本文關(guān)鍵詞： 異種移植 離體肺灌注 超急性免疫排斥反應(yīng) GalTKO.hcD46.hTM轉(zhuǎn)基因豬 n-3多不飽和脂肪酸 mfat-1轉(zhuǎn)基因小鼠 Solexa測(cè)序 血清循環(huán)miRNAs 癌癥　出處：《南京醫(yī)科大學(xué)》2014年博士論文　論文類(lèi)型：學(xué)位論文

【摘要】：肺移植術(shù)是治療各種晚期阻塞性、限制性、感染性肺部疾病和肺血管病等終末期肺病的有效方法。隨著臨床醫(yī)學(xué)的發(fā)展以及免疫抑制劑的廣泛利用,同種器官移植已獲得顯著成效。但器官資源缺乏嚴(yán)重阻礙其廣泛應(yīng)用,因而異種器官移植包括異種肺移植受到了高度重視。豬因其器官形狀和大小適宜,易進(jìn)行基因調(diào)控和控制病毒傳播等因素成為異種移植的理想供體。然而,異種移植雖然可以有效解決器官來(lái)源的問(wèn)題,但卻也面臨比同種異體移植更為復(fù)雜的免疫排斥反應(yīng),這一難題成為擺在研究者們面前的第一道屏障。異種移植免疫排斥反應(yīng)中最先發(fā)生的也是需要首先解決的就是超急性免疫排斥反應(yīng),會(huì)導(dǎo)致移植物出血和血小板血栓形成,從而破壞內(nèi)皮功能。它的發(fā)生是由于人體內(nèi)預(yù)存的異種反應(yīng)性天然抗體與異種器官血管內(nèi)皮細(xì)胞表面的抗原結(jié)合,從而引發(fā)的補(bǔ)體系統(tǒng)鏈?zhǔn)郊せ钏�。被天然抗體識(shí)別的異種抗原主要是Gala(1,3)Gal,也稱(chēng)Gal表位,其在α1,3-半乳糖苷轉(zhuǎn)移酶(al,3galactosyltransferase, al,3-GT)作用下合成。因而從理論上我們需要從異種移植物抗原和補(bǔ)體方面考慮克服超急性免疫排斥反應(yīng)的發(fā)生。 我們的課題組建立了GalTKO.hCD46.hTM轉(zhuǎn)基因豬模型,這一轉(zhuǎn)基因豬敲除了α1,3-半乳糖苷轉(zhuǎn)移酶基因,并通過(guò)進(jìn)一步人源化修飾,轉(zhuǎn)入人膜輔助調(diào)節(jié)蛋白(membrane cofactor protein, MCP/CD46)和在凝血機(jī)制中發(fā)揮重要作用的人血栓調(diào)節(jié)蛋白(thrombomodulin, TM),使轉(zhuǎn)基因豬的血管內(nèi)皮細(xì)胞表面表達(dá)人源化的補(bǔ)體調(diào)節(jié)蛋白和血栓調(diào)節(jié)蛋白,從而抑制補(bǔ)體活化反應(yīng)和凝血反應(yīng),進(jìn)一步降低異種移植排斥反應(yīng)。 利用GalTKO.hCD46.hTM轉(zhuǎn)基因豬模型,我們采用多種研究手段對(duì)異種肺移植進(jìn)行了有效研究,包括離體肺灌注、檢測(cè)人源化修飾基因在細(xì)胞水平的表達(dá)及功能、體外細(xì)胞灌流等,以驗(yàn)證這些表型的轉(zhuǎn)入對(duì)異種移植肺在移植后抗器官損傷、抗免疫排斥反應(yīng)以及抗凝血反應(yīng)方面的作用。 在離體肺灌注實(shí)驗(yàn)中,我們利用體外循環(huán)系統(tǒng)采用肝素化的新鮮人血對(duì)GalTKO.hCD46.hTM轉(zhuǎn)基因豬(n=14)、野生型豬(n=16)、Ga1TKO轉(zhuǎn)基因敲除豬(n=16)進(jìn)行了異種灌注,對(duì)12只豬肺進(jìn)行了自體血液灌注,并監(jiān)測(cè)了灌注過(guò)程中的各項(xiàng)生理、血液和生化指標(biāo)。結(jié)果顯示,GalTKO.hCD46.hTM的轉(zhuǎn)基因豬肺平均生存期為175分鐘(生存期范圍：15-240分鐘),Ga1TKO豬肺為120分鐘(生存期范圍：15-240分鐘,p=0.24),而野生型豬肺平均灌注存活時(shí)間僅為10分鐘(p0.001)。與GalTKO豬相比,GalTKO.hCD46.hTM異種灌注豬肺中的補(bǔ)體激活水平、血小板活化水平顯著降低,在240分鐘時(shí),ΔC3a：238±22VS572±162, p=0.03; ACD62P:11.9±6VS25.4±7.4, p=0.03。 GalTKO.hCD46.hTM豬肺在抑制凝血級(jí)聯(lián)活化反應(yīng)和中性粒細(xì)胞封存等方面也顯示出了明顯的優(yōu)勢(shì)。此外通過(guò)免疫組化結(jié)果顯示在灌注過(guò)程中,肺組織始終有血栓調(diào)節(jié)蛋白的表達(dá)。 