甲真菌病中几丁质合酶基因表达的研究

发布时间：2018-03-03 12:46

本文选题：甲真菌病　切入点：几丁质合酶　出处：《安徽医科大学》2016年硕士论文　论文类型：学位论文

【摘要】：目的：甲真菌病(Onychomycosis)是指由皮肤癣菌、酵母菌和条件致病霉菌侵犯甲板和(或)甲下组织引起的一类疾病。国内患病率高,具有一定的传染性,临床治疗困难且系统抗真菌治疗费用高、有一定的副作用。目前甲真菌病的发病机制尚未完全清楚。真菌细胞壁几丁质的合成是一个复杂的过程,其关键酶为几丁质合酶(chitin synthnase,CHS)。在不同的真菌中存在着一种至多种类别的CHS同工酶。这些同工酶在维持细胞壁完整性,菌丝正常生长和无性孢子产生等方面起着重要的作用。在致病真菌中还发现一些CHS基因的缺失会使菌株的生长速率和感染能力同时减弱。由于在动物和植物中不存在几丁质,因此CHS被认为是抗真菌药物的理想靶标。本课题拟选取甲真菌病中最常见的皮肤癣菌致病菌红色毛癣菌,通过建立红色毛癣菌感染甲板的甲真菌病体外模型,采用实时荧光定量PCR技术检测红色毛癣菌几丁质合酶CHS1 mRNA和CHS2 mRNA的表达情况,探讨CHS在真菌感染甲板中的作用,为研究真菌CHS作为抗真菌药物有效靶标及其抑制剂提供参考,且能更好地理解甲真菌病的发病机制,更有效地指导新型抗真菌药物的研制与开发,进一步指导临床相关治疗。方法：1、采用猪甲板构建红色毛癣菌感染的甲真菌病体外模型。2、运用实时荧光定量PCR技术建立几丁质合酶(CHS2 mRNA)的标准品和标准曲线。3、采用上述构建的标准品检测不同时间点(1、2、3、4、5周)红色毛癣菌感染猪甲板后几丁质合酶(CHS1 mRNA、CHS2 mRNA)的表达水平。结果：1、采用猪甲板构建红色毛癣菌感染的甲真菌病体外模型,密封性较好,易于观察红色毛癣菌不同时间点穿透甲板的大体形态。2、相对定量PCR构建几丁质合酶(CHS2 mRNA)的标准品和标准曲线,检测结果显示Total RNA反转录得到的cDNA作为标准品可以得到较好的扩增曲线、熔解曲线和标准曲线,标准曲线显示线性关系良好(管家基因GAPDH:R2=0.999,目的基因CHS2:R2=0.999R20.98),可以在宽广的范围内进行准确定量；扩增效率较好(管家基因GAPDH:E=89.18%,目的基因CHS2：E=96.08%,0.8E1.2)。3、与对照组相比,实验组红色毛癣菌CHS1 mRNA表达在1周前无明显变化(P0.05),但在2周后表达明显增加(P0.05),而CHS2 mRNA在各时间点表达均明显增加(P0.05)。结论：1、甲真菌病体外模型构建良好,经济可行,适用于评估真菌对甲板的致病作用。2、相对定量方法构建的目的基因CHS2与管家基因GAPDH标准曲线呈现良好的线性关系和重复性3、从荧光定量PCR检测目的基因(CHS1 mRNA、CHS2 mRNA)的相对值可以看出,实验组与对照组存在明显差异(P0.05),说明几丁质合酶在红色毛癣菌感染甲板的过程中对甲的致病有重要的作用。
[Abstract]:Objective: Onychomycosisis is a class of diseases caused by dermatophytes, yeasts and opportunistic fungi invading decks and / or subarachnoid tissues. Clinical treatment is difficult and systemic antifungal therapy is expensive and has some side effects. At present, the pathogenesis of onychomycosis is not completely clear. The synthesis of chitin in fungal cell wall is a complicated process. Its key enzyme is chitin synthysm CHS. There is one or more classes of CHS isozymes in different fungi. These isozymes maintain cell wall integrity. Hyphal normal growth and asexual spore production play an important role. In pathogenic fungi, it was also found that the absence of some CHS genes weakens both the growth rate and infection ability of the strain, since chitin does not exist in animals and plants. Therefore, CHS is considered to be an ideal target for antifungal drugs. In this study, the most common dermatophytes, Trichophyton rubrum, which is the most common pathogen in onychomycosis, was selected to establish an in vitro model of onychomycosis on the infection deck of Trichophyton rubrum. The expression of chitinase CHS1 mRNA and CHS2 mRNA in Trichophyton rubrum was detected by real-time fluorescence quantitative PCR technique, and the role of CHS in fungal infection deck was discussed, which provided a reference for the study of fungal CHS as an effective target of antifungal drugs and its inhibitor. And it can better understand the pathogenesis of onychomycosis and guide the research and development of new antifungal drugs more effectively. Methods: 1 was used to construct an in vitro model of onychomycosis infected by Trichophyton rubrum, and real-time fluorescence quantitative PCR was used to establish the standard product and standard curve of chitinase CHS2 mRNA.3. The expression level of chitin synthase CHS1 mRNA-CHS2 mRNAs at different time points was detected at different time points. Results: the in vitro model of onychomycosis infected by Trichophyton rubrum was constructed. It is easy to observe the gross shape of Trichophyton rubrum through deck at different time points. The relative quantitative PCR was used to construct the standard product and standard curve of chitinase ChS 2 mRNAs. The results showed that cDNA obtained by reverse transcription of Total RNA could obtain better amplification curve, melting curve and standard curve. The standard curve showed a good linear relationship (GAPDH: R2N 0.999, target gene CHS2: R2O0.999R20.98), which could be accurately quantified in a wide range, and had a better amplification efficiency (GAPDH: E96.080.8E1.2N. 3, compared with the control group), the standard curve showed a good linear relationship (GAPDH: R2N 0.999, CHS2: R2O0.999R20.98), and the amplification efficiency was better (GAPDH: E96.080.8E1.2k.3.Compared with the control group. The expression of CHS1 mRNA of Trichophyton rubrum in the experimental group did not change significantly before 1 week, but increased significantly after 2 weeks, while the expression of CHS2 mRNA increased significantly at each time point. Conclusion: 1: 1, the in vitro model of onychomycosis is well constructed and feasible. It can be used to evaluate the pathogenicity of fungi on deck. 2. The standard curve of target gene CHS2 and housekeeper gene GAPDH is linear and reproducible 3. The detection of target gene CHS1 mRNA-CHS2 mRNAs from fluorescent quantitative PCR). As you can see from the relative value, There was a significant difference between the experimental group and the control group, indicating that chitin synthase plays an important role in the process of infection deck of Trichophyton rubrum.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R756

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