CRK1基因缺失影响白念珠菌与肠粘膜相互作用的研究

发布时间：2018-05-24 07:44

本文选题：白念珠菌 + CRK1基因缺失　；参考：《第二军医大学》2014年硕士论文

【摘要】：白念珠菌是人体常见的条件致病真菌，2010年，美国的一项调查研究表明[1]：念珠菌属已经占了医院获得性系统性感染的第四位，其死亡率上升至50%，其中白念珠菌毒力最强、临床最常见，目前已成为国内外学者的研究热点。众多学者认为[2][3]：人类的胃肠道是白念珠菌的主要储存库，同时也是系统性白念珠菌感染主要来源。当人体抵抗力下降时，如：HIV感染、长期使用广谱抗生素、肿瘤放化疗后，白念珠菌就表现为很强的侵袭力，能穿透肠粘膜屏障、引起系统性白念珠菌感染，常常危及生命[4]。因此，以白念珠菌与肠粘膜上皮相互作用为出发点，研究其致病机制，对预防和治疗肠道来源的系统性白念珠菌感染具有重要意义。早在1996年，上海生物化学研究所陈江野教授及其团队利用寡核苷酸探针技术筛选得到了CRK1基因，它是一种细胞周期蛋白2（CDC2）相关性蛋白激酶。研究发现[5]：Crk1蛋白与酿酒酵母的Sgv1蛋白及人类Pk11/Cdk9蛋白相似，在多种培养条件下，如：YPD培养基、Lee's培养基等，CRK1基因缺失影响到白念珠菌的生长速度和形态发生。因此，我们拟在前人研究的基础上，通过比较CRK1基因缺失前后（即：标准菌SC5314及ΔCRK1菌），菌株在生物被膜的形成、黏附、侵袭以及由此引发的宿主免疫反应等方面的差异，了解CRK1基因缺失对白念珠菌与肠粘膜上皮相互作用的影响，进一步阐明肠道来源的念珠菌病的发病机制。生物被膜的形成常常被认为是白念珠菌重要的毒力因素之一。为比较ΔCRK1菌及标准菌SC5314形成生物被膜的差异，我们通过扫描电镜，从形态学上观察两者之间的差异，ΔCRK1菌：可见大量伸长的菌丝、相互交织、排列紧密并铺满瓶底，但与标准菌SC5314相比，形成的生物被膜较疏松，中央仍依稀可见散在的空隙；而标准菌SC5314：可见大量伸长的菌丝和酵母细胞在细胞外基质中相互交织，排列紧密、呈现多层膜状结构。同时，我们应用MTT法（波长为490nm）及结晶紫法（波长为620nm），通过比较各个波长处吸收峰的大小，评价生物被膜中细胞数目及细胞外高分子物质的多少，从而比较ΔCRK1菌及标准准SC5314形成生物被膜的差异，，吸光度越大，说明形成的生物被膜越好，结果两种方法均表明：ΔCRK1菌形成的生物被膜的吸光度小于标准菌SC5314，两者之间有统计学差异。实验结果说明：CRK1基因缺失不利于白念珠菌生物被膜的形成。 Caco-2细胞是一种来源于人结直肠腺癌细胞株，在普通培养条件下，能分化成小肠上皮细胞单层，具有微绒毛等结构。为研究CRK1基因缺失对白念珠菌黏附于肠粘膜上皮的影响，本实验利用Caco-2细胞体外构建肠粘膜上皮，应用PAS染色（糖原染色）于显微镜下观察白念珠菌与肠粘膜相互作用的不同时间点（30、60、90、120min），ΔCRK1菌及标准菌SC5314黏附于肠粘膜上皮的形态特点；同时，应用菌落计数法比较两者之间的差异，结果表明：随着时间的延长，ΔCRK1菌及标准菌SC5314黏附于肠粘膜的菌落数逐渐增多，均在90min时达到最多，之后逐渐减少，且在60、90min时，SC5314菌的黏附数均多于ΔCRK1菌，两者之间有统计学意义。为进一步研究CRK1基因缺失对白念珠菌穿透肠粘膜的影响，我们通过菌落计数法，比较标准菌SC5314及ΔCRK1菌与肠粘膜相互作用6h、24h时穿透肠粘膜的菌落数，结果表明：当白念珠菌与肠粘膜相互作用6h时，ΔCRK1菌及标准菌SC5314穿透肠粘膜的菌落数均很少，两者之间无统计学差异，而在24h时，ΔCRK1菌穿透肠粘膜的菌落数明显少于标准菌SC5314，两者比较有统计学差异。结果说明：CRK1基因缺失导致白念珠菌穿透肠粘膜的能力下降。当白念珠菌作用于肠粘膜上皮时，会诱发宿主细胞产生一系列的炎症反应，导致一系列细胞因子的释放，因此，为研究CRK1基因缺失导致肠粘膜上皮细胞因子的表达差异，我们采用ELISA方法从蛋白水平对常见的促炎细胞因子（IFN-γ，IL-8，TNF-α）和抗炎细胞因子（IL-10，IL-12，TGF-β）进行分析比较，实验结果表明：与标准菌SC5314相比，ΔCRK1菌作用于宿主细胞后，诱发产生的促炎细胞因子TNF-α、IL-8、IFN-γ的水平相对较高，而对于抗炎细胞因子IL-12，TGF-β、IL-10，则标准菌SC5314多于ΔCRK1菌，两者比较均有统计学差异。促炎细胞因子在病原菌入侵时发挥抗菌作用，使病原菌更易于被宿主抵抗，我们可以推断：相对于标准菌SC5314，ΔCRK1菌易于被宿主抵抗，因而其对宿主的致病力降低。总之，我们从生物被膜的形成、黏附并侵袭肠粘膜上皮以及宿主细胞因子的释放等几个方面,对CRK1基因缺失影响白念珠菌与肠粘膜上皮相互作用进行了研究。我们发现：CRK1基因缺失影响白念珠菌生物被膜的形成，同时使白念珠菌黏附、穿透肠粘膜上皮的能力下降，当ΔCRK1菌作用于宿主细胞后，诱发产生的促炎细胞因子TNF-α、IL-8、IFN-γ的水平相对较高，而对于抗炎细胞因子IL-12，TGF-β、IL-10，相对较少，与标准菌SC5314相比，两者之间有统计学差。结果说明了：由于CDC2家族的蛋白激酶在真核生物周期的调控中起着关键作用，CRK1基因作为CDC2相关的蛋白激酶，可能也参与细胞周期的调控。本研究证实了CRK1基因从多个方面影响了白念珠菌与肠粘膜细胞的相互作用。
