【摘要】：引言 主要組織相容復(fù)合物I類相關(guān)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,是參與機(jī)體免疫的重要免疫分子,具有較豐富的多態(tài)性,其編碼的蛋白作為活化性受體NKG2D的配體,與NKG2D/DAP10結(jié)合,參與移植免疫、腫瘤免疫及多種自身免疫疾病,并在免疫監(jiān)視作用及腫瘤逃逸免疫中發(fā)揮重要作用。國內(nèi)外多項(xiàng)研究表明MICA基因由于較高的多態(tài)性與其發(fā)揮的免疫學(xué)效應(yīng)尤其是移植免疫聯(lián)系緊密,目前已發(fā)現(xiàn)68個(gè)等位基因,并且存在著廣泛的地域及人種差別。關(guān)于MICA基因多態(tài)性的研究逐漸成為對MICA基因研究的熱點(diǎn),目前越來越多的研究證明,MICA基因存在多態(tài)性現(xiàn)象,MICA基因及其抗體在多種器官移植中參與術(shù)后移植物抗宿主反應(yīng)(GVHR)及宿主抗移植物反應(yīng)(HVGR)。目前關(guān)于MICA等位基因配型及其抗體在器官移植中產(chǎn)生的免疫學(xué)效應(yīng)國內(nèi)外尚未見報(bào)道,我們旨在建立一套多態(tài)性MICA基因同種異體移植瘤模型,在受供體HLA配型完全一致的情況下,能夠?qū)Ｒ恍苑从巢煌琈ICA等位基因及其配型在移植免疫中所起到的作用。 目的 通過病毒轉(zhuǎn)化人外周血單個(gè)核細(xì)胞(PBMC),并通過序列特異性PCR(PCR-SSP)分析,建立特定HLA及MICA基因型的B淋巴母細(xì)胞系(BLCL)。建立中國人群常見MICA等位基因的真核表達(dá)載體。應(yīng)用人源化免疫NOD/SCID鼠方案,建立可模擬體內(nèi)免疫環(huán)境的小鼠模型,進(jìn)而建立多態(tài)性MICA基因同種異體移植瘤模型,為后續(xù)研究不同MICA等位基因在器官移植中所起的免疫學(xué)作用提供理論依據(jù)及試驗(yàn)基礎(chǔ)。 方法 本實(shí)驗(yàn)包含四個(gè)部分。第一部分,制備EBV病毒上清,并通過EBV轉(zhuǎn)化人外周血單個(gè)核細(xì)胞,建立B淋巴母細(xì)胞系。第二部分,合成可用于鑒定MICA等位基因的PCR混合引物對,通過PCR-SSP分析MICA基因特異性位點(diǎn),進(jìn)而確定試驗(yàn)對象的MICA基因型,為MICA等位基因篩選及MICA配型提供試驗(yàn)依據(jù)。第三部分,同過PCR-SSP對本實(shí)驗(yàn)室常見細(xì)胞系MICA基因特異性位點(diǎn)進(jìn)行分析,預(yù)篩選出特定MICA等位基因的目的細(xì)胞系,提取RNA,逆轉(zhuǎn)錄PCR獲取包含特定MICA等位基因的cDNA全長片段,連接到真核表達(dá)載體,構(gòu)建特定等位基因的MICA質(zhì)粒。第四部分,通過NOD/SCID小鼠腹腔注射與移植瘤HLA及MICA相同配型的PBMC,建立人源化免疫NOD/SCID小鼠多態(tài)性MICA基因同種異體移植瘤模型。 結(jié)果 1.建立了特定HLA及MICA基因型的B淋巴母細(xì)胞系,細(xì)胞分裂增殖良好,鏡下成團(tuán)簇狀懸浮于培養(yǎng)液,每次傳代后凍存數(shù)支于液氮中,并可復(fù)蘇及繼續(xù)傳代。 2.合成了可供分析中國人群9種常見MICA等位基因的PCR混合引物對,對目的DNA產(chǎn)物進(jìn)行PCR-SSP試驗(yàn),通過特異性位點(diǎn)的凝膠電泳條帶,鑒定了建系細(xì)胞系BLCL的MICA等位基因?qū)儆贛ICA*008,并完成了本實(shí)驗(yàn)室常見細(xì)胞系特定MICA基因預(yù)篩選,確定7901胃癌細(xì)胞系包含目的基因MICA*008。 3.在MICA等位基因預(yù)篩選的基礎(chǔ)上,成功構(gòu)建包含MICA*008基因完整外顯子的真核表達(dá)載體pcDNA3(+)/MICA*008,轉(zhuǎn)染后成功表達(dá)由MICA*008基因所編碼的MICA膜蛋白,驗(yàn)證了應(yīng)用PCR-SSP技術(shù)對細(xì)胞系中對特定MICA等位基因預(yù)篩選及構(gòu)建質(zhì)粒的可行性。 4.通過動物實(shí)驗(yàn)論證,人免疫源性的NOD/SCID小鼠對特定HLA及MICA基因型移植瘤組與未經(jīng)人源化免疫的NOD/SCID小鼠組對比,移植物成形時(shí)間及生長速度上,人源化組均慢于對照組,說明包含多態(tài)性MICA基因的移植瘤模型的建立是成功的。 結(jié)論 本研究通過PCR-SSP的方法,鑒定了常見細(xì)胞系及BLCL的MICA基因特異性位點(diǎn),由此建立了包含特定MICA等位基因的細(xì)胞系BLCL,建立了包含特定MICA等位基因的真和表達(dá)載體pcDNA3(+)/MICA*008。通過與建系細(xì)胞系相同個(gè)體的外周血單個(gè)核細(xì)胞免疫NOD/SCID小鼠,使NOD/SCID小鼠的人源化免疫環(huán)境與BLCL移植瘤的HLA配型完全相合,為單一性研究不同MICA等位基因在移植免疫中發(fā)揮的作用及機(jī)制,提供了良好的HLA配型環(huán)境。本實(shí)驗(yàn)將臨床上樣本量非常稀少的HLA全相合器官移植病例,在動物模型上得以體現(xiàn),為分析多態(tài)性MICA基因配型及其抗體在器官移植中的免疫學(xué)作用奠定了基礎(chǔ)。
[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.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R392

【共引文獻(xiàn)】

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2 張s，

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