多态性MICA基因同种异体移植瘤模型的建立与研究

发布时间：2018-08-14 18:42

【摘要】：引言主要组织相容复合物I类相关基因A(major histocompatibility complex class I chain-ralated gene A,MICA)是MIC基因家族的功能性基因,是参与机体免疫的重要免疫分子,具有较丰富的多态性,其编码的蛋白作为活化性受体NKG2D的配体,与NKG2D/DAP10结合,参与移植免疫、肿瘤免疫及多种自身免疫疾病,并在免疫监视作用及肿瘤逃逸免疫中发挥重要作用。国内外多项研究表明MICA基因由于较高的多态性与其发挥的免疫学效应尤其是移植免疫联系紧密,目前已发现68个等位基因,并且存在着广泛的地域及人种差别。关于MICA基因多态性的研究逐渐成为对MICA基因研究的热点,目前越来越多的研究证明,MICA基因存在多态性现象,MICA基因及其抗体在多种器官移植中参与术后移植物抗宿主反应(GVHR)及宿主抗移植物反应(HVGR)。目前关于MICA等位基因配型及其抗体在器官移植中产生的免疫学效应国内外尚未见报道,我们旨在建立一套多态性MICA基因同种异体移植瘤模型,在受供体HLA配型完全一致的情况下,能够专一性反映不同MICA等位基因及其配型在移植免疫中所起到的作用。目的通过病毒转化人外周血单个核细胞(PBMC),并通过序列特异性PCR(PCR-SSP)分析,建立特定HLA及MICA基因型的B淋巴母细胞系(BLCL)。建立中国人群常见MICA等位基因的真核表达载体。应用人源化免疫NOD/SCID鼠方案,建立可模拟体内免疫环境的小鼠模型,进而建立多态性MICA基因同种异体移植瘤模型,为后续研究不同MICA等位基因在器官移植中所起的免疫学作用提供理论依据及试验基础。方法本实验包含四个部分。第一部分,制备EBV病毒上清,并通过EBV转化人外周血单个核细胞,建立B淋巴母细胞系。第二部分,合成可用于鉴定MICA等位基因的PCR混合引物对,通过PCR-SSP分析MICA基因特异性位点,进而确定试验对象的MICA基因型,为MICA等位基因筛选及MICA配型提供试验依据。第三部分,同过PCR-SSP对本实验室常见细胞系MICA基因特异性位点进行分析,预筛选出特定MICA等位基因的目的细胞系,提取RNA,逆转录PCR获取包含特定MICA等位基因的cDNA全长片段,连接到真核表达载体,构建特定等位基因的MICA质粒。第四部分,通过NOD/SCID小鼠腹腔注射与移植瘤HLA及MICA相同配型的PBMC,建立人源化免疫NOD/SCID小鼠多态性MICA基因同种异体移植瘤模型。结果 1.建立了特定HLA及MICA基因型的B淋巴母细胞系,细胞分裂增殖良好,镜下成团簇状悬浮于培养液,每次传代后冻存数支于液氮中,并可复苏及继续传代。 2.合成了可供分析中国人群9种常见MICA等位基因的PCR混合引物对,对目的DNA产物进行PCR-SSP试验,通过特异性位点的凝胶电泳条带,鉴定了建系细胞系BLCL的MICA等位基因属于MICA*008,并完成了本实验室常见细胞系特定MICA基因预筛选,确定7901胃癌细胞系包含目的基因MICA*008。 3.在MICA等位基因预筛选的基础上,成功构建包含MICA*008基因完整外显子的真核表达载体pcDNA3(+)/MICA*008,转染后成功表达由MICA*008基因所编码的MICA膜蛋白,验证了应用PCR-SSP技术对细胞系中对特定MICA等位基因预筛选及构建质粒的可行性。 4.通过动物实验论证,人免疫源性的NOD/SCID小鼠对特定HLA及MICA基因型移植瘤组与未经人源化免疫的NOD/SCID小鼠组对比,移植物成形时间及生长速度上,人源化组均慢于对照组,说明包含多态性MICA基因的移植瘤模型的建立是成功的。结论本研究通过PCR-SSP的方法,鉴定了常见细胞系及BLCL的MICA基因特异性位点,由此建立了包含特定MICA等位基因的细胞系BLCL,建立了包含特定MICA等位基因的真和表达载体pcDNA3(+)/MICA*008。通过与建系细胞系相同个体的外周血单个核细胞免疫NOD/SCID小鼠,使NOD/SCID小鼠的人源化免疫环境与BLCL移植瘤的HLA配型完全相合,为单一性研究不同MICA等位基因在移植免疫中发挥的作用及机制,提供了良好的HLA配型环境。本实验将临床上样本量非常稀少的HLA全相合器官移植病例,在动物模型上得以体现,为分析多态性MICA基因配型及其抗体在器官移植中的免疫学作用奠定了基础。
[Abstract]:Introduction
The major histocompatibility complex class I-chain-ralated gene A (MICA) is a functional gene of the MIC gene family and an important immune molecule involved in immunity. It has rich polymorphisms. Its encoded protein acts as a ligand of NKG2D and binds to NKG2D/DAP10. It plays an important role in immune surveillance and tumor escape immunity. Many studies have shown that the MICA gene is closely related to its immunological effects, especially transplantation immunity, because of its high polymorphism. At present, 68 alleles have been found and exist widely. The study of MICA gene polymorphism has gradually become a hot spot in the study of MICA gene. More and more studies have proved that there is a polymorphism of MICA gene. MICA gene and its antibodies are involved in graft versus host response (GVHR) and host versus graft response (HVGR) after multiple organ transplantation. The immunological effects of MICA alleles and their antibodies in organ transplantation have not been reported at home and abroad. We aim to establish a polymorphic allograft tumor model of MICA gene, which can specifically reflect different MICA alleles and their matches in transplantation immunity when the HLA matches of recipients are identical. Played a role.
objective
