發(fā)布時間：2018-04-20 12:58

  本文選題：GGTA1基因 + 小型豬�。� 參考：《甘肅農(nóng)業(yè)大學》2017年碩士論文

【摘要】：豬是人異種器官移植的理想供體,由α-1,3半乳糖苷(α-1,3Gal)引起的豬到人器官移植的超急性排斥反應已經(jīng)基本解決,但是隨之而來的細胞性排斥反應以及血栓和炎癥成為目前亟待解決的問題,豬白細胞抗原(swine leukocyte antigen,SLA)是引起異種移植細胞性免疫排斥的主要因素之一,轉人內皮蛋白C受體(endothelial cell protein C receptor,EPCR)可以有效降低豬到人器官移植引起的血栓和炎癥。本研究主要探討了α-1,3半乳糖苷轉移酶(α-1,3-galactosyltransferase,GGTA1)基因敲除巴馬小型豬(Bama minipig,BMP)的健康水平以及SLA等位基因和單倍型為異種器官移植研究篩選細胞排斥低的供體,制備可以降低異種器官移植引起的炎癥和血栓反應的轉hEPCR基因豬。本論文共有三部分研究內容,分別為:1.GGTA1基因敲除巴馬小型豬的繁育及健康水平檢測。本課題組制備的GGTA1基因敲除(α-1,3-galactosyltransferase knockout,GTKO)巴馬小型豬已經(jīng)繁育到F3代。本研究跟蹤了GTKO巴馬小型豬的遺傳、窩產(chǎn)仔數(shù)和血常規(guī)血生化指標,評估其繁殖和健康狀況。通過PCR產(chǎn)物測序鑒定仔豬GGTA1基因敲除類型,FITC-GSIB4與仔豬外周血單個核細胞(peripheral blood mononuclear cells,PBMC)孵育后流式細胞術檢測α-1,3-Gal表達,以窩產(chǎn)仔數(shù)測定繁殖力,以血常規(guī)和血生化檢測反映健康狀況。結果顯示,GGTA1基因的遺傳符合孟德爾分離定律;GGTA1敲除純合子(GGTA1-/-)仔豬的流式檢測熒光強度低;GTKO巴馬豬母豬頭胎窩產(chǎn)仔數(shù)為7.38±1.64頭,經(jīng)產(chǎn)母豬窩產(chǎn)仔數(shù)為9.43±1.68頭,與已有報道的普通巴馬小型豬繁殖力無明顯差異;血常規(guī)血生化檢測的各項指標與普通巴馬豬基本無差異�？傊�,連續(xù)3代GTKO巴馬仔豬遺傳穩(wěn)定,繁殖力正常,生理健康。GTKO巴馬小型豬可作為異種器官移植研究的可靠供體。2.GGTA1基因敲除巴馬小型豬SLA單倍型分型。本研究主要對SLA基因上5個位點(SLA-1、SLA-2、SLA-3、SLA-DRB1和SLA-DQB1)進行研究。采用轉錄本PCR產(chǎn)物直接測序的方法獲得GTKO巴馬小型豬SLA等位基因和單倍型,補體依賴的淋巴細胞毒試驗(Complementdependent cytotoxicity,CDC)檢測不同SLA單倍型豬的PBMC與人血清反應的細胞死亡率。結果發(fā)現(xiàn),GGTA1基因敲除巴馬小型豬群的5個SLA位點共發(fā)現(xiàn)15個等位基因,其中SLA-2*05:07(序列號:KX022946)和SLA-3*03:10(序列號:KX022947)2條序列為新發(fā)現(xiàn)序列,GGTA1基因敲除巴馬小型豬群共有5個SLA單倍型,分別為Hp-80.27、Hp-81.27、Hp-83a.45、Hp-82.44和Hp-83b.10c。4種雜合單倍型(Hp-80.27/81.27,Hp-82.44/83b.10c,Hp-80.27/83b.10c和Hp-83a.45/83b.10c)豬CDC結果細胞死亡率都小于27.5%,Hp-80.27/81.27和Hp-82.44/83b.10c單倍型的豬CDC結果細胞死亡率都小于17%。結果表明,不同SLA單倍型豬PBMC與人血清CDC試驗結果不同,Hp-80.27/81.27和Hp-82.44/83b.10c單倍型的豬CDC結果細胞死亡率更低,更適合做異種器官移植研究供體。3.轉hEPCR基因豬的制備。本研究構建廣泛性表達啟動子CAG調控的hEPCR基因表達載體pCAGGS-hEPCR-Puro,轉染巴馬五指山雜交豬胎兒成纖維細胞,通過體細胞克隆技術制備轉基因克隆豬。利用PCR技術對克隆仔豬進行轉基因鑒定,同時通過實時熒光定量PCR(qRT-PCR)、Western Blot及免疫組化方法分析hEPCR在轉基因豬的各個器官中的表達。本研究成功構建了pCAGGS-hEPCR-Puro載體,轉染細胞后篩選出71個有效克隆點,共得到2頭轉hEPCR陽性仔豬,qRT-PCR檢測發(fā)現(xiàn)hEPCR基因在克隆仔豬肝臟、腎臟、心臟、脾臟、胰臟、肺臟、主動脈等主要器官都有表達,在主動脈、胰臟、肺臟中的表達水平較高。Western Blot和免疫組化結果表明hEPCR蛋白在耳朵、主動脈,胰臟、心臟和腎臟都有表達。本研究成功制備了轉hEPCR基因豬,并且hEPCR基因在克隆豬的主要器官組織都有較高的表達水平,為異種器官移植研究制備了良好供體。
[Abstract]:Porcine is an ideal donor for xenotransplantation. The hyperacute rejection of porcine to human organ transplantation caused by alpha -1,3 galactoside (alpha -1,3Gal) has been basically solved, but the attendant cellular rejection and thrombus and inflammation are the problems to be solved urgently. The swine leukocyte antigen (SLA) is the leading factor. One of the main factors for xenograft rejection