抑制肠上皮细胞O型糖链的合成减少细菌的粘附及其MUC2表达

发布时间：2018-09-07 15:01

【摘要】：研究背景和目的：肠道在机体中是独特的，从出生开始就被大量的微生物定植，尤其是细菌。粘液作为宿主-细菌相互作用的关键介质，，是管腔微生物与底层的免疫细胞之间的重要屏障。肠道粘液主要成分是粘蛋白2（MUC2），粘蛋白家族的一个重要组成成员。小鼠缺乏Muc2将导致结肠粘液层缺乏，但不会限制细菌附着到粘膜组织，这可能在小鼠的成长中导致严重的自发性结肠炎和结直肠癌。高分子质量的O-连接糖蛋白作为粘蛋白的主要成分，广泛表达于人体各个部位，并在生理和病理过程中扮演着许多重要的角色。粘蛋白型O-glycans主要包含Core1，Core2，Core3和Core4四种不同的类型。Core1O-glycans是主要的O-glycans，在大多数组织中表达。缺失Core1O-glycans严重损坏了小鼠肠道粘液层的形成，并诱发自发性肠道炎症。但是O-glycans如何促进粘液屏障的完整性，以及与肠道菌群相互作用维持肠道内环境的稳态仍不清楚。本研究用苄基-N-乙酰基-α-D-半乳糖胺（benzyl-α-GalNAc）来处理结肠癌上皮细胞HT-29及其分化型细胞HT-29-Gal，观察肠出血性大肠杆菌(enterohemorrhagicEscherichia coli serotvpe O157:H7or EHEC O157:H7)和肠致病性大肠杆菌(enteropathogenic Escherichia coli or EPEC)对肠上皮细胞粘附行为的改变；对HT-29细胞进行Core1合酶（C1GALT1）的RNA干扰，并建立稳定转染的细胞系，为进一步观察Core1合酶抑制的肠上皮细胞与细菌的粘附创造必要的条件。通过研究粘蛋白分子上O-glycans与病原微生物之间的相互作用，帮助我们理解目前患病率日益增加的炎症性肠病的发生机制。方法： 1.将结肠癌上皮细胞HT-29进行半乳糖诱导，得到分化型细胞HT-29-Gal。 2．将HT-29和促分化型HT-29-Gal细胞用benzyl-α-GalNAc处理，得到O型糖链合成抑制的命名为HT-29-OBN和HT-29-Gal-OBN的细胞。 3．采用Western blot和Real-time PCR方法检测肠分化细胞HT-29-Gal和benzyl-α-GalNAc处理得到的HT-29-OBN和HT-29-Gal-OBN细胞中MUC2的表达情况。 4．将HT-29，HT-29-Gal，HT-29-OBN和HT-29-Gal-OBN四种细胞分别与大肠杆菌EHEC O157:H7和EPEC共培养，采用系列稀释菌落计数法和免疫荧光染色法观察细菌在上述四种细胞表面的粘附情况。 5.构建Core1合酶（C1GALT1）的干扰质粒，转染Core1合酶高表达的HT-29细胞，并进行G418筛选。结果： 1. Western blot和Real-time PCR结果显示，相对于HT-29和HT-29-Gal细胞，经benzyl-α-GalNAc处理后的HT-29-OBN和HT-29-Gal-OBN细胞中MUC2的蛋白和mRNA表达水平均明显减低。 2. HT-29-Gal-OBN和HT-29-OBN细胞与大肠杆菌EHEC O157:H7或EPEC的粘附明显少于对应的HT-29-Gal和HT-29细胞。 3.获得稳定转染的C1GALT1沉默的HT-29细胞。结论：抑制肠上皮细胞O型糖链的合成，减少了细胞MUC2的表达及其细菌的粘附。
[Abstract]:Background and objective: the gut is unique in the body and has been colonized by a large number of microorganisms, especially bacteria. As the key medium of host-bacteria interaction, mucus is an important barrier between lumen microbes and underlying immune cells. Intestinal mucus is mainly composed of mucin 2 (MUC2), an important member of mucin family. Lack of Muc2 in mice leads to mucous layer deficiency in colon, but does not restrict bacterial adhesion to mucous tissue, which may lead to severe spontaneous colitis and colorectal cancer in mice. As the main component of mucin, polymer mass O-ligand glycoprotein is widely expressed in various parts of human body and plays many important roles in physiological and pathological processes. Mucin type O-glycans mainly contains four different types of Core1,Core2,Core3 and Core4. Core1O-glycans is the main O-glycans and expressed in most tissues. The absence of Core1O-glycans seriously damaged the formation of mucus layer and induced spontaneous intestinal inflammation in mice. However, it is unclear how O-glycans promotes the integrity of mucus barrier and maintains the homeostasis of intestinal environment by interacting with intestinal flora. In this study, benzyl- 伪 -galactosamine (benzyl- 伪 -GalNAc) was used to treat HT-29 of colon cancer epithelial cells and differentiated HT-29-Gal, cells to observe (enterohemorrhagicEscherichia coli serotvpe O157:H7or EHEC O157:H7 and (enteropathogenic Escherichia coli or EPEC) of enterogenic Escherichia coli. The change of cell adhesion behavior; The RNA interference of Core1 synthase (C1GALT1) was carried out on HT-29 cells and a stable transfected cell line was established to create necessary conditions for further observation of the adhesion of intestinal epithelial cells inhibited by Core1 synthase to bacteria. By studying the interaction between O-glycans and pathogenic microorganisms on mucin molecules, we can understand the pathogenesis of inflammatory bowel disease (IBD), which has a growing prevalence. Methods: 1. HT-29 of colon cancer epithelial cells was induced by galactose to obtain differentiated cells HT-29-Gal. 2. HT-29 and HT-29-Gal cells were treated with benzyl- 伪 -GalNAc. 3. The expression of MUC2 in HT-29-Gal and benzyl- 伪 -GalNAc treated with HT-29-Gal and benzyl- 伪 -GalNAc was detected by Western blot and Real-time PCR methods. 4. Four kinds of cells of HT-29,HT-29-Gal,HT-29-OBN and HT-29-Gal-OBN were co-cultured with Escherichia coli EHEC O157:H7 and EPEC, respectively. A series of dilution colony counting and immunofluorescence staining methods were used to observe the adhesion of bacteria to the above four cell surfaces. 5. 5. The interfering plasmid of Core1 synthase (C1GALT1) was constructed and transfected into HT-29 cells with high expression of Core1 synthase and screened by G418. Results: 1. The results of Western blot and Real-time PCR showed that the expression of MUC2 protein and mRNA in HT-29-OBN and HT-29-Gal-OBN cells treated with benzyl- 伪 -GalNAc was significantly lower than that in HT-29 and HT-29-Gal cells. The adhesion of HT-29-Gal-OBN and HT-29-OBN cells to Escherichia coli EHEC O157:H7 or EPEC was significantly lower than that of corresponding HT-29-Gal and HT-29 cells. Stable transfected C1GALT1 silenced HT-29 cells were obtained. Conclusion: inhibiting the synthesis of O-type sugar chain in intestinal epithelial cells and reducing the expression of MUC2 and the adhesion of bacteria.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R574

【共引文献】