【作者】 余灏；

【导师】 汪荣泉；

【作者基本信息】 第三军医大学 ， 内科学， 2010， 硕士

【摘要】 背景与目的:研究发现,结肠黏液由两层性质不同的黏液构成。靠近肠腔侧的疏松层可以被轻吸去除,而紧密粘附于肠上皮的致密层需轻刮去除,说明两层黏液在物理性质上存在差异。有意思的是,小鼠结肠石蜡切片的荧光原位杂交(fluorescence in situ hybridization, FISH)和免疫荧光染色显示两层黏液截然不同的细菌分布模式,即疏松层内分布着大量细菌,而致密层内是无菌的,提示两黏液层不同的抗菌特性。导致这种性质差异的分子机制还不清楚。但是蛋白质组学分析表明,疏松层和致密层黏液的蛋白质组成成分是相同的,包括黏液的结构性成分MUC2和Fcγ连接蛋白(Fc gamma binding protein,Fcgbp/ IgGFcγBP)、钙满激活氯离子通道3(chloride channel calcium activated 3, Clca3)等杯状细胞来源的分子均存在于疏松层和致密层黏液内,因此成分的差异不足以解释两层黏液性质差异。在黏液的蛋白质组学研究中,我们没有找到同为杯状细胞来源的肠三叶因子TFF3的信息。三叶因子(trefoil factor family, TFF)据认为是一类黏蛋白相关分子,不同的TFF成员与相应的黏蛋白家族共定位于特异的组织,影响黏蛋白的分泌、稳定及粘弹性。此外,两种分子在黏膜上皮保护方面还具有协同效应。研究表明,rTFF3与IgGFcγBP共同参与肠上皮细胞的保护。在DSS(葡聚糖硫酸钠)诱导的大鼠结肠炎模型中,免疫组化染色显示IgGFcγBP在发病期、活动期和肠上皮再生阶段的表达水平与rTFF3相似。已知rTFF3是参与肠上肠皮快速修复的重要分子,提示IgGFcγBP具有保护肠上皮的作用,其功能可能与TFF3相关。最近,Johansson等人运用蛋白质组学方法,发现小鼠天然结构的MUC2与IgGFcγBP与通过共价键连接,共同参与黏液的形成。另一方面,既有观点认为,上皮黏液内黏蛋白与三叶因子相互作用,从而影响黏液的粘弹性。鉴于此,我们设想通过研究rTFF3在黏液层中有无分布及rTFF3与Muc2、rIgGFcγBP在黏液中的分子存形式,进一步通过免疫共沉淀和Western blot检测方法,分析rTFF3、rMuc2、rIgGFcγBP三种蛋白之间的相互作用关系,寻找与不同黏液层性质差异相关的潜在线索。材料与方法根据文献,我们利用真空泵和载玻片依次对大鼠结肠黏液进行收集,然后用Carnoy固定液对黏液全层及轻吸、轻刮后的结肠黏膜进行固定。通过对石蜡切片进行AB-PAS染色及黏液层形态学变化的观察,我们对大鼠两层黏液收集方法的可行性进行验证;分别用rTFF3、rMuc2和rIgGFcγBP的抗体对大鼠结肠全层进行FITC荧光染色,以此验证rTFF3在黏液中有无分布以及与rMuc2和rIgGFcγBP的定位关系;通过非还原(不加还原剂DTT)或还原(加DTT)SDS-PAGE,我们分别对来源于黏膜疏松层、致密层和黏膜层的黏液蛋白样品进行rTFF3、rMuc2和rIgGFcγBP分子存在形式分析,以期望通过分析不同黏液层内三种蛋白在量或分子存在形式上的差异,探寻它们与不同黏液层性质差异的线索;最后,我们用rTFF3、rMuc2、rIgGFcγBP抗血清和免疫前血清分别对大鼠结肠黏液蛋白样品进行免疫共沉淀,再用rTFF3、rMuc2和rIgGFcγBP抗血清分别对上述四种免疫沉淀样品进行检测,分析上述三种蛋白质之间的作用。结果1.AB-PAS染色显示,完整黏液层与轻吸后的黏液层厚度具有明显差异,轻刮黏液后未见黏液,证明黏液收集方法可行。2.免疫荧光实验表明,rTFF3在黏液层中有分布,与rMuc2和rIgGFcγBP均定位于黏液层及杯状细胞的黏液滴内。3.Western blot结果显示,rTFF3主要以250kD的分子形式分布于疏松层和致密层黏液,其6kD单体见于疏松层、致密层和黏膜层。250kD主要分布于疏松层,致密层内仅有痕量印迹,而黏膜层内没有检测到该分子形式。非还原条件下,rMuc2均以分子量约大于250kD的复合物的形式分布于疏松层、致密层和黏膜层。还原后,疏松层、致密层和黏膜层内均检测到164kD的rMuc2单体,此外,疏松层内还可检测到278kD的rMuc2条带。rIgGFcγBP在疏松层、致密层和黏膜层蛋白样品内均检测到还原前的大于214kD条带,还原后则呈258kD,214kD,140kD的亚单位。4.免疫共沉淀及免疫印迹检测结果表明,rTFF3抗体除检测到其本身的免疫沉淀样品中的6kD条带外,未在rMuc2和rIgGFcγBP免疫沉淀样品中检测到任何蛋白条带;而rMuc2抗血清和rIgGFcγBP抗血清均能在这两种抗血清对应的黏液蛋白免疫沉淀样品中检测到rMuc2和rIgGFcγBP对应的蛋白条带,在rTFF3抗血清对应的免疫沉淀样品中则未能检测到阳性条带。结论rTFF3与MUC2、rIgGFcγBP 3种杯状细胞特异性蛋白在黏液层均有分布。rTFF3主要以通过二硫键形成的复合物形式分布于疏松层黏液,该分子形式可能与不同黏液层的性质有关系;rMUC2、rIgGFcγBP均以复合物的形式存在于两层黏液及黏膜内。免疫共沉淀及Western blot表明,与rTFF3形成复合物的分子不包括rMUC2和rIgGFcγBP,但是rMUC2与rIgGFcγBP之间是连结在一起,与已有的研究结果一致。更多还原

【Abstract】 Background and Purpose:It was revealed that the colon mucus of mice was composed of two layers with different characteristics. The loose layer on the lumen side could be removed by gentle suction, while the inner layer firmly attached to the epithelia was removed by gentle scraping, indicating the different physical properties between the two layers. Interestingly, FISH (fluorescence in situ hybridization) combined with immunofluorescence assay on the paraffin-embed colon sections of mice displayed the absolutely different distribution patterns of bacteria in the two layers. The loose layer was contaminated with bacteria, whereas the firm layer was free of bacteria, implying the