is that the endothelial cell protein C receptor (EPCR) C receptor (EPCR) can effectively reduce the thrombosis and inflammation caused by human organ transplantation. This study mainly discusses the alpha galactosidase (alpha -1,3-galactosyltransferase, GGTA1) gene knockout in Bama. The health level of Bama Minipig (BMP) and the SLA allele and haplotype for xenotransplantation screening the low cell rejection donor for the preparation of hEPCR transgenic pigs that can reduce the inflammatory and thrombus reaction caused by xenotransplantation. This paper has three parts: 1.GGTA1 gene knockout Bama miniature pig GGTA1 gene knockout (alpha -1,3-galactosyltransferase knockout, GTKO) Bama miniature pigs have been bred to the F3 generation. This study traced the inheritance, litter size and blood biochemical indexes of GTKO Bama miniature pigs, and evaluated their reproductive and health status. The offspring were sequenced and identified by PCR products. GGTA1 gene knockout type, FITC-GSIB4 and peripheral blood mononuclear cells (peripheral blood mononuclear cells, PBMC) of piglets were incubated by flow cytometry to detect the expression of alpha -1,3-Gal. The fecundity was measured by litter size, and the health status was reflected by blood routine and blood biochemical tests. The results showed that the inheritance of GGTA1 gene conforms to the Mendel law of separation. The flow detection fluorescence intensity of GGTA1 knockout homozygote (GGTA1-/-) piglets was low, the number of litter size in the head litter of GTKO Bama sows was 7.38 + 1.64, the number of litter size of the sows was 9.43 + 1.68 heads, and there was no significant difference from the common Bama miniature pig's fecundity, and the indexes of blood routine blood biochemistry were basically no different from that of normal Bama pigs. In a word, the 3 generations of GTKO Bama piglets have been genetically stable, and the reproductive capacity is normal. The physiological healthy.GTKO Bama miniature pig can be used as a reliable donor.2.GGTA1 gene for SLA haplotyping of the Bama miniature pig. This study mainly studies the 5 loci of the SLA gene (SLA-1, SLA-2, SLA-3, SLA-DRB1 and SLA-DQB1). The GTKO Bama miniature pig SLA allele and haplotype were obtained by direct sequencing of the PCR product, and the complement dependent lymphocyte toxicity test (Complementdependent cytotoxicity, CDC) was used to detect the cell death rate of PBMC and human serum reaction in different SLA haplotype pigs. The results showed that the GGTA1 gene knocked out 5 SLA loci of the Bama miniature pig. 15 alleles were found, including 2 sequences of SLA-2*05:07 (sequence number: KX022946) and SLA-3*03:10 (sequence number: KX022947). There were 5 SLA haplotypes of GGTA1 gene knockout Bama miniature pigs, Hp-80.27, Hp-81.27, Hp-83a.45, Hp-82.44 and Hp-83b.10c.4 hybrid haplotypes. 