白假丝酵母菌对人脐静脉内皮细胞株ECV304细胞增殖的诱导作用

目的 研究白假丝酵母菌(S.albicans)对人脐静脉内皮细胞株ECV304细胞增殖及细胞周期的影响.方法 体外培养ECV304,实验分为S.albicans上清液组、S.albicans灭活菌液组、上清液和灭活菌液混合组、对照组.采用MTT法、细胞计数法、倒置显微镜及流式细胞术分别观察各组对ECV304细胞增殖及细...     

嗜酸乳杆菌对人舌癌细胞增殖的影响

目的研究嗜酸乳杆菌对人舌癌细胞Tca8113增殖及细胞周期的影响。方法体外培养Tca8113细胞,分别将不同稀释度（原液和4、16倍稀释）的嗜酸乳杆菌上清液、灭活菌液和无细胞提取物与Tca8113细胞共培养,采用倒置显微镜观察细胞形态并行细胞计数,磺酰罗丹明B（SRB）法测定细胞增殖率,流式细胞术分析嗜酸乳杆菌各组分对Tca8113细胞增殖及细胞周期的影响,激光扫描共聚焦显微镜（CLSM）检测细胞内自由基和Ca2+含量。结果嗜酸乳杆菌各组分作用于Tca8113细胞48 h后,在倒置显微镜下观察,细胞由菱形、多角形、铺路石状变为细长形。细胞计数与SRB实验分析：在不同稀释度同一培养时间与不同培养时间同一稀释度培养条件下,嗜酸乳杆菌各组分均可明显抑制Tca8113细胞增殖,抑制力随稀释度增加而降低,随培养时间延长而增强。流式细胞术分析：嗜酸乳杆菌各组分作用Tca8113细胞48 h后,细胞增殖指数降低（P<0.01）。CLSM检测：嗜酸乳杆菌各组分作用Tca8113细胞48 h后细胞内自由基和Ca2+含量均升高（P<0.01）。结论嗜酸乳杆菌代谢产物、灭活菌液、无细胞提取物均可抑制Tca8113细胞增殖,可能与菌体及其代谢产物引起细胞内自由基含量增多、Ca2+超载有关。     

不同清洗方式对义齿白色假丝酵母菌生物膜清除效果的影响

目的比较不同清洁方式对义齿白色假丝酵母菌生物膜的清除能力。方法制作并消毒树脂片,在其表面培养白色假丝酵母菌生物膜,分别以化学(浸泡)、机械(刷洗)和复合(浸泡与刷洗并用)等方式处理覆有白色假丝酵母菌生物膜的树脂片,再将其浸入新鲜培养液进行残留生物膜的恢复培养,分别在0、6、24h后利用菌落计数进行效果评价。结果机械清除效果与牙膏种类密切相关,单纯的化学处理没有明显的清除作用,保丽净义齿清洁片复合处理清除白色假丝酵母菌生物膜的效果最佳,其处理后的树脂片培养24h仍未检出白色假丝酵母菌。结论不同的义齿清洗方法对附着在义齿表面的白色假丝酵母菌生物膜的清除能力有差异。其中,以保丽净义齿清洁片复合效果最好且能够持续地发挥抑菌作用。     

与口腔黏膜病相关的假丝酵母菌代谢组学鉴定的初步研究

目的 分析3种假丝酵母菌的磁共振代谢物图谱,探讨用代谢组学方法快速鉴定口腔黏膜致病菌的可行性。方法 在沙保培养基中分别接种白色假丝酵母菌ATCC 10231、热带假丝酵母菌ATCC 13803和光滑假丝酵母菌ATCC 15126,绘制生长曲线,选择3种菌生长稳定期的培养液检测其磁共振图谱,用主成分分析法进行数据分析。结果 主成分分析法显示：每两种细菌在主成分得分图中有组间分散、组内聚集的特点,可以区分不同的假丝酵母菌。结论 代谢组学分析技术是一种有良好发展前景的口腔细菌快速鉴定技术。     