體外實(shí)驗(yàn)結(jié)果顯示,Ga1TKO.hCD46.hTM的轉(zhuǎn)基因豬的血管內(nèi)皮細(xì)胞具有極高的hCD46及血栓調(diào)節(jié)蛋白表達(dá)率,且針對(duì)血栓調(diào)節(jié)蛋白的功能檢測(cè)結(jié)果也顯示,血管內(nèi)皮細(xì)胞表達(dá)的血栓調(diào)節(jié)蛋白可以有效激活蛋白C,其所輔助活化的蛋白C含量是Ga1TKO組的14倍,是陽(yáng)性對(duì)照組人臍靜脈內(nèi)皮細(xì)胞(HUVEC)的5.5倍。在體外細(xì)胞灌流過(guò)程中,Ga1TKO.hCD46.hTM組的內(nèi)皮細(xì)胞發(fā)揮了良好的抗凝血功能,與Ga1TKO組相比,使血栓體積降低了84%(P=0.0001),使黏附和聚集率降低了58%和65%(P0.001)。 以上均說(shuō)明我們對(duì)于轉(zhuǎn)基因豬的人源化修飾是有效的,這些表型的轉(zhuǎn)入對(duì)移植后抗器官損傷、抗免疫排斥反應(yīng)以及抗凝血反應(yīng)均產(chǎn)生了積極的作用。我們將進(jìn)一步探討不同表型轉(zhuǎn)入對(duì)于異種移植物抗免疫排斥反應(yīng)的影響,并通過(guò)藥物干預(yù)等合理的方式對(duì)其進(jìn)行進(jìn)一步修飾與補(bǔ)充。 研究證實(shí),機(jī)體內(nèi)多不飽和脂肪酸的含量與構(gòu)成對(duì)癌細(xì)胞的分化、增殖和凋亡有著復(fù)雜而重要的作用,尤其是n-3多不飽和脂肪酸被認(rèn)為在細(xì)胞的功能和分化中發(fā)揮功能,可起到降血脂、舒張血管、抑制過(guò)敏與炎癥反應(yīng)、抑癌及預(yù)防心血管疾病等作用,但其作用的分子機(jī)制尚不明確。 microRNA (miRNA)是一類(lèi)長(zhǎng)約19-23個(gè)核苷酸的小分子單鏈RNA,它由一段具有發(fā)夾結(jié)構(gòu)的單鏈RNA前體經(jīng)Drosha酶和Dicer酶的剪切后生成。miRNA通過(guò)與目標(biāo)mRNA分子的3’端非編碼區(qū)域(3'-UTR)互補(bǔ)配對(duì),使目標(biāo)mRNA分子的翻譯受到抑制或引起特異性的針對(duì)mRNA分子的切割,從而在轉(zhuǎn)錄后水平對(duì)靶基因的表達(dá)進(jìn)行調(diào)控。自在秀麗隱桿線(xiàn)蟲(chóng)中發(fā)現(xiàn)第一個(gè)miRNA以來(lái),越來(lái)越多的miRNA在其他物種中被發(fā)現(xiàn)。保守估計(jì)miRNA調(diào)控著基因組中30%以上基因的表達(dá)。大量研究成果表明,miRNA參與著生物體中包括癌癥的發(fā)生等基本生命過(guò)程的調(diào)控,在生命活動(dòng)中起著非常重要的作用。 miRNA在各種組織中的表達(dá)失調(diào)已經(jīng)被證實(shí)與多種疾病相關(guān),如癌癥和糖尿病等。血清中的循環(huán)miRNA以其良好的個(gè)體間穩(wěn)定性和重復(fù)性,被認(rèn)為是各種癌癥和其他疾病檢測(cè)的潛在生物標(biāo)志物。課題組建立了mfat-1轉(zhuǎn)基因小鼠動(dòng)物模型,特異性的表達(dá)來(lái)源于秀麗隱桿線(xiàn)蟲(chóng)的fat-1基因,這一基因編碼n-3多不飽和脂肪酸脫氫酶,可以n-6多不飽和脂肪酸為底物,將其轉(zhuǎn)換為n-3多不飽和脂肪酸。采用Solexa基因芯片測(cè)序的方法,我們分析了野生型小鼠與mfat-1轉(zhuǎn)基因小鼠血清中miRNA的表達(dá)譜差異,并通過(guò)定量RT-PCR驗(yàn)證確認(rèn)了12種miRNA在mfat-1轉(zhuǎn)基因小鼠中高表達(dá)。針對(duì)這12種miRNA所調(diào)控的靶基因的進(jìn)一步分析顯示,其靶標(biāo)基因均為癌癥發(fā)生發(fā)展相關(guān)的多種信號(hào)通路中的關(guān)鍵基因,包括PI3K, MAPK, mTOR等多條信號(hào)通路,揭示了n-3多不飽和脂肪酸對(duì)于癌癥發(fā)生發(fā)展的影響。
[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.

【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：R655.3

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