80.27/83b.10c and Hp-83a.45/83b.10c) the mortality of pig CDC cells was less than 27.5%. The results of CDC results in Hp-80.27/81.27 and Hp-82.44/83b.10c haplotype pigs were less than that of 17%., and the results of PBMC in different SLA haplotype pigs were different from those of CDC in human serum, and the result cells died in Hp-80.27/81.27 and Hp-82.44/83b.10c haplotypes. The death rate is lower, and it is more suitable for the preparation of the donor.3. transgenic hEPCR gene pig. This study constructs a hEPCR gene expression vector pCAGGS-hEPCR-Puro regulated by the promoter CAG, which is widely used to transfect the fetal fibroblasts of Bama Five Fingers Group hybrid pig, and the transgenic cloned pig is prepared by somatic cell clon technology. The PCR technology is used. The cloning of piglets was genetically modified, and the expression of hEPCR in various organs of transgenic pigs was analyzed by real-time quantitative PCR (qRT-PCR), Western Blot and immunohistochemical method. The pCAGGS-hEPCR-Puro vector was successfully constructed. After transfecting the cells, 71 effective clones were screened and 2 hEPCR positive piglets were obtained, qRT-PCR was obtained. The expression of hEPCR gene in the liver, kidney, kidney, heart, spleen, pancreas, pancreas, lung and aorta of piglets was expressed. The high expression level of.Western Blot and immunohistochemistry in aorta, pancreas and lung showed that hEPCR protein was expressed in the ear, active vein, pancreas, heart and kidney. Transgenic hEPCR pigs and hEPCR genes have higher expression level in the main organs and tissues of the cloned pigs, and have prepared good donors for xenotransplantation.

【學位授予單位】：甘肅農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R617;Q78

【參考文獻】

相關期刊論文 前3條

1 FENG Chong;LI Xi-rui;CUI Hui-ting;LONG Chuan;LIU Xia;TIAN Xing-hua;PAN Deng-ke;LUO Yu-zhu;;Highly efficient generation of GGTA1 knockout pigs using a combination of TALEN m RNA and magnetic beads with somatic cell nuclear transfer[J];Journal of Integrative Agriculture;2016年07期

2 潘登科;張莉;周艷榮;馮沖;龍川;劉曉;董恩球;王樹臣;萬榮;張健;陳紅星;;體細胞核移植生產(chǎn)轉ω-3脂肪酸去飽和酶基因sFat-1克隆豬[J];中國科學(C輯:生命科學);2009年03期

3 楊述林;任紅艷;王恒;馮書堂;甘世祥;王愛德;李奎;;中國實驗用小型豬種群血液生理指標分析[J];中國畜牧獸醫(yī);2007年02期

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本文編號：1777840