3种树脂基托试件微生物黏附的研究

目的比较BPS注塑树脂、热凝基托树脂和不碎胶树脂表面的微生物黏附能力。方法将BPS注塑树脂、热凝基托树脂和不碎胶树脂试件进行微生物体外黏附实验,采用菌落形成单位计数法测定血链球菌、黏性放线菌和白色假丝酵母菌黏附量的大小。结果血链球菌和白色假丝酵母菌黏附实验中,培养24、48、168h后,热凝基托树脂组与BPS注塑树脂组和不碎胶树脂组间微生物黏附量差异有统计学意义（P〈0.001）,BPS注塑树脂组和不碎胶树脂组间微生物黏附量差异无统计学意义（P〉0.05）。在黏性放线菌黏附实验中,培养24h时,热凝基托树脂组和BPS注塑树脂组间微生物黏附量差异有统计学意义（P〈0.05） 培养48、168h时,热凝基托树脂组与不碎胶树脂组和BPS注塑树脂组间微生物黏附量差异有统计学意义（P〈0.05）。结论BPS注塑树脂和不碎胶树脂较热凝基托树脂更能减少血链球菌、黏性放线菌和白色假丝酵母菌在其表面的黏附。     

口腔白色假丝酵母菌毒力因子与随机扩增多态性DNA条带的多元回归分析

目的探讨白色假丝酵母菌毒力因子与随机扩增多态性DNA（RAPD）电泳条带之间的关系,构建多元回归模型。方法采用体外法对92株白色假丝酵母菌的细胞外磷脂酶活性、分泌性蛋白酶活性、芽管生成率、对口腔黏膜细胞的黏附力进行检测;并通过RAPD方法进行扩增,电泳后分析扩增条带。对上述毒力因子和电泳条带进行多元回归分析。结果白色假丝酵母菌的细胞外磷脂酶活性与RAPD扩增后350、450、650和1 300 bp 4个条带明显相关（P<0.05）;分泌性蛋白酶活性与350、1 200 bp 2个条带明显相关（P<0.05）;芽管生成率与400、550 bp 2个条带明显相关（P<0.05）。结论白色假丝酵母菌RAPD部分电泳条带与部分毒力因子的强弱有关,其所含基因信息可能参与该菌毒力因子的调节。     

口腔念珠菌病患者口内菌株的检出和药敏性观察

目的通过对健康人和口腔念珠菌病患者口内假丝酵母菌（即念珠菌）株的检测和药物敏感性试验,探讨假丝酵母菌的种类及药敏性,并结合制霉菌素局部疗效的观察,初步探讨最小抑菌浓度（MIC）值与临床疗效的关系,为临床用药提供参考。方法选择61例口腔念珠菌病患者为试验组,43例健康自愿者为对照组,含漱法收集口腔假丝酵母菌标本,采用CHROMagar假丝酵母菌显色培养基对其进行分离鉴定,然后采用NCCLSM27- A微量稀释法测定假丝酵母菌分离株对制霉菌素、酮康唑和氟康唑的MIC值。试验组中选择31例进行制霉菌素治疗,1周后观察临床疗效,并与患者的MIC值作比较。结果①试验组和对照组假丝酵母菌检出率分别为78.69%和30.23%,其中白色假丝酵母菌分别占80.70%和92.31%。②白色假丝酵母菌对氟康唑和酮康唑的MIC值均数间无统计学差异（P>0.05）,但唑类药物的MIC值小于制霉菌素。③白色假丝酵母菌对氟康唑、酮康唑和制霉菌素的敏感率分别为95.65%、80.43%和89.13%,少数菌株存在耐药现象。④制霉菌素局部治疗口腔念珠菌病有效率为87.10%,存在少数MIC值与临床疗效结果不一的病例。结论目前口腔假丝酵母菌感染患者口内菌株的耐药现象并不突出,白色假丝酵母菌对氟康唑、酮康唑、制霉菌素的敏感率均较高;酮康唑和氟康唑MIC值较小,提示临床上用制霉菌素治疗疗效欠佳时可换用唑类药物。MIC值与临床疗效存在一定相关性,但MIC值高低与临床疗效并非完全一致。     

白色假丝酵母菌pACT1-GFP质粒的构建及表达

目的构建能够稳定表达绿色荧光蛋白（GFP）的白色假丝酵母菌菌株,以便对目的基因进行示踪。方法构建pACT1-GFP质粒,以白色假丝酵母菌CAI4菌株作为感受态进行转化培养后,分别观察绿色荧光蛋白在两相型下的表达情况。结果含有pACT1-GFP的白色假丝酵母菌菌株,99%在两相型下均有较高水平的荧光蛋白表达,且荧光强度没有明显差别。结论pACT1-GFP能够在白色假丝酵母菌体内稳定表达。     

分泌型天冬氨酸蛋白酶基因表达差异及其影响因素

分泌型天冬氨酸蛋白酶(SAP)是白色假丝酵母菌最重要的毒力因子之一,由sap基因家族编码.在体外培养和体内外白色假丝酵母菌感染过程中,sap基因家族各成员间存在着表达和调控上的差异.本文就白色假丝酵母菌sap基因家族分子及其结构特点、sap基因表达差异及其影响因素作一综述.     

