【作者】 向志光；

【导师】 夏咸柱； 张连峰； 李万波；

【作者基本信息】 中国协和医科大学 ， 免疫学， 2010， 博士

【摘要】 白细胞介素33(Interleukin 33, IL-33)是近年发现的IL-1类细胞因子家族的成员。它的受体是早于它被发现的孤儿受体ST2。作为细胞表面受体的ST2主要表达于Th2辅助性淋巴细胞、肥大细胞等细胞表面,IL-33具有促进Th2细胞免疫的作用。而在临床上IL-33和ST2与自身免疫病、变态反应性疾病、心血管疾病和神经退行性病变等的发生发展相关,从而引起人们的关注。小鼠腹腔注射重组的IL-33分子,动物表现为呼吸道和消化道的炎症损伤,脾脏肿大。而对于动物体内内源性的IL-33表达上升的研究目前未有报道。在分子结构上IL-33分子具有核定位功能域和IL-1类细胞因子活性功能域。IL-33分子本身缺乏分泌信号,目前对于IL-33细胞因子的分泌释放和它作为活性细胞因子的分泌形式等都存在着争议。为了研究IL-33在小鼠体内的内源性表达及其对动物的影响,我们首先从新生小鼠的组织中克隆了小鼠的IL-33基因,并将该基因构建到以CMV为启动子的真核表达载体pCDNA3.1上,通过显微注射的方式建立了CMV-mIL-33的转基因小鼠。通过免疫印迹分析检测到在转基因小鼠的肺脏、心脏、肾脏和胰腺组织中IL-33表达提高。高表达的IL-33分子在肺脏组织以18 kDa的形式分泌到呼吸道。通过双向电泳分析18 kDa分子的等电点性质,发现高表达的18 kDa的IL-33分子更接近于小鼠IL-33分子中有功能活性的109-266 aa肽段的肽段等电点性质。mIL-33转基因对小鼠的出生率、性别比率、体重等并没有产生影响。大体解剖和组织学分析没有发现转基因动物的脑、心脏、肝脏、脾脏、肾脏、小肠等受到影响。病理学分析发现IL-33转基因小鼠的肺脏出现炎症性反应：在支气管和小血管周围出现了包括嗜酸性粒细胞在内的炎性细胞浸润；在转基因小鼠的呼吸道出现了杯状细胞的高反应性和粘液性物质的聚集；对肺组织灌洗液的分析发现灌洗液中的嗜酸性粒细胞、嗜中性粒细胞、单核细胞和淋巴细胞等各类炎性细胞数目增加,炎性细胞因子IL-5、IL-8、IL-13的水平提高,IgE水平提高,同时炎症抑制性细胞因子IL-10和可溶性ST2的水平也提高。这些结果表明IL-33在肺脏组织的过表达诱发了小鼠自发的肺部炎症反应。我们通过转基因的方式研究分析了内源性高表达IL-33,发现了IL-33在体内确实存在剪切,而且剪切出的分子的性质更接近活性序列的性质。内源性转基因的IL-33在肺脏组织得以高表达,促发了肺部的炎症反应,印证了IL-33是一种促炎症因子。物种的进化是长期自然选择的结果,在进化过程中物种的遗传物质相应也会发生改变。在本质上物种的进化是遗传物质——基因组的进化,在基因水平包括基因的融合、倒位、易位,基因的丢失和新基因的出现。人类作为灵长类动物他们在1.2亿年前和啮齿类动物具有共同的祖先。但是长期的进化使得他们的基因组发生了变化。根据目前对部分灵长类和啮齿类动物基因组测序的结果,在基因水平每类基因组中大约有1%的基因在另外一类动物的基因组中是找不到的。这样一类基因被称为种属特异基因。我们通过比较灵长类和啮齿类动物的基因组找到这样一些种属特异基因。XAGE-3是灵长类种属特异的一个基因。在本论文工作中,我们首先在生物信息学的水平验证类人类XAGE-3基因在灵长类动物相对啮齿类动物的种属特异性。随后从人类胎盘组织中克隆了该基因。将该基因插入真核表达载体pCDNA3.1构建了以CMV为启动子的表达载体。通过显微注射的方法得到了XAGE-3的转基因小鼠。分析了转基因小鼠部分组织的反转录产物中该基因的表达,发现该基因在转基因小鼠的小肠、胸腺和睾丸组织有较高表达,利用Brdu标记3周龄的雄性转基因小鼠,结果发现XAGE-3转基因小鼠有更多的发育晚期的精母细胞被标记,人XAGE-3转基因影响到了小鼠生精细胞的发育。MAN2C1是一种α-甘露糖苷酶,调节蛋白质的糖基化。既往的研究表明MAN2C1和肿瘤的发生发展相关。本论文的部分工作研究了hMAN2C1转基因小鼠肝脏和脾脏组织的α-甘露糖苷酶活性,制备了兔抗人MAN2C1的多克隆抗体并以之分析了所转MAN2C1基因在两个转基因品系中表达。以此转基因动物验证了MAN2C1基因对于移植肿瘤的生长和迁移的影响。为了进一步研究MAN2C1的功能,我们制备了MAN2C1基因表达抑制的基因沉默小鼠。在转录水平MAN2C1基因表达被抑制,但组织酶活分析却没有发生明显改变。更多还原

