GalTKO.hCD46.hTM转基因猪异种肺移植的初步研究及mfat-1转基因小鼠血清中miRNAs的表达分析

发布时间：2018-03-01 18:53

本文关键词： 异种移植离体肺灌注超急性免疫排斥反应 GalTKO.hcD46.hTM转基因猪 n-3多不饱和脂肪酸 mfat-1转基因小鼠 Solexa测序血清循环miRNAs 癌症　出处：《南京医科大学》2014年博士论文　论文类型：学位论文

【摘要】：肺移植术是治疗各种晚期阻塞性、限制性、感染性肺部疾病和肺血管病等终末期肺病的有效方法。随着临床医学的发展以及免疫抑制剂的广泛利用,同种器官移植已获得显著成效。但器官资源缺乏严重阻碍其广泛应用,因而异种器官移植包括异种肺移植受到了高度重视。猪因其器官形状和大小适宜,易进行基因调控和控制病毒传播等因素成为异种移植的理想供体。然而,异种移植虽然可以有效解决器官来源的问题,但却也面临比同种异体移植更为复杂的免疫排斥反应,这一难题成为摆在研究者们面前的第一道屏障。异种移植免疫排斥反应中最先发生的也是需要首先解决的就是超急性免疫排斥反应,会导致移植物出血和血小板血栓形成,从而破坏内皮功能。它的发生是由于人体内预存的异种反应性天然抗体与异种器官血管内皮细胞表面的抗原结合,从而引发的补体系统链式激活所致。被天然抗体识别的异种抗原主要是Gala(1,3)Gal,也称Gal表位,其在α1,3-半乳糖苷转移酶(al,3galactosyltransferase, al,3-GT)作用下合成。因而从理论上我们需要从异种移植物抗原和补体方面考虑克服超急性免疫排斥反应的发生。我们的课题组建立了GalTKO.hCD46.hTM转基因猪模型,这一转基因猪敲除了α1,3-半乳糖苷转移酶基因,并通过进一步人源化修饰,转入人膜辅助调节蛋白(membrane cofactor protein, MCP/CD46)和在凝血机制中发挥重要作用的人血栓调节蛋白(thrombomodulin, TM),使转基因猪的血管内皮细胞表面表达人源化的补体调节蛋白和血栓调节蛋白,从而抑制补体活化反应和凝血反应,进一步降低异种移植排斥反应。利用GalTKO.hCD46.hTM转基因猪模型,我们采用多种研究手段对异种肺移植进行了有效研究,包括离体肺灌注、检测人源化修饰基因在细胞水平的表达及功能、体外细胞灌流等,以验证这些表型的转入对异种移植肺在移植后抗器官损伤、抗免疫排斥反应以及抗凝血反应方面的作用。在离体肺灌注实验中,我们利用体外循环系统采用肝素化的新鲜人血对GalTKO.hCD46.hTM转基因猪(n=14)、野生型猪(n=16)、Ga1TKO转基因敲除猪(n=16)进行了异种灌注,对12只猪肺进行了自体血液灌注,并监测了灌注过程中的各项生理、血液和生化指标。结果显示,GalTKO.hCD46.hTM的转基因猪肺平均生存期为175分钟(生存期范围：15-240分钟),Ga1TKO猪肺为120分钟(生存期范围：15-240分钟,p=0.24),而野生型猪肺平均灌注存活时间仅为10分钟(p0.001)。与GalTKO猪相比,GalTKO.hCD46.hTM异种灌注猪肺中的补体激活水平、血小板活化水平显著降低,在240分钟时,ΔC3a：238±22VS572±162, p=0.03; ACD62P:11.9±6VS25.4±7.4, p=0.03。 GalTKO.hCD46.hTM猪肺在抑制凝血级联活化反应和中性粒细胞封存等方面也显示出了明显的优势。此外通过免疫组化结果显示在灌注过程中,肺组织始终有血栓调节蛋白的表达。体外实验结果显示,Ga1TKO.hCD46.hTM的转基因猪的血管内皮细胞具有极高的hCD46及血栓调节蛋白表达率,且针对血栓调节蛋白的功能检测结果也显示,血管内皮细胞表达的血栓调节蛋白可以有效激活蛋白C,其所辅助活化的蛋白C含量是Ga1TKO组的14倍,是阳性对照组人脐静脉内皮细胞(HUVEC)的5.5倍。在体外细胞灌流过程中,Ga1TKO.hCD46.hTM组的内皮细胞发挥了良好的抗凝血功能,与Ga1TKO组相比,使血栓体积降低了84%(P=0.0001),使黏附和聚集率降低了58%和65%(P0.001)。以上均说明我们对于转基因猪的人源化修饰是有效的,这些表型的转入对移植后抗器官损伤、抗免疫排斥反应以及抗凝血反应均产生了积极的作用。我们将进一步探讨不同表型转入对于异种移植物抗免疫排斥反应的影响,并通过药物干预等合理的方式对其进行进一步修饰与补充。研究证实,机体内多不饱和脂肪酸的含量与构成对癌细胞的分化、增殖和凋亡有着复杂而重要的作用,尤其是n-3多不饱和脂肪酸被认为在细胞的功能和分化中发挥功能,可起到降血脂、舒张血管、抑制过敏与炎症反应、抑癌及预防心血管疾病等作用,但其作用的分子机制尚不明确。 microRNA (miRNA)是一类长约19-23个核苷酸的小分子单链RNA,它由一段具有发夹结构的单链RNA前体经Drosha酶和Dicer酶的剪切后生成。miRNA通过与目标mRNA分子的3’端非编码区域(3'-UTR)互补配对,使目标mRNA分子的翻译受到抑制或引起特异性的针对mRNA分子的切割,从而在转录后水平对靶基因的表达进行调控。自在秀丽隐杆线虫中发现第一个miRNA以来,越来越多的miRNA在其他物种中被发现。保守估计miRNA调控着基因组中30%以上基因的表达。大量研究成果表明,miRNA参与着生物体中包括癌症的发生等基本生命过程的调控,在生命活动中起着非常重要的作用。 miRNA在各种组织中的表达失调已经被证实与多种疾病相关,如癌症和糖尿病等。血清中的循环miRNA以其良好的个体间稳定性和重复性,被认为是各种癌症和其他疾病检测的潜在生物标志物。课题组建立了mfat-1转基因小鼠动物模型,特异性的表达来源于秀丽隐杆线虫的fat-1基因,这一基因编码n-3多不饱和脂肪酸脱氢酶,可以n-6多不饱和脂肪酸为底物,将其转换为n-3多不饱和脂肪酸。采用Solexa基因芯片测序的方法,我们分析了野生型小鼠与mfat-1转基因小鼠血清中miRNA的表达谱差异,并通过定量RT-PCR验证确认了12种miRNA在mfat-1转基因小鼠中高表达。针对这12种miRNA所调控的靶基因的进一步分析显示,其靶标基因均为癌症发生发展相关的多种信号通路中的关键基因,包括PI3K, MAPK, mTOR等多条信号通路,揭示了n-3多不饱和脂肪酸对于癌症发生发展的影响。
[Abstract]:Lung transplantation is the treatment of various advanced obstructive, restrictive, effective methods of infectious lung disease and pulmonary vascular disease and end-stage lung disease. With the extensive use of the development of clinical medicine and immunosuppression after organ transplantation, has obtained remarkable results. But the organ resources lack its wide application and serious obstacles, including xenotransplantation xenogeneic lung transplantation has attracted more attention. Because of the pig organ shape and suitable size, easy for gene regulation and control the spread of the virus and other factors become the ideal donor for xenotransplantation. However, although xenotransplantation can effectively solve the problem of the source of organs, but also face more