白色念珠菌对口腔上皮细胞细胞周期的影响

目的:研究白色念珠菌对口腔上皮细胞增殖及细胞周期的影响。从另一种角度证明白色念珠菌感染与癌发展的关系。方法:在培养的口腔上皮细胞(KB细胞)中,加入菌丝相、孢子相的白色念珠菌,共同培养48h,采用流式细胞仪观察白色念珠菌对KB细胞增殖及细胞周期的影响。结果:与对照组相比,菌丝组G0/G1期细胞所占比例降低,S期、G2/M期所占百分比显著升高,反映细胞增殖活力的增殖指数PI增高,差异有显著性(P<0.05)。而孢子组的KB细胞PI值与对照组相比无明显变化,P>0.05。结论:白色念珠菌可引起KB细胞细胞周期的改变;白色念珠菌对KB细胞周期的改变有赖于该菌的毒性及对细胞的感染。     

木糖醇对四型常见念珠菌生长和产酸的影响

目的通过分析念珠菌培养基中OD鲫和pH值变化,探讨木糖醇对念珠菌生长和产酸的影响,为临床念珠菌感染的防治提供实验依据。方法将白色念珠菌、光滑念珠菌、克柔念珠菌、热带念珠菌4型常见念珠菌加入后沙堡弱培养基,记录24、48、72、96、120h的pH值;同时利用木糖醇逐步替代培养基中的葡萄糖,测定不同木糖醇浓度培养基的0D600和pH值。结果白色念珠菌和光滑念珠菌在培养72h时pH值明显降低;热带念珠菌培养96h时pH值明显降低;克柔念珠菌培养基pH值无明显变化,但总体呈下降趋势;4型念珠菌在全木糖醇培养基中的0D600值最低,pH值最高;随着培养基中木糖醇浓度的升高,白色念珠菌和克柔念珠菌pH值呈上升趋势,热带念珠菌和光滑念珠菌培养基pH值随着木糖醇浓度升高而降低。结论木糖醇对念珠菌生长和产酸的有抑制作用,可为临床念珠菌感染的防治提供参考。     

Human laminin-332 degradation by Candida proteinases

Background:  Human laminin-332 (Lm-332) degradation by 12 Candida strains and effects of synthetic proteinase inhibitors [Ilomastat (ILM), EDTA, chemically modified tetracycline-3(CMT-3), CMT-308, synthetic peptide CTT-2, and Pefabloc] were studied.
Materials and methods:  Laminin-332 was incubated with sonicated cell fractions and 10 times concentrated cell-free fractions of reference and clinical strains of C. albicans, C. dubliniensis , C. guilliermondii , C. glabrata, C. krusei , and C. tropicalis . Proteolysis, pH effects, and inhibitors were analyzed by fluorography and zymography.
Results:  Cell fractions of all species except C. guilliermondii and cell-free fractions of C. albicans , and C. dubliniensis showed 20–70 kDa gelatinases at pH 5.0 and 6.0. At pH 7.6, C. glabrata , C. krusei , and C. tropicalis cell fractions and C. tropicalis cell-free fractions showed 55–70 kDa gelatinases. CMT-3, CMT-308, and CTT-2 inhibited Candida gelatinases slightly better than Pefabloc, ILM, and EDTA. No Candida fractions degraded Lm-332 at pH 7.6, but at pH 5.0, 100 kDa bands were generated by cell fractions of C. dubliniensis and C. tropicalis ; C. albicans and C. glabrata clinical strains; and C. guilliermondii reference strain. C. krusei reference strain yielded three 100–130 kDa bands. C. albicans , C. dubliniensis , and C. tropicalis reference and clinical strain's cell-free fractions generated 100 kDa band .
Conclusions:  Laminin-332 degradation is pH-dependent and differences exist between studied Candida strains. Lm-332 degradation can exert functional disturbances on basement membrane integrity, possibly aiding Candida cell invasion into tissues. Certain synthetic matrix metalloproteinase inhibitors (CMTs, CTT) can inhibit Candida proteinases and may be therapeutically useful in future.     

