【作者】 高琪；

【导师】 张金廷；

【作者基本信息】 河北医科大学 ， 口腔临床医学， 2003， 硕士

【摘要】 目的：富组蛋白（HRPs）是一族存在于灵长类动物腮腺和頜下腺分泌物中富含组氨酸的小分子、阳离子多肽。在宿主非免疫防御系统中起重要作用。特别是1984年Pollock等首次报道了HRPs混合物抗口腔白念珠菌的作用，其对念珠菌病的预防作用尤为引人瞩目。迄今为止，Troxler等已从腮腺和頜下腺中分离出至少12种富组蛋白，分别命名为HRPs1-12。HRPs1、3、5是其主要成份，这三种蛋白质之和占富组蛋白总含量的85%-90%，且HRPs5抗白念珠菌作用最强。因此学者们常以HRPs5及其残基片断代表HRPs来研究抗口腔白念珠菌的作用。本实验从利用不同浓度的HRPs5在体外测定其杀灭白念珠菌孢子、芽管的作用及抑制孢子形成芽管的能力三个方面来深入探讨HRPs5抗标准白念珠菌（ATCC22019）的作用,及其抗菌机制。方法：1 将一定数量的标准白念珠菌（ATCC22019）孢子和芽管分别与不同浓度的HRPs5混合，用沙氏培养基培养，置于400倍的倒置显微镜下观察菌落数，得出杀孢子率、杀芽管率和抑制孢子形成芽管率。2 实验组：100μg/ml的HRPs5和对数生长期念珠菌菌悬液（2×10个/ml）各0.1ml混合培养3小时。对照组：10mM的PPB和对数生长期念珠菌菌悬液（2×10个/ml）各0.1ml混合培养3小时。在扫描电镜下分析对照组与实验组菌细胞的变形率有无显著性差异。并且在扫描电镜和透射电镜下观察白念珠菌孢子超微结构的变化。结果：1 HRPs5在体外具有较强杀灭白念珠菌孢子、芽管的作用及抑制孢子形成芽管的作用，且随着HRPs5浓度的升高其杀灭白念珠菌孢子、芽管的作用及抑制孢子形成芽管的作用而增强。其中当25μg/ml或更高浓度的HRPs5和白念珠菌孢子作用后，可使90%以上的孢子丧失活力；100μg/ml的HRPs5和白念珠菌芽管作用后，可使90%以上的孢子丧失活力；400μg/ml的HRPs5和白念珠菌孢子作用后，可使100%孢子受到抑制而不形成芽管；而50μg/ml的HRPs5对<WP=4>白念珠菌孢子形成芽管仅有轻微的抑制作用。2 扫描电镜观察经过HRPs5处理过白念珠菌孢子表面不光滑并有明显的凹陷和穿孔。5000倍扫描电镜下分析对照组与实验组菌细胞的变形率有极显著性差异（P＜0.001）。透射电镜观察经过HRPs5处理过的白念珠菌孢子可见细胞质内有空泡变性等改变。结论：1 HRPs5对标准白念珠菌（ATCC22019）有较强的抗菌活性，且随着HRPs5浓度的升高其抗菌活性增强。在针对白念珠菌的三方面的试验中，HRPs5抑制或杀灭白念珠菌孢子的能力最强；其次为抑制或杀灭白念珠菌芽管的能力；抑制孢子形成芽管的作用最弱。2 电镜观察提示：富组蛋白能破坏白念珠菌的细胞膜，诱导胞内物质丧失，从而导致生物体死亡。更多还原

【Abstract】 Objective: HRPs are a group of micro-molecular cation and histidine-rich polypeptides,present in primate parotid and submandibular secrtions,which play a main part in non-immunity system of host,especially in 1984 Pollock reported the anticandidal activity of the mixture of HRPs for the first time.As the same time their preventive effection to candidal diseasefous attention.Troxler has separated 12 kinds of HRPs from parotid and submandibular secrtions so far named HRPs1-12,respectively.the main compositions are HRPs1.3.5,the sun of which take 85%-90% of HRPs and anticandidal activity of HRPs5 is strongest,so scholars reseach their anticandidal activity with HRPs5 or its residues representing HRPs.The goal of the present investigation was a detail function characterization of these anticandidal activities of HRPs5 at the levels of killing blastoconidia,killing of germinated cell and inhibition of germination and HRPs5 mechanism.Methods:Some of blastoconidia and germination of standard candida albicans(ATCC22019) were mixed with HRPs5 on different concentrations and cultured by<WP=6>Sabouraud dextrose agar.And the determined the percentage of killing blastoconidia,killing of germinated cell and inhibition of germination under inverted microscope at 400×To compare the difference of the percentage of deformation between the test group and contrast group under SEM.As the same time,micro-structure changes of blastoconidia after infected by HRPs5 obzerved with SEM and TEM.Results:HRPs5 has strong function on killing blastoconidia,killing of germinated cell and inhibition of germination,the functions become stronger with rising the levels of concertration.More then 90% blastoconidia have died after infected by 25μg/ml HRPs5 or higher. More then 90% blastoconidia have died after germination infected by 100μg/ml HRPs5 or higher.100% blastoconidia inhibited and coundnt form germination after infected by 400μg/ml HRPs5 or higher.But 50μg/ml HRPs5 has a slight function of inhibiting germination.Blastoconidia infected by HRPs5 hase rough surface with obvious hollow and perforation with SEM.There are significant different between test group and contrast group with SEM of 5000 times(P<0.001).Organell were unclear and vacuoles formed with TEM.Conclusion:HRPs5 has anticandidal activities which stronger with rising the level of concertration,farmore HRPs5，ability inhibiting and killing Blastoconidia is the strongest;secondly,the ability of inhibiting and killing germination;the last is ability of inhibiting germination<WP=7>in the three fields test.Micrographs suggest that HRPs5 damage cell membrane and induce the loss of intracellular materials,leading to death of organism.更多还原