complex immune rejection than the allogeneic transplantation. A problem has become the first barrier in front of the researchers. Xenotransplantation immune rejection occurred in the first reaction is needed is to first solve hyperacute Immune rejection leads to graft bleeding and platelet thrombus formation, resulting in the destruction of endothelial function. Its occurrence is due to the combination of internal prestored xenoreactive natural antibodies and xeno vascular endothelial cell surface antigen, complement system activation induced by causing a chain type. By heterologous antigen antibody recognition of natural Gala (1,3 Gal), also known as the Gal epitope of the 1,3- transferase in alpha galactosidase (al, 3galactosyltransferase, Al, 3-GT) under the action of synthesis. So in theory we need from the xenograft antigen and complement aspects to overcome hyperacute rejection.
Our group established GalTKO.hCD46.hTM transgenic pig model, the transgenic pig knockout alpha 1,3- galactose transforase gene, and through further humanized modification to human membrane cofactor protein (membrane cofactor, protein, MCP/CD46) and thrombosis play an important role in the blood coagulation mechanism in regulating protein (thrombomodulin, TM) so, the surface of transgenic porcine endothelial cells to express humanized complement regulatory protein and thrombomodulin, thereby inhibiting complement activation and blood coagulation reaction, further reduce xenograft rejection.
The use of GalTKO.hCD46.hTM transgenic pig model, we used a variety of research tools for lung transplantation were dissimilar effective research, including isolated lung perfusion, expression and functional characterization of humanized modified gene at the cellular level, in vitro perfusion, to verify the phenotype of resistance into organ damage xenotransplantation in lung transplantation. Anti immune response and anti coagulation effect of rejection.
In isolated lung perfusion experiments, we use the extracorporeal circulation system using heparinized fresh human blood of GalTKO.hCD46.hTM transgenic pig (n=14), wild type pigs (n=16), Ga1TKO gene knockout pigs (n=16) were different in 12 lung perfusion of autologous blood perfusion, and physiological monitoring in the process of reperfusion, blood and biochemical indexes. The results showed that the average survival of transgenic porcine GalTKO.hCD46.hTM was 175 minutes (survival time range: 15-240 minutes to 120 minutes), Ga1TKO lung (survival time range: 15-240 minutes, p=0.24), while the wild type lung perfusion average survival time was 10 minutes (p0.001). Compared with GalTKO pigs, the level of activated GalTKO.hCD46.hTM perfusion in porcine xenogeneic complement, platelet activation was significantly lower, in 240 minutes, a C3a:238 + 22VS572 + 162, p=0.03; ACD62P: 11.9 + 6VS25.4 + 7.4, p=0.03. GalTKO.hCD4 6.hTM lung in the inhibition of the coagulation cascade activation and neutrophil sequestration also showed a clear advantage. The immunohistochemistry results showed that during perfusion, lung tissue has expression of thrombomodulin.
In vitro experiments showed that hCD46 transgenic pig Ga1TKO.hCD46.hTM and thrombosis of vascular endothelial cells is highly regulated protein expression rate, and for the thrombomodulin function test results also showed that the expression of vascular endothelial cell thrombomodulin can effectively activate protein C, the protein content of C assisted activation is 14 times higher than that of Ga1TKO group that is the positive control group of human umbilical vein endothelial cells (HUVEC) of 5.5 times. In vitro perfusion process, Ga1TKO.hCD46.hTM group of endothelial cells play a good anti coagulation function, compared with the Ga1TKO group, the thrombus volume decreased by 84% (P=0.0001), the adhesion and aggregation rate decreased by 58% and 65% (P0.001).