The in vitro proteolytic and saccharolytic activity of Candida species cultured in human saliva

The proteolytic and saccharolytic activity of 4 Candida species was investigated in batch cultures of pooled, human mixed saliva supplemented with glucose. All the Candida species investigated ( Candida albicans, Candida tropicalis, Candida glabrata and Candida krusei ) demonstrated a marked growth in saliva with a concomitant reduction in pH from about 7.5 to 3.3, within 72 h. Isotachophoretic analysis of the culture supernatant revealed the presence of a variety of acid anions of which pyruvate and acetate were the most abundant. Proteolysis of salivary components, evaluated by a biochemical assay and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was exhibited by all 4 Candida species, although there was inter-species variation. Despite the similarity in growth rates, C. tropicalis and C. krusei demonstrated greater proteolytic activity than C. albicans and C. glabrata. Neither candidal growth nor proteolysis was observed in glucose-free control saliva samples. In contrast, the degree of saccharolytic and proteolytic activity of a single isolate of C. albicans in glucose-supplemented parotid saliva appeared to be relatively weak compared with mixed saliva. As the oral cavity provides ideal low pH niches periodically supplemented with dietary carbohydrates, the acidic proteinases of Candida species may play a role in the pathogenesis of oral candidiasis.     

The in vitro post-antifungal effect of nystatin on Candida species of oral origin

The post-antifungal effect (PAFE) is defined as the suppression of growth that persists following limited exposure of yeasts to antimycotics and subsequent removal of the drug. Although limited data are available on the PAFE of nystatin on oral isolates of C albicans, there is no information on non-albicans Candida species. As nystatin is the commonest antifungal agent prescribed in dentistry, the main aim of this investigation was to measure the PAFE of oral isolates of Candida belonging to six different species (five isolates each of C. albicans, C. tropicalis, C. krusei, C. parapsilosis, C. glabrata and C. guilliermondii) following limited exposure (1 h) to nystatin. The yeasts were examined for the presence of the PAFE after 1 h exposure to the minimum inhibitory concentration (MIC) of nystatin. The PAFE was determined as the difference in time (h) required for the growth of the drug-free control and the drug-exposed test cultures to increase to the 0.05 absorbance level following removal of the antifungal agent. The mean duration of nystatin-elicited PAFE was lowest for C. albicans (6.85 h) and greatest for C. parapsilosis (15.17 h), while C. krusei (11.58 h), C. tropicalis (12.73 h), C. glabrata (8.51 h), and C. guilliermondii (8.68 h) elicited intermediate values. These findings clarify another intriguing possibility for the persistent, chronic recurrence of oral C. albicans infections despite apparently adequate antifungal drug regimens. The significant variations in nystatin-induced PAFE amongst non-albicans species may also have clinical implications, in terms of nystatin regimens used in the management of these fungal infections.     

Susceptibility of oral Candida species to calcium hydroxide in vitro.

AIM: The susceptibility of common oral Candida species to saturated aqueous calcium hydroxide solution was studied. METHODOLOGY: The yeast species tested were C. albicans (16 strains). C. glabrata (three strains), C. guilliermondii (three strains), C. krusei (two strains), and C. tropicalis (two strains). At least one reference strain of each species was used; the others were clinical isolates either from persistent apical periodontitis or from marginal periodontitis. The susceptibility of Enterococcus faecalis (ATCC 29212) was studied for comparative purposes. Standardized inocula of the strains were incubated in aqueous calcium hydroxide solution, pH 12.4, for time-periods ranging from 5 min to 6 h. Volumes of 0.1 mL of the test suspension were cultured directly on Brucella blood agar and incubated in air at 37C. The plates were inspected for growth at 24 and 48 h and the colonies were counted. The time required to reduce the number of colony-forming units to less than 0.1% of the initial number was determined for each strain. RESULTS: The sensitivity of the C. albicans strains was generally low, with 16 h of incubation required to kill 99.9% of the colony-forming units. No differences in susceptibility between C. albicans strains isolated from root-canal infections and from periodontitis were found. Both strains of C. tropicalis were killed between 3 and 6 h of incubation, whilst strains of C. guilliermondii were killed after only 1020 min of incubation. All strains of C. glabrata survived 20 min, but not 1 h, of incubation, whilst 13 h were required to kill C. krusei. Compared with E. faecalis, all Candida spp. showed either equally high or higher resistance to aqueous calcium hydroxide. CONCLUSIONS: This study indicates that Candida spp. are resistant to calcium hydroxide in vitro, which may explain the isolation of yeasts from cases of persistent apical periodontitis.     