【Abstract】 Interleukin-33 is a recent found cytokine which belongs to the cytokine family of Interleukin-1. The orphan receptor ST2 which was found in 1989, is ragarded as IL-33 receptor on target cell surface. ST2 was mainly expressed on the surface of Th2 helper lymphocytes and mast cells. IL-33 was regarded as a pro-inflammatory factor to promote Th2 cells functions. IL-33 and ST2 play an important role in inflammatory diseases including hypersensitive diseases like asthma, autoimmune diseases like rheumatoid arthritis, cardiovascular diseases like heart failure and neurodegenerative diseases like Alzheimer’s disease. The exogenous administration of recombinant IL33 induced splenomegaly changes and severe airway and digestive trace inflammation in mice. Currently there were no report on endogenous up-regulation of IL-33 in animals. IL-33 contains an IL-1-like cytokine fold in its carboxyl-terminus and chromatin binding domain in amino-terminus. Without a signal peptide for secretion, it is necessary to understand the functional structure of IL-33, that is the cleavage and secretion of this cytokine. A lot of researchers had tried to solve this question, but there are conflicts now.Here we reported the generation of an IL-33 transgenic mouse, in which mouse IL-33 full length cDNA was cloned out of neonate mouse tissue, inserted into euocaryotic expression vector pCDNA3.1, controlled under the CMV promoter. Immuno-blot results showed that the transgenic IL-33 was released as a cleaved form with molecular weight of 18 kDa in pulmonary, nephritic, cardiac and pancreatic tissues in transgenic mice and the pⅠof 18 kDa peptide was about pH3-5 on the 2-D PAGE, which was similar with the activated IL-33 peptide of 109-266 aa.The birth-rates, body weights, and sex ratios of ltransgenic mice were recorded and there were no difference compared with that of C57BL/6 wild-type mice. The gross anatomic and histological analyses were performed on the tissues of heart, liver, spleen, kidney, pancreas, intestine and brain of IL-33 transgenic mice at 2-month-old. No obvious changes were observed. Histological analysis showed massive pulmonary inflammation with infiltration of eosinophils around bronchi and small blood vessels, hyperplasia of goblet cells and accumulation of mucus-like material in pulmonary tissue of transgenic mice. An increase of IL-5, IL-8, IL-13 and IgE was detected in bronchoalveolar lavage fluid (BALF) of transgenic mice, which are inflammatory factors. sST2 and the anti-inflammatory factor IL-10 were also up-regulated to conterbalance the inlfamatroy damage of IL-33. These findings suggest transgenic IL-33 could be cleaved and secreted in an activated form and play an important role in the pathogenesis of pulmonary inflammation. Through our IL-33 transgenic research, the endogenous over-expression of mouse IL-33 was achived in mouse pulmonary tissue. And we detected the endogenous cleavage of IL-33 in mouse, and the character of the cleaved IL-33 was similar to the peptide with cytokine activity. The endogenous over-expression of IL-33 leads to spontaneous pulmonary inflammation, which proved the pro-inflammatory character of IL-33. The evolution of species is the result of nature selection. The genetic material would also changes with the evolution of species. In fact the evolution of species is also the evolution of the genomes. There were different kinds of changes of the gene, including the appearance of new gene.120 million years ago, the primate like human being had the same ancestor with the rodent. With the evolution of the two kinds of animals, there were significant changes in their genomes. Based on the genomic sequence analysis of these two kinds of animals, there were about 1%genes, which only exist in one kind, not in the other. These genes were regarded as species specific genes.We’ve found some species specific genes through the sequence comparison of these two kinds of animals. XAGE-3 is one of the primate species specific gene. In this study, we firstly confirm the species specific character of XAGE-3 by bio-information method. Subsequently we cloned this gene out of human placenta, and inserted this gene into eukaryotic expression vector pCDNA3.1. The XAGE-3 transgenic mice were got by micro-injection. We analyzed the XAGE-3 expression in transcripts from different tissues. Over-expression of XAGE-3 can be detected in intestine, thymus and testis in transgenic mice.3-week-old male mice were labeled with Brdu. More spermatocytes were labeled inXAGE-3 transgenic mice compared with negative control. The results suggests the human XAGE-3 transgene may affect the proliferation and development of spermatocytes. MAN2C1 is an a-mannosidase, which regulates protein glycosylation. Previous studies have showed its effect on tumor proliferation. In this study, the enzyme activity of a-mannosidase in liver and spleen of MAN2C1 transgenic mice were analysed. A rabbit polyclonal antibody against hMAN2Cl was raised to detect the transgenic MAN2C1 expression in 2 lines of hMAN2Cl transgenic mice. The transgenic model was used to prove the effect of MAN2C1 on proliferation and migration of grafted tumors. For better understanding the biological function of this gene, a transgenic gene silence mouse model was made. The expression of the targeted gene had been inhibited in transcripts, but there no no changes in the enzyme activity in the tissue from gene silenced mice.更多还原