 主要在 哪些组织 发挥 重要的功能,这些 重要组织 将是 我们 进一步 研究 该基因功能 以及 进行 基因敲除 和 基因修饰 研究的 靶组织。构建了 CMAH、OXSR1、CD80、ASGR1和B4GALNT2基因 和 绿色荧光蛋白AcGFP1 真核 融合表达载体 并 将其 转染 猪肾上皮PK15细胞 进行 瞬时表达,均 检测到了 绿色荧光表达。在此基础上 为了 研究 这些 猪的基因 与 人免疫排斥反应的 相关性,将这些 真核融合表达载体 转染 人脐静脉内皮细胞HUVEC 进行 表达,均检测到了 稳定表达的 绿色荧光,表明 CMAH、OXSR1、CD80、ASGR1和B4GALNT2基因 与 猪→人异种器官免疫排斥反应 相关。为 这些 基因在 猪→人异种移植排 斥反应中 分子机制 的 阐明奠定了 基础。
Result showed that the full-length CDS sequences of pig CMAH, OXSR1, CD80, ASGR1 and B4GALNT2 genes were 1734 bp, 1590 bp, 894 bp, 861 bp, and 1509 bp, respectively. This result was submitted to GenBank and verification was realized. qPCR analysis and Western Blot verification showed that CMAH mRNA achieved the highest expression in submandibular gland tissue; OXSR1 mRNA achieved the highest expression in testis;CD80 mRNA achieved the highest expression in the tissue of
spleen;ASGR1 mRNA achieved specific expression in liver, which achieved no expression in other tissues;B4GALNT2 mRNA achieved the highest expression in tonsilla;moreover it also revealed the phenomena of differential expression under
the conditions of the same gene in different tissues as well as different genes in the same tissue. It also showed the tissues in which these genes mainly exerted significant functions, which will be regarded as target tissues for gene function research and gene knockout and modification research. In this work, we established the eukaryotic fusion expression vectors for gene CMAH、OXSR1、CD80、ASGR1, B4GALNT2 respectively and green fluorescent protein AcGFP1 and then transfected it
to pig kidney epithelial cell PK15 for transient expression, and green fluorescence expression was detected. On this basis, to research the correlation between these pig genes and human immunological rejection reaction, these eukaryotic fusion
expression vectors were transfected to human umbilical vein endothelial cell (HUVEC) for expression, and the stably expressed green fluorescence can be detected, indicating that the pig genes of CMAH, OXSR1, CD80, ASGR1 and B4GALNT2 are related with pig→human xenotransplantation rejection reaction. This experiment lays a foundation for illustrating the molecular mechanism in pig→human xenotransplantation rejection reaction.
为深入了解 基因编码的 蛋白质的 相关功能性质,我们 对相应蛋白质 进行了 功能生物信息学分析,包括 分析 其 氨基酸组成、分子式、分子量、等电点、消光系数、半衰期、原子组成、不稳定系数、脂肪指数、亲水系数 等蛋白质 基本信息;疏水性、跨膜结构、卷曲螺旋、信号肽、固有无序性、亮氨酸富集的 核输出信号、亚细胞定位 等 蛋白质的特征信息;结构域 和 功能位点(磷酸化和糖基化);二级结构 和 三级结构等。这些蛋白质的 基本信息、特征信息、结构域、功能位点 以及 高级结构的 生物信息学分析 为 CMAH、OXSR1、CD80、ASGR1和B4GALNT2 5个 蛋白质的 进一步 功能研究 和 验证 提供了 理论依据,也为 下一步 进行 猪→人异种器官免疫排斥反应 相关基因 的 联合敲除 和 基因修饰 奠定了 实验和临床试验 基础。
In order to graso an in-depth knowledge on functional properties of gene encoding proteins, we analyzed the functional bioinformation of related proteins, including basic informations such as amino acid composition, molecular formula, molecular
weight, isoelectric point, extinction coefficient, half-life period, atomic composition, instability coefficient, fat index, and hydrophilic coefficient; characteristic informations such as hydrophobicity, transmembrane structure, coiled coil, signal peptide, inherent disorder, leucine-rich nuclear export signal, and subcellular localization; structural domain and function site (phosphorylation and glycosylation); secondary structure and tertiary structure. All these information provide theoretical basis for further functional research and verification of 5 proteins of CMAH, OXSR1, CD80, ASGR1 and B4GALNT2, and also lay foundation for clinical trails of gene knockout and modification related to pig→human xenotransplantation rejection reactiono in the next stage.
本研究 填补了 CMAH、OXSR1、CD80、ASGR1和B4GALNT2基因在 猪→人异种器官移植 非超急性排斥反应 α1,3Gal基因研究 之外的 空白,本研究结果 不仅可为 BMI 猪→人异种器官移植 利用 提供 基础数据资料,同时为 促进其 开发和利用 提供 科学依据,也可以为 猪→人异种器官 的早日实现 奠定理论 和 实验基础。
This research fills the blank research area on the correlation between 5 pig genes (CMAH, OXSR1, CD80, ASGR1 and B4GALNT2, other than α1,3Gal gene) and pig→human xenotransplantation non-hyperacute rejection reaction. The research results not only provide basic data material for the application of BMI in pig→human xenotransplantation, but also provides scientific basis for the development and utilization of BMI, more importantly lays theoretical and experimental basis for early realization of pig→human xenotransplantation. |
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