本文選題：白念珠菌 + CRK1基因缺失　； 參考：《第二軍醫(yī)大學》2014年碩士論文

【摘要】：白念珠菌是人體常見的條件致病真菌，2010年，美國的一項調(diào)查研究表明[1]：念珠菌屬已經(jīng)占了醫(yī)院獲得性系統(tǒng)性感染的第四位，其死亡率上升至50%，其中白念珠菌毒力最強、臨床最常見，目前已成為國內(nèi)外學者的研究熱點。眾多學者認為[2][3]：人類的胃腸道是白念珠菌的主要儲存庫，同時也是系統(tǒng)性白念珠菌感染主要來源。當人體抵抗力下降時，如：HIV感染、長期使用廣譜抗生素、腫瘤放化療后，白念珠菌就表現(xiàn)為很強的侵襲力，能穿透腸粘膜屏障、引起系統(tǒng)性白念珠菌感染，常常危及生命[4]。因此，以白念珠菌與腸粘膜上皮相互作用為出發(fā)點，研究其致病機制，對預防和治療腸道來源的系統(tǒng)性白念珠菌感染具有重要意義。 早在1996年，上海生物化學研究所陳江野教授及其團隊利用寡核苷酸探針技術篩選得到了CRK1基因，它是一種細胞周期蛋白2（CDC2）相關性蛋白激酶。研究發(fā)現(xiàn)[5]：Crk1蛋白與釀酒酵母的Sgv1蛋白及人類Pk11/Cdk9蛋白相似，在多種培養(yǎng)條件下，如：YPD培養(yǎng)基、Lee's培養(yǎng)基等，CRK1基因缺失影響到白念珠菌的生長速度和形態(tài)發(fā)生。因此，我們擬在前人研究的基礎上，通過比較CRK1基因缺失前后（即：標準菌SC5314及ΔCRK1菌），菌株在生物被膜的形成、黏附、侵襲以及由此引發(fā)的宿主免疫反應等方面的差異，了解CRK1基因缺失對白念珠菌與腸粘膜上皮相互作用的影響，進一步闡明腸道來源的念珠菌病的發(fā)病機制。 生物被膜的形成常常被認為是白念珠菌重要的毒力因素之一。為比較ΔCRK1菌及標準菌SC5314形成生物被膜的差異，我們通過掃描電鏡，從形態(tài)學上觀察兩者之間的差異，ΔCRK1菌：可見大量伸長的菌絲、相互交織、排列緊密并鋪滿瓶底，但與標準菌SC5314相比，形成的生物被膜較疏松，中央仍依稀可見散在的空隙；而標準菌SC5314：可見大量伸長的菌絲和酵母細胞在細胞外基質(zhì)中相互交織，排列緊密、呈現(xiàn)多層膜狀結(jié)構(gòu)。同時，我們應用MTT法（波長為490nm）及結(jié)晶紫法（波長為620nm），通過比較各個波長處吸收峰的大小，評價生物被膜中細胞數(shù)目及細胞外高分子物質(zhì)的多少，從而比較ΔCRK1菌及標準準SC5314形成生物被膜的差異，，吸光度越大，說明形成的生物被膜越好，結(jié)果兩種方法均表明：ΔCRK1菌形成的生物被膜的吸光度小于標準菌SC5314，兩者之間有統(tǒng)計學差異。實驗結(jié)果說明：CRK1基因缺失不利于白念珠菌生物被膜的形成。 Caco-2細胞是一種來源于人結(jié)直腸腺癌細胞株，在普通培養(yǎng)條件下，能分化成小腸上皮細胞單層，具有微絨毛等結(jié)構(gòu)。為研究CRK1基因缺失對白念珠菌黏附于腸粘膜上皮的影響，本實驗利用Caco-2細胞體外構(gòu)建腸粘膜上皮，應用PAS染色（糖原染色）于顯微鏡下觀察白念珠菌與腸粘膜相互作用的不同時間點（30、60、90、120min），ΔCRK1菌及標準菌SC5314黏附于腸粘膜上皮的形態(tài)特點；同時，應用菌落計數(shù)法比較兩者之間的差異，結(jié)果表明：隨著時間的延長，ΔCRK1菌及標準菌SC5314黏附于腸粘膜的菌落數(shù)逐漸增多，均在90min時達到最多，之后逐漸減少，且在60、90min時，SC5314菌的黏附數(shù)均多于ΔCRK1菌，兩者之間有統(tǒng)計學意義。 為進一步研究CRK1基因缺失對白念珠菌穿透腸粘膜的影響，我們通過菌落計數(shù)法，比較標準菌SC5314及ΔCRK1菌與腸粘膜相互作用6h、24h時穿透腸粘膜的菌落數(shù)，結(jié)果表明：當白念珠菌與腸粘膜相互作用6h時，ΔCRK1菌及標準菌SC5314穿透腸粘膜的菌落數(shù)均很少，兩者之間無統(tǒng)計學差異，而在24h時，ΔCRK1菌穿透腸粘膜的菌落數(shù)明顯少于標準菌SC5314，兩者比較有統(tǒng)計學差異。結(jié)果說明：CRK1基因缺失導致白念珠菌穿透腸粘膜的能力下降。 當白念珠菌作用于腸粘膜上皮時，會誘發(fā)宿主細胞產(chǎn)生一系列的炎癥反應，導致一系列細胞因子的釋放，因此，為研究CRK1基因缺失導致腸粘膜上皮細胞因子的表達差異，我們采用ELISA方法從蛋白水平對常見的促炎細胞因子（IFN-γ，IL-8，TNF-α）和抗炎細胞因子（IL-10，IL-12，TGF-β）進行分析比較，實驗結(jié)果表明：與標準菌SC5314相比，ΔCRK1菌作用于宿主細胞后，誘發(fā)產(chǎn)生的促炎細胞因子TNF-α、IL-8、IFN-γ的水平相對較高，而對于抗炎細胞因子IL-12，TGF-β、IL-10，則標準菌SC5314多于ΔCRK1菌，兩者比較均有統(tǒng)計學差異。促炎細胞因子在病原菌入侵時發(fā)揮抗菌作用，使病原菌更易于被宿主抵抗，我們可以推斷：相對于標準菌SC5314，ΔCRK1菌易于被宿主抵抗，因而其對宿主的致病力降低。 總之，我們從生物被膜的形成、黏附并侵襲腸粘膜上皮以及宿主細胞因子的釋放等幾個方面,對CRK1基因缺失影響白念珠菌與腸粘膜上皮相互作用進行了研究。我們發(fā)現(xiàn)：CRK1基因缺失影響白念珠菌生物被膜的形成，同時使白念珠菌黏附、穿透腸粘膜上皮的能力下降，當ΔCRK1菌作用于宿主細胞后，誘發(fā)產(chǎn)生的促炎細胞因子TNF-α、IL-8、IFN-γ的水平相對較高，而對于抗炎細胞因子IL-12，TGF-β、IL-10，相對較少，與標準菌SC5314相比，兩者之間有統(tǒng)計學差。結(jié)果說明了：由于CDC2家族的蛋白激酶在真核生物周期的調(diào)控中起著關鍵作用，CRK1基因作為CDC2相關的蛋白激酶，可能也參與細胞周期的調(diào)控。本研究證實了CRK1基因從多個方面影響了白念珠菌與腸粘膜細胞的相互作用。
[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.
【學位授予單位】：第二軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R519

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