All the above shows that we are effective for the humanized modification of transgenic pigs, the phenotype of organ damage after transplantation into the anti, anti rejection and anti coagulation reaction to produce positive effect. We will further explore the different phenotype effect on xenograft compound anti immune rejection, and through drug intervention the reasonable way for further modification and complement it.
The research confirmed that the body of polyunsaturated fatty acids content and composition on the differentiation of cancer cells, is a complex and important role in cell proliferation and apoptosis, especially n-3 polyunsaturated fatty acids are thought to function in cell differentiation and function, can play a role in reducing blood fat, blood vessels, inhibiting inflammation and allergy reaction of tumor suppressor role and prevention of cardiovascular disease, but its mechanism is not clear.
MicroRNA (miRNA) is a kind of single stranded RNA molecules of about 19-23 nucleotides in length, which is composed of a section with.MiRNA to generate single stranded RNA precursor hairpin structure by shear Drosha and Dicer enzyme and mRNA molecular target by 3 'non encoding region (3'-UTR) complementary, so that target mRNA molecules the translation is inhibited or induced by specific for mRNA molecular cutting, to regulate expression of transcription of the target gene. Since the first miRNA found itself in Caenorhabditis elegans, more and more miRNA was found in other species. The conservative estimate miRNA regulates expression of over 30% genes in the genome. A large number of research results show that miRNA plays a role in the regulation of cancer including basic life processes in organisms, plays a very important role in life activities.
The expression of miRNA in various tissues of disorders have been confirmed with a variety of diseases, such as cancer and diabetes. Circulating miRNA in serum with good stability and repeatability between individuals, are considered to be potential biomarkers for detection of various cancers and other diseases. Subject to establish mfat-1 transgenic mice animal model, expression the source of specificity in Caenorhabditis elegans fat-1 gene, the gene encoding n-3 polyunsaturated fatty acid dehydrogenase, n-6 polyunsaturated fatty acid as substrate, convert it to n-3 polyunsaturated fatty acid. Using Solexa gene chip sequencing method, we analyzed the differential expression of miRNA in serum of wild type mice with mfat-1 transgenic mice in the spectrum, and validated by quantitative RT-PCR confirmed that 12 miRNA high expression in mfat-1 transgenic mice. Further target genes regulated by the 12 kinds of miRNA. Analysis shows that the target genes are all the key genes in various signaling pathways related to the development and progression of cancer, including PI3K, MAPK, mTOR and many other signaling pathways, revealing the influence of n-3 polyunsaturated fatty acids on the occurrence and development of cancer.

【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R655.3

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