[Abstract]:Candida albicans is a common pathogenic fungus in the human body. In 2010, a study in the United States showed that [1]: Candida has accounted for fourth of the hospital acquired systemic infection, and its mortality rate is up to 50%. The virulence of Candida albicans is the strongest and most common in clinic. At present, it has become a hot spot of research at home and abroad. Many scholars believe that the Candida albicans are the most popular. [2][3]: the human gastrointestinal tract is the main repository of Candida albicans. It is also the main source of systemic Candida albicans infection. When human resistance is decreased, such as HIV infection, long spectrum antibiotics are used for a long time. After radiotherapy and chemotherapy of the tumor, Candida albicans is very aggressive and can penetrate the intestinal mucosal barrier and cause systemic Candida albicans. Infection, often endangering life [4]., is the starting point of the interaction between Candida albicans and intestinal mucosa epithelium, and the study of its pathogenesis is of great significance for the prevention and treatment of systemic Candida albicans infection.
As early as 1996, Professor Chen Jiangye of the Shanghai Institute of Biochemistry and his team screened the CRK1 gene by using oligonucleotide probe technique. It was a cyclin 2 (CDC2) related protein kinase. The study found that [5]:Crk1 protein was similar to the Sgv1 protein of Saccharomyces cerevisiae and human Pk11/Cdk9 protein in a variety of culture conditions, such as YPD culture medium, Lee's medium and so on, CRK1 gene deletion affects the growth speed and morphogenesis of Candida albicans. Therefore, on the basis of previous studies, we intend to compare the formation, adhesion, invasion and host immune response of the biofilm by comparison of the CRK1 gene deletion (i.e., standard bacteria SC5314 and Delta CRK1 bacteria) before and after the deletion of the gene (i.e., standard bacteria SC5314 and delta CRK1 bacteria). To understand the effect of CRK1 gene deletion on the interaction between Candida albicans and intestinal mucosal epithelium and further elucidate the pathogenesis of candidiasis in the intestinal tract.
