外阴阴道念珠菌病的病因研究及体外药敏检测

目的了解福州地区阴道念珠菌病的病因、致病菌的菌种特征。方法采用科玛嘉念珠菌显色培养基和YBC鉴定卡及API-20C AUX酵母菌鉴定系统对门诊就诊661例患者阴道分泌物分离到的332株致病真菌进行鉴定,并采用ROSCO纸片扩散法对4种抗真菌药进行药物敏感检测。结果总感染率为69.45%共分离菌种6种。339株念珠菌,其中白念珠菌为288株占85%,光滑念珠菌23株占6.8%,热带念珠菌12株占3.54%,克柔念珠菌7株占2%,近平滑念珠菌4株占1.18%,光滑假丝酵母菌4株占1.18%,酿酒酵母菌1株占0.29%。结论本地区念珠菌性阴道炎主要的病原菌以白色念珠菌为主,其次是光滑念珠菌、热带念珠菌。     

临床疑诊甲真菌病1036例真菌学分析

目的　了解近 5年本院甲真菌病病原菌的种类和构成 ,观察流行病学特点。方法　对近 5年临床疑诊的10 3 6例甲真菌病患者的真菌学实验室检查情况进行系统分析、总结。结果　共培养出真菌 63 1株 ,其中酵母菌占49.60 % ,以克柔念珠菌、红酵母、近平滑念珠菌为主 ;皮肤癣菌占 2 1.71% ,主要菌种为须癣毛癣菌和红色毛癣菌 ;其他丝状真菌占 19.81% ,主要菌种为曲霉和青霉 ;污染真菌占 8.87%。结论　本院近 5年甲真菌病患者病原菌依次为酵母菌、皮肤癣菌、其他丝状真菌 ,排前 5位的真菌分别是须癣毛癣菌 (11.2 5 % ) ,克柔念珠菌 (10 .14 % ) ,红酵母 (9.98% ) ,红色毛癣菌 (9.5 1% ) ,曲霉 (8.87% )。     

伏立康唑与其他5种抗真菌药体外抗念珠菌活性的比较研究

目的比较伏立康唑与其他5种抗真菌药在体外抗深部致病念珠菌的活性。方法按NCCLS推荐的M27A方案(肉汤微量稀释法)检测伏立康唑等6种抗真菌药对6种共52株深部致病念珠菌临床分离株在体外的抗真菌活性。结果对受试菌株总体而言,伏立康唑的活性最高(MIC90≤0.5μg/ml)。伏立康唑对6种念珠菌的MIC90从小到大依次为白念珠菌、热带念珠菌、近平滑念珠菌、克柔念珠菌、光滑念珠菌和法氏念珠菌。伏立康唑对各受试菌种的MIC90均低于氟康唑和特比萘芬;对白念珠菌、热带念珠菌、光滑念珠菌和近平滑念珠菌的MIC90低于伊曲康唑;对克柔念珠菌和法氏念珠菌的MIC90低于两性霉素B。结论伏立康唑在体外能有效抑制深部致病念珠菌的生长,其抗菌谱较广,尤其对唑类药耐药的两种念珠菌——克柔念珠菌和光滑念珠菌也具有较好的抗真菌活性,部分菌种抗真菌活性甚至优于氟康唑和伊曲康唑。     

不同部位及不同年龄小儿皮肤念珠菌病菌种培养分析

皮肤念珠菌病为儿童皮肤科常见病之一,主要由白念珠菌、近平滑念珠菌、克柔念珠菌、热带念珠菌、星型念珠菌/高里念珠菌等引起,1念珠菌培养及菌种鉴定对临床诊断及抗真菌药物的选用至关重要.     

