發(fā)布時間：2018-04-15 10:06

  本文選題：超急性排斥反應(yīng) + 補體調(diào)節(jié)蛋白　； 參考：《武漢大學(xué)》2005年博士論文

【摘要】：異種器官移植成為解決器官短缺的有效途徑正受到越來越多的關(guān)注。然而免疫排斥反應(yīng),特別是超急性排斥反應(yīng)(hyperacute rejection,HAR)很大程度上制約了它的發(fā)展。補體系統(tǒng)激活是異種器官移植超急性排斥反應(yīng)發(fā)生的主要原因,抑制受體的補體系統(tǒng)就可以阻止超急性排斥反應(yīng)的發(fā)生。膜補體調(diào)節(jié)蛋白衰變加速因子(decay accelerating factor,DAF),膜輔蛋白(membrane cofactor protein,MCP)和保護(hù)素(protectin,CD59)是補體系統(tǒng)中具有明顯的同種限制作用的3種補體下調(diào)因子,在補體攻擊時能提供區(qū)分“自我”與“非我”的方式,保護(hù)宿主細(xì)胞不受同源性補體的傷害,從而延長移植物的存活時間,克服超急性排斥反應(yīng)的發(fā)生,在異種器官移植方面有重大的應(yīng)用價值。因此人補體調(diào)節(jié)蛋白(complement regulator proteins,CRPs)是目前異種移植界的研究熱點之一。 本研究根據(jù)文獻(xiàn)報道的衰變加速因子DAF cDNA的序列,設(shè)計并合成特異引物,以中國人胚胎mRNA為模板,采用RT-PCR方法擴增出1684 bpDAF cDNA。序列分析表明,該cDNA含DAF全長編碼區(qū),共編碼381個氨基酸,沒有編碼Alu家族的序列,為GPI錨連型DAF。 將DAF全長cDNA序列插入真核表達(dá)載體pcDNA3,成功地構(gòu)建了重組真核表達(dá)載體pcDNA3-DAF,以磷酸鈣沉淀法轉(zhuǎn)染NIH/3T3細(xì)胞,用G418篩選獲得NIH/3T3DAF,PCR實驗結(jié)果顯示DAF基因整合在轉(zhuǎn)化的NIH/3T3細(xì)胞的染色體上,RT-PCR和Western印跡實驗分別從RNA水平和蛋白質(zhì)水平證實了人補體調(diào)節(jié)蛋白DAF在NIH/3T3DAF細(xì)胞系中獲得表達(dá)。檢測連續(xù)傳代30次NIH/3T3DAF結(jié)果表明人補體調(diào)節(jié)蛋白基因DAF仍穩(wěn)定整合并高效表達(dá),并未隨著傳代而丟失,以上結(jié)果顯
[Abstract]:More and more attention has been paid to xenotransplantation as an effective way to solve organ shortage.However, immune rejection, especially hyperacute rejection (HARs), restricts its development to a large extent.The activation of complement system is the main cause of hyperacute rejection in xenotransplantation. The suppressive complement system can prevent the occurrence of hyperacute rejection.The decay accelerating factor-dafs, membrane coprotein membrane cofactor protein (MCPs) and protectinin CD59) are three kinds of complement down-regulation factors which have the same limiting effect in the complement system.During complement attack, it can provide a way to distinguish "self" from "non-self" and protect host cells from the damage of homologous complement, thus prolonging the survival time of grafts and overcoming the occurrence of hyperacute rejection.It has great application value in xenogeneic organ transplantation.Therefore, human complement regulator proteins (CRPs) is one of the hotspots in the field of xenotransplantation.Based on the sequence of decay acceleration factor (DAF cDNA) reported in the literature, a specific primer was designed and synthesized. 1684 bpDAF cDNA was amplified by RT-PCR using Chinese embryonic mRNA as a template.Sequence analysis showed that the cDNA contained a full-length coding region of DAF, encoding 381 amino acids, but not encoding the sequence of Alu family. It was a GPI anchor type.The full-length cDNA sequence of DAF was inserted into eukaryotic expression vector pcDNA3. The recombinant eukaryotic expression vector pcDNA3-DAF was successfully constructed and transfected into NIH/3T3 cells by calcium phosphate precipitation.The results of NIH / 3T3DAFN PCR showed that the expression of human complement regulatory protein DAF in NIH/3T3DAF cell line was confirmed by RT-PCR and Western blotting from the RNA level and protein level, respectively.The results of 30 successive passages of NIH/3T3DAF showed that the human complement regulatory protein gene DAF was still stable and highly expressed, and was not lost with passage.
【學(xué)位授予單位】：武漢大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2005
【分類號】：Q78;R392

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本文編號：1753639