The human peripheral blood mononuclear cells (PBMC) were transformed by virus, and the specific HLA and MICA genotypes of B lymphoblasts (BLCL) were established by sequence specific PCR-SSP analysis. The eukaryotic expression vectors of common MICA alleles in Chinese population were established. Mouse models of immune environment in vivo were established by humanized immunization of NOD/SCID mice. Furthermore, a polymorphic MICA gene allograft tumor model was established to provide a theoretical basis and experimental basis for the follow-up study of the immunological role of different MICA alleles in organ transplantation.
Method
In the first part, the supernatant of EBV was prepared and transformed into human peripheral blood mononuclear cells to establish B lymphoblastic cell lines. In the second part, the PCR mixed primer pairs were synthesized to identify the alleles of MICA. The specific sites of MICA gene were analyzed by PCR-SSP, and then the MICA genotype was determined as MIC. In the third part, with PCR-SSP, we analyzed the specific sites of MICA gene in the common cell lines in our laboratory, pre-screened the target cell lines with specific MICA alleles, extracted RNA, and obtained full-length fragments of the cDNA containing specific MICA alleles by reverse transcription PCR, which were linked to eukaryotic expression vectors. In the fourth part, the allograft tumor model of human immunized NOD/SCID mice with polymorphic MICA gene was established by intraperitoneal injection of PBMC with the same matching as HLA and MICA in NOD/SCID mice.
Result
1. B-lymphoblastoid cell lines with specific HLA and MICA genotypes were established. The cells divide and proliferate well. Under microscope, the cells are suspended in culture medium in clusters. After each passage, several branches are frozen in liquid nitrogen and can be resuscitated and passed on.
2. A pair of PCR mixed primers was synthesized for the analysis of nine common MICA alleles in Chinese population. PCR-SSP test was carried out on the target DNA products. The MIA allele of BLCL cell line was identified as MICA*008 by gel electrophoresis of specific loci. Pre-screening of the specific MICA gene of the common cell line in our laboratory was completed and 79. 01 gastric cancer cell line contains target gene MICA*008.
3. Based on the pre-screening of MICA alleles, the eukaryotic expression vector pcDNA3 (+) / MICA * 008 containing the complete exon of MICA * 008 gene was successfully constructed. After transfection, the MICA membrane protein encoded by MICA * 008 gene was successfully expressed, which verified the feasibility of using PCR-SSP technology to pre-screen specific MICA alleles and construct plasmids in cell lines.
4. Human immunogenic NOD/SCID mice showed slower graft formation time and growth rate than the control group in the specific HLA and MICA genotype transplanted tumor group and the non-human immunogenic NOD/SCID mice group, indicating that the establishment of transplanted tumor model containing polymorphic MICA gene was successful.
conclusion
In this study, PCR-SSP was used to identify ICA gene specific sites in common cell lines and BLCL, and a cell line BLCL containing specific MICA alleles was established. The true and expression vector pcDNA3 (+) / MICA * 008 containing specific MICA alleles was established. OD/SCID mice, which completely matched the humanized immune environment of NOD/SCID mice with the HLA matching of BLCL transplanted tumor, provided a good HLA matching environment for the study of the role and mechanism of different MICA alleles in transplantation immunity. The model can be used to analyze the polymorphic MICA genotype and its antibody in organ transplantation.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392

【共引文献】