念珠菌碱溶性β-葡聚糖水解生成葡萄糖含量的分类鉴定意义

目的 :　探讨 8种医学上重要的念珠菌酵母相胞壁碱溶性 β -葡聚糖水解生成的D -葡萄糖占菌体干重含量百分率的分类鉴定意义。方法 :　采用双波长薄层扫描法 ,测定 6 5株 8种念珠菌及 8种酿酒酵母的标准菌株上述含量值 ,并检测 46株念珠菌临床分离株 ,且与标准株结果相比较。结果 :实验结果经Tukey法统计处理 ,可将 9种菌分为 3个同质性亚群 ,亚群 1:酿酒酵母、克柔、季也蒙、光滑、热带念珠菌 (P =0 .0 6 5 ) ,亚群 2 :克柔、季也蒙、光滑、热带、近平滑、伪热带、星形念珠菌 (P =0 .336 ) ,亚群 3:白念珠菌 (P =1.0 0 0 )。此外 ,酿酒酵母与 8种念珠菌经t检验 ,均有显著性差异。临床分离株与标准株均无显著性差异。结论 :　该指标能在属水平区分念珠菌和酿酒酵母 ,可将 8种念珠菌分为 3个亚群 ;且重复性较好。因此似能作为一种念珠菌分类鉴定研究的化学分类法指标。     

山苍子油抗念珠菌的敏感性及作用机理的电镜研究

目的应用美国国家临床试验标准化委员会(NCCLS)推荐的微量法测定山苍子油(Liseacubebaoil)对5种医学重要念珠菌的最小抑菌浓度,并在电镜下观察山苍子油作用后,耐唑类药物的克柔念珠菌(Candidakrusei)超微结构的改变,探讨其作用机制,从而为耐唑类药物的抗念珠菌中药开发提供依据。方法参照NCCLS(NCCLS-M27-T文件)推荐的抗真菌药物敏感试验方案微量稀释法检测5种念珠菌标准菌株,并以氟康唑作为质控药物。电镜下观察山苍子油作用前后克柔念珠菌超微结构的改变。结果山苍子油对5种标准菌株的最小抑菌浓度(MIC)分别为:白念珠菌(14.14±3.64)μg/mL,热带念珠菌(23.22±2.85)μg/mL,光滑念珠菌(31.24±2.88)μg/mL,近平滑念珠菌(76.19±4.40)μg/mL,克柔念珠菌(28.30±2.54)μg/mL。电镜观察发现,氟康唑处理组克柔念珠菌结构完整,其细胞壁、细胞膜结构清晰;山苍子油处理组见克柔念珠菌细胞壁溶解破坏,细胞膜连续性破坏,细胞器肿胀溶解乃至坏死溶解。结论山苍子油不仅对白念珠菌有抗菌作用,对其他致病菌种,特别是耐唑类药物的菌种如克柔念珠菌也有着类似的抗菌作用。山苍子油可能通过破坏克柔念珠菌的细胞壁、细胞膜的结构而抗真菌。     

“酵母鉴定系统”对鉴定医学上重要酵母的评价

作者利用新近市售的“酵母鉴定”(Yeast-IDENT,Y-I)系统对9属医学重要酵母91株进行了评价。该鉴定系统中含有酵母尿素、磷酸对硝基酚等,另以一般鉴定法包括光镜形态学、碳原同化、发酵试验、其他营养试验及(或)API20C 试验等进行对照。结果显示:Y-I 鉴定系统对白念珠菌、伪热带念珠菌,白色隐球菌、新生隐球菌、红色隐球酵母,绿色掷孢酵母、念珠隐球菌菌光滑球拟酵母、二组符合率为100%。对其他菌如克柔念     

93例女性生殖道感染念珠菌菌种分布及药敏结果分析

目的:明确93例女性生殖道念珠菌感染菌种分布及药敏。方法:收集93例女性生殖道感染患者分泌物,采用Sabouraud琼脂培养基进行菌落培养,血清芽管试验及CHROMagar显色培养基进行菌株的筛选,DL-96 FUNGUS念珠菌鉴定系统进行念珠菌鉴定及药敏试验。结果:菌种构成分布依次为白念珠菌81株(87.10%),光滑念珠菌8株(8.60%),都柏林念珠菌、热带念珠菌、克柔念珠菌、乳酒念珠菌各1株(1.08%)。白念珠菌对两性霉素B和5-氟胞嘧啶敏感率为100.00%,其它依次为伏立康唑(93.83%)、氟康唑(85.19%)和伊曲康唑(62.96%)。光滑念珠菌对两性霉素B和5-氟胞嘧啶敏感率为100.00%,其它依次为伏立康唑(87.50%)、氟康唑(62.50%)和伊曲康唑(25.00%)。结论:女性生殖道感染的念珠菌主要菌种是白念珠菌,敏感率最高的药物为两性霉素B和5-氟胞嘧啶。     