平阳霉素对人脐静脉内皮细胞系ECV304的抑制作用及其机制的实验研究

目的：探讨平阳霉素对人脐静脉内皮细胞系ECV304的抑制作用及其相关机制。方法：用不同浓度的平阳霉素作用ECV304细胞不同时间,以MTT法比较平阳霉素作用24、48及72h后的细胞抑制率,应用DNA电泳实验、流式细胞术分析细胞凋亡、坏死情况、细胞周期及Fas、Bcl-2蛋白的表达。结果：MTT法显示随平阳霉素浓度升高和作用时间的延长,细胞生长抑制越明显DNA电泳证实合适浓度平阳霉素作用下细胞发生凋亡,过高浓度细胞则发生坏死;流式细胞术显示细胞凋亡率和坏死率随药物浓度和作用时间增加而增高,过高浓度细胞坏死占主导;平阳霉素各浓度作用24h后,G2—M期百分率增高,S期百分率降低,G0—G1期百分率无明显变化;Fas表达随药物浓度增加而递减,Bcl-2表达随药物浓度增加而升高。结论：平阳霉素能明显抑制ECV304细胞生长,并具有浓度和时间依赖性;平阳霉素可能通过诱导凋亡和坏死作用以抑制ECV304细胞的体外生长和增殖;细胞凋亡发生在G2—M期,是通过上调Fas蛋白并下调Bcl-2蛋白的表达来实现的。     

In vitro antifungal effect of amine fluoride-stannous fluoride combination on oral Candida species

OBJECTIVE: The combination of amine fluoride and stannous fluoride (AmF/SnF2) was, by chance, found to be antifungal in a clinical trial. This study investigated its effect on pathogenic Candida species with the hypothesis that the antifungal action on different species is variable. MATERIALS AND METHODS: Growth inhibition effect of Meridol mouth rinse which contains 250 ppm AmF/SnF2 was evaluated on 43 reference and clinical strains of Candida albicans, C. dubliniensis, C. glabrata, C. guilliermondii, C. krusei, C. parapsilosis, and C. tropicalis. Meridol base solution without AmF/SnF2 was used as a negative control. RESULTS: Undiluted Meridolmouth rinse killed most study strains within a few minutes. In ascending order, C. parapsilosis, C. tropicalis, C. albicans, C. glabrata, C. krusei and C. dubliniensis showed higher resistance against AmF/SnF2 than C. guilliermondii. CONCLUSION: AmF/SnF2 could be used as a potent adjunct to antifungal therapy for oral yeasts. Although different Candida species demonstrated variable sensitivity the most prevalent oral yeast C. albicans appeared sensitive to the AmF/SnF2 combination.     

Identification of oral yeasts by polyacrylamide gel electrophoresis

Protein profiles of sonicated cells for 9 species of yeasts isolated from oral samples were obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and visualized with a silver stain. The profiles obtained on 12% acrylamide gels were distinct, and characteristic for 6 species of Candida , and Torulopsis glabrata, Yarrowia lipolytica , and Saccharomyces cerevisiae. Using this method, 79 fresh isolates of yeasts from saliva samples were identified; 58 as Candida albicans , 9 as Candida parapsilosis , 1 as Candida tropicalis , and 11 as T. glabrata. SDS-PAGE offers a rapid, convenient alternative or adjunct to yeast identification systems based on carbohydrate assimilation tests.     

Prevalence of Candida species in necrotic pulp with chronic periapical processes

The aim of this study was to identify species of the genus Candida in mucosa of oral cavity and in single-rooted teeth with pulp necrosis with chronic endodontic periapical processes, with radiographic images 2+/-4 mm and without clinical symptomatology, in immunocompetent patients. The study included 82 immunocompetent patients of both sexes aged 18-70 years with a clinical dental diagnosis of septic pulp necrosis. Samples were taken from root canals with sterile # 25 paper points and from oral mucosa with a sterile swab. Seven different Candida species were identified (C. albicans, C. dubliniensis, C. guilliermondii, C. krusei, C. parapsilopsis, C. tropicalis and C. glabrata). All of them were present in oral mucosa, while two of them (C. parapsilopsis and C. glabrata) were not identified in the periapical zone of necrotic canals. Considering all the samples isolated from oral mucosa, there was a significantly greater frequency of C. albicans than there was in the periapical zone of necrotic canals.