The formation of the biofilm is often considered as one of the important virulence factors of Candida albicans. In order to compare the differences between the delta CRK1 bacteria and the standard bacteria SC5314 to form the biofilm, we observe the differences between the two bacteria by scanning electron microscope, and we can see that a large number of elongated fungi are interwoven, closely arranged and covered with the bottom of the bottle by scanning electron microscope. Compared with the standard bacteria SC5314, the biofilm formed was looser and the center still could not be seen in the gap, while the standard bacteria SC5314: a large number of elongated mycelia and yeast cells interlaced in the extracellular matrix, arranged closely and presented multilayer membrane structure. At the same time, we should use the MTT method (wavelength 490nm) and crystal violet (wavelength 620nm). By comparing the size of the absorption peaks at various wavelengths, the number of cells in the biofilm and the amount of extracellular polymeric substances are evaluated, and the difference between the delta CRK1 bacteria and the standard quasi SC5314 to form the biofilm is compared. The greater the absorbance, the better the formation of the biofilm. The results of the two methods all indicate that the biofilm formed by the delta CRK1 bacteria The absorbance was less than that of the standard SC5314. There was a significant difference between the two. The results showed that the deletion of CRK1 gene was not conducive to the formation of Candida albicans biofilm.
Caco-2 cells, which are derived from human colorectal adenocarcinoma cell lines, can differentiate into small intestinal epithelium monolayers and have microvilli structure under common culture conditions. In order to study the effect of CRK1 gene deletion on the adhesion of Candida albicans to intestinal mucosa epithelium, this experiment uses Caco-2 cells to construct intestinal mucosa in vitro and PAS staining (Tang Yuanran). The different time points (30,60,90120min) of the interaction between Candida albicans and intestinal mucosa were observed under microscope. The morphological characteristics of delta CRK1 bacteria and standard bacteria SC5314 adhered to the intestinal mucosa epithelium. At the same time, the difference between them was compared with the colony counting method. The results showed that, with the prolongation of time, the delta CRK1 bacteria and the standard bacteria SC5314 adhered to the intestines. The number of colonies in the mucous membrane increased gradually and reached the maximum at 90min, and then decreased gradually. At the time of 60,90min, the number of adhesion of SC5314 bacteria was more than that of delta CRK1 bacteria, and there was a statistical significance between them.
In order to further study the effect of CRK1 gene deletion on the penetration of Candida albicans through intestinal mucosa, we compare the interaction of standard bacteria SC5314 and delta CRK1 with intestinal mucosa by colony counting method, and the number of colonies that penetrate the intestinal mucosa at 24h. The results show that when Candida albicans and intestinal mucosa are used to interact with the intestinal mucosa, 6h, Delta CRK1 bacteria and standard bacteria SC5314 penetrate the intestinal mucosa. The number of colonies was very small, and there was no statistical difference between them. At 24h, the number of delta CRK1 bacteria that penetrated the intestinal mucosa was significantly less than that of the standard bacteria SC5314. The results showed that the loss of CRK1 gene led to the decrease of the ability of Candida albicans to penetrate the intestinal mucosa.
When Candida albicans act on intestinal mucosa, it induces a series of inflammatory reactions in the host cells and causes a series of cytokines release. Therefore, in order to study the difference in the expression of intestinal epithelial cytokines caused by the deletion of CRK1 gene, we use the ELISA method from the protein level to the common proinflammatory cytokines (IFN- gamma, IL-8, TNF-). Alpha) and anti-inflammatory cytokines (IL-10, IL-12, TGF- beta) were compared and compared. The experimental results showed that, compared with the standard bacteria SC5314, the level of the pro-inflammatory cytokines TNF- a, IL-8, IFN- gamma induced by the delta CRK1 bacteria acting on the host cells was relatively high, while the standard bacteria SC5314 was more than the Delta, two There are statistical differences. Proinflammatory cytokines play an antibacterial role in the invasion of pathogens and make the pathogen more susceptible to host resistance. We can infer that, relative to the standard bacteria SC5314, Delta CRK1 bacteria are susceptible to host resistance, thus reducing the pathogenicity of the host.
In a word, we have studied the interaction between Candida albicans and intestinal mucosal epithelium from the formation of biofilm, adhesion and invasion of the intestinal mucosa and the release of cytokine in the host. We found that the deletion of CRK1 gene affects the formation of the biofilm of Candida albicans and the adhesion of Candida albicans to CRK1. The ability to penetrate the intestinal mucosal epithelium decreased, and when the delta CRK1 bacteria acted on the host cells, the induced pro-inflammatory cytokines TNF- alpha, IL-8, and IFN- gamma were relatively high, while the anti inflammatory cytokines IL-12, TGF- beta, and IL-10 were relatively less, compared with the standard bacteria SC5314, there were statistical differences between the two. The results were: the eggs of the CDC2 family. White kinase plays a key role in the regulation of the eukaryotic cell cycle. As a protein kinase associated with CDC2, the CRK1 gene may also be involved in the regulation of cell cycle. This study confirms that the CRK1 gene affects the interaction between Candida albicans and intestinal mucosa cells from many aspects.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R519

【共引文献】