性病门诊女性患者外阴阴道念珠菌病的病原学研究

目的了解性病门诊外阴阴道念珠菌病(vulvovaginal candidiasis,VVC)的发病率,致病菌种及体外对抗真菌药物敏感情况.方法对性病门诊1 040例妇女进行VVC发病情况调查,对分离的念珠菌进行菌种鉴定和体外抗真菌药物敏感试验,并同时检查淋球菌(Ng)、解脲脲原体(Uu)、沙眼衣原体(Ct).结果性病门诊女性VVC的患病率为30.77%(320/1040).共检出念珠菌324株,白念珠菌、热带念珠菌、克柔念珠菌、光滑念珠菌、和近平滑念珠菌分别占:95.99%、1.54%、1.54%、0.62%、0.31%.311株白念珠菌对二性霉素B、制霉菌素、酮康唑、咪康唑、氟康唑和伊曲康唑的敏感率依次为100%、100%、92.93%、84.24%、83.60%和82.32%.在混合感染中,在VVC患者中念珠菌合并Uu感染率为35%(112/320),与非VVC组相比有相关性(OR=2.54,x2=38.41,P＜0.05).念珠菌合并Ng、Ct感染率为15.31%(49/320).结论VVC是性病门诊的常见病,白念珠菌仍然是主要致病菌,其次为克柔念珠菌和热带念珠菌.常用抗真菌药物对引起VVC的白念珠菌存在不同程度的耐药率,Uu感染与VVC有相关性.     

DNA芯片鉴定念珠菌种和氟康唑耐药基因ERG11突变

目的 建立DNA芯片技术鉴定念珠菌种和氟康唑耐药基因ERG11突变。方法 根据6种常见念珠菌内转录间隔（ITS2）区种特异性序列和白念珠菌ERG11基因中已证实可导致对氟康唑耐药的6种突变序列设计探针,制备DNA芯片,鉴定12条50 bp的念珠菌种特异性序列和ERG11突变序列及34株念珠菌（其中白念珠菌29株,热带念珠菌、光滑念珠菌、都柏林念珠菌、近平滑念珠菌和克柔念珠菌各1株）。结果 ①芯片正确鉴定12条人工合成序列;②正确鉴定34株试验菌株的菌种;③正确鉴定29株白念珠菌ERG11基因中可致耐药的已知突变。敏感性和特异性均为100%。结论 用DNA芯片进行念珠菌菌种鉴定和白念珠菌ERG11突变筛查,结果可靠。     

致病隐球菌DNA中鸟嘌呤加胞嘧啶摩尔百分比含量的研究

用热变性法测定了致病隐球菌DNA核普酸组成中的硷基比率.结果罗伦隐球菌最低,新型隐球菌最高,新型隐球菌上海变种介于罗伦隐球菌和新型隐球菌格特变种之间.其变异范围为47.82-61.98.用蜗牛酶辅以十二烷基磺酸钠(SLS)破壁,克服了隐球菌壁厚且有夹膜难以提取DNA的问题.     

DNA impulse cytophotometric studies of the modification of the proliferation behavior of the outer hair root sheath by topical glucocorticoid administration

A corticoid preparation (C) was topically applied on the scalps of 47 healthy test persons for 7 or 14 days, resp. After this treatment, we plucked anagen hairs under standardized conditions both from the area treated with C and the contralateral, untreated area. The proliferative activity of plucked anagen hairs with root sheaths was analyzed by means of DNA flow cytometry. Already a 7 days treatment with topical C resulted in kinetic changes of the outer root sheath: The percentage of the cells in the G1/10-phase of the cell cycle was increased, whereas that of the cells in the S-phase and the G2+M-phase was decreased.     

国内首株从病人体内分离的阴道加德纳菌的鉴定

阴道加德纳菌是引起人细菌性,非特异性阴道炎和尿道炎的病原菌之一,患者多为多个性伴者,国内还未见报道。我们从尿道炎患者体内分离到首株阴道加德纳菌,并进行了生长特性、细胞形态、生化反应、抗生素敏感性、G+cMo1%,扫描电镜及菌体脂肪酸气相色谱分析等方面的鉴定。其分解尿素和过氧化氢酶阳性的特点说明该菌可能为一新种。     