different antimicrobial effects of the two layers.The mechanisms associated with the different properties remain unknown. However, proteomic analyses revealed the identity of proteins in both mucus layers. The Muc2 mucin and other goblet cell-derived molecules such as Fc gamma binding protein(Fcgbp/ IgGFcγBP) and chloride channel calcium activated 3 (clca3) were present in both the loose layer and the firm layer. The MUC2 mucin is the structural constituent of intestinal mucus, and IgGFcγBP is postulated to be involved in immune protection of intestinal epithelia, thus the difference of constituents was not capable of explaining the different characteristics of the two mucus layer convincingly. In the result of proteomics analysis of two mucus layers, the information of TFF3 secreted by goblet cell was missing. As we know, TFFs(trefoil factor family) were believed to be a group of mucin-associated molecules, and different members were found to be co-localized with specific mucins in proper tissues, thus affecting the secretion, stability or viscoelasticity of mucins. In addition, the two molecules were involved in protection of epithelia synergistically. It was showed that rTFF3 and IgGFcγBP were involved in the protection of intestinal epithelia. In DSS-induced colitis of rats, It was revealed, with histoimmunochemistry assay, that the expression level of rIgGFcγBP was similar to that of rTFF3 during the onset, active or regeneration phase respectively. It’s known that rTFF3 is an important molecule involved in restitution of epithelia, implying the ability of rIgGFcγBP to protect epithelia and functional association of rIgGFcγBP with rTFF3.Recently, Johansson et al showed that the intact Muc2 was bound to IgGFcγBP with covalent linkage in mice colon mucus. However, it’s believed that mucus covering epithelia interacts with trefoil factor family. Here, we plan to find the potential clues associated with the different properties of the two mucus layers via identification of presence, molecular forms of rTFF3, rMuc2 and rIgGFcγBP. The further study is to analyze the interaction of the three molecules above with Co-immunoprecipitation and Western blot assay.Method and material:A mini vacuum pump and glass slides were sequentially used to collect rat colon mucus samples of different layers according to the document. The intact mucosa, post-suction mucosa or post-scraping mucosa was fixed immediately in Carnoy fixtitive at 4℃for 2 hours. Paraffin-embedded sections were stained with AB-PAS so as to validate the reliability of the methods of sample collection. In order to identify the presence of rTFF3 in mucus and its localization pattern compared with rMuc2 and rIgGFcγBP, the antibodies against the proteins above combined with SABC-FITC were used to detect the corresponding antigens distributed in the intact mucus layer. Non-reducing SDS-PAGE or reducing SDS-PAGE followed by Western blot were used to analyze the molecular forms of rTFF3、rMuc2 or rIgGFcγBP present in loose layer, firm layer or single mucosa respectively so as to find the potential clues relevant to different properties of the two mucus layers by identifying the difference of amount or molecular forms of the three proteins. Anti-rTFF3 serum,anti-rMuc2 serum, anti-rIgGFcγBP serum or preimmune serum were used to co-immunoprecipitate the corresponding antigens presensent in rat colon mucus, and each of the co-immunoprecipitated samples was tested by Anti-rTFF3 serum,anti-rMuc2 serum, anti-rIgGFcγBP serum respectively with immunoblot assay to analyze the interaction of rTFF3, rMuc2 and rIgGFcγBP.Resμlt1. AB-PAS staining showed apparent difference of mucus thickness between the intact mucosa and post-suction mucosa, while no mucus was observed after the scraping of post-suction mucosa. The results proved the reliability of the method of sample collection.2. The Immunofluoresence assay showed localization of rTFF3 in the mucus. It was localized in mucus and granules of goblet cell together with rMuc2 and rIgGFcγBP.3.Western blot assay showed the presence of rTFF3 complex(250kD) in both the loose layer and the firm layer, while the monomer(6kD) was present in the loose layer, the firm layer and the single mucosa layer. Besides, the complex seemed to be localized mainly in the loose layer, whereas the trace of the complex was found in the firm layer. Under non-reducing condition, the complex of rMuc2 with a molecular weight of above 250kD was tested in the samples from the loose layer, firm layer and mucosa. Under reducing condition, such a complex shifted to a subunit of 164kD as well as the 278kD subunit in the loose layer and firm layer. rIgGFcγBP were present in form of complex under non-reducing condition, However, the complex shifted to the subunits of 258kD,214kD,140kD respectively when reduced.4. Western blot combined with co-immunoprecipitation showed that anti-rTFF3 serum failed to test the corresponding antigen in the samples co-immunoprecipitated with anti-rMuc2 serum or anti-rIgGFcBP serum, whereas the antigen was tested in the sample co-immunopricipitated with anti-rTFF3 serum itself. anti-rMuc2 serum or anti-rIgGFcBP serum tested the corresponding antigens in both samples co-immunoprecipitated with anti-rMuc2 serum or anti-rIgGFcBP serum, while failed to tested the corresponding antigens in the sample co-immunopricipitated with anti-rTFF3 serum. All anti-serums above failed to test the corresponding antigens in the sample co-immunoprecipitated with the pre-immune serum.ConclusionThe goblet cell-derived proteins rTFF3, rMuc2 or rIgGFcγBP were all localized in the mucus layer of rat colon. rTFF3 was present in the loose mucus layer in form of complex, which may be associated with the different characteristics of different mucus layers; rMuc2, rIgGFcγBP were present in both mucus layers in form of complex and shifted to subunits when reduced. rIgGFcγBP, but not rTFF3 was shown to interact with rMuc2, which was in line with the previous result based on proteomics analyses.更多还原