IL36RN基因突变与掌跖脓疱病的相关性研究

目的：明确IL36RN基因突变与掌跖脓疱病的相关性。方法：提取96例掌跖脓疱病患者(PPP)与144名健康对照外周血DNA,采用聚合酶链反应(PCR)技术扩增 IL36RN 基因所有外显子及其侧翼序列并进行Sanger测序。结果：96例PPP患者中有3例患者携带c.115+6T>C(p.Arg10ArgX1),3例携带c.140A>G (p.Asn47Ser)。144名对照者中有2名携带c.115+6T>C,4名携带c.140A>G。PPP组与健康对照组IL36RN突变率比较,差异无统计学意义(P=0.67,OR=0.65,95% CI: 0.20~2.09)。结论：PPP与IL36RN基因突变不相关。     

UV fingerprints predominate in the PTCH mutation spectra of basal cell carcinomas independent of clinical phenotype

Basal cell carcinoma (BCC) shows a wide interpatient variation in lesion accrual. To determine whether certain tumorigenic fingerprints and potentially predisposing patched (PTCH) tumor suppressor single-nucleotide polymorphisms (SNPs) are distributed differently among sporadic BCC patients, we compared the PTCH mutation spectra in early-onset BCC (first lesion at age < 35 years), regular BCC (first lesion at age > or = 35 years and < 10 lesions), and multiple BCC (> or = 10 lesions). The PTCH gene was mutated in 29 of 60 cases (48%). Most of the PTCH mutations bore the UV fingerprint (i.e., C --> T or tandem CC --> TT transitions at dipyrimidine sites). However, neither the proportion nor the spectra of exonic PTCH mutations differed significantly among the three groups. A large number of SNPs (IVS10+99C/T, IVS11-51G/C, 1665T/C, 1686C/T, IVS15+9G/C, IVS16-80G/C, IVS17+21G/A, and 3944C/T or its combinations) were also detected, but again their incidence did not differ significantly among the groups. Interestingly, expression of the IVS16-80G/C and the IVS17+21G/A genotype did not achieve the Hardy-Weinberg equilibrium in patients with regular and/or early-onset BCC. These data suggest that a (UV-) mutated PTCH gene is important for sporadic BCC formation independent of clinical phenotype and that the IVS16-80G/C and/or IVS17+21G/A SNP site might be important for tumorigenesis in certain BCC patients.     

Cell kinetic effects of crude coal tar application plus long wave ultraviolet radiation on normal and hyperproliferative epidermis of guinea pig skin

This study investigated the cell kinetic effects of combined treatment with crude coal tar and long wave ultraviolet (UVA) radiation on the normal and n-hexadecane-induced hyperproliferative epidermis of guinea pig skin. Flow cytometry was used to determine the proportion of cells in S phase (S fraction) and G2 + M phase (G2 + M fraction). Bromodeoxyuridine incorporation was used to determine the labeling index. Conventional histologic techniques were used to observe the mitotic index. In the normal epidermis after a single treatment with tar and UVA (1 J/cm2) or tar alone, the labeling index showed an initial decrease of 4 hr duration followed by moderate increase. The initial decrease was more pronounced in the tar-UVA-treated epidermis than in the tar-treated epidermis. The mitotic index was depressed during the first 12 hr. S- and G2 + M fraction showed no changes during the first 12 to 18 hour, and then increased in varying degrees. In the hyperproliferative epidermis after two applications of tar and UVA (1 and 4J/cm2) or tar alone, the labeling index was depressed during the first 12 hr, and mitotic index was below the control level until the 36 hr. The inhibitory effects on DNA synthesis and mitosis were more pronounced in the tar-UVA-treated epidermis than in the tar-treated epidermis. The S- and G2 + M fraction exceeded the control level in varying degrees during the whole experimental period. The results indicate that tar inhibits the epidermal DNA synthesis and mitosis by itself, and that the inhibitory effects of tar are intensified by the radiation of UVA.     

Substance P induces inositol 1,4,5-trisphosphate and intracellular free calcium increase in cultured normal human epidermal keratinocytes

Abstract Substance P is a neuropeptide which is present in peripheral C nerve endings and released from them. Free nerve endings of C nerve are present in human epidermis. The effects of substance P on the transmembrane signaling system of pig epidermal sheets were previously reported. In these studies, a small amount of cells other than keratinocytes contaminated the epidermal sheets and the species difference from human was also noticed. Therefore we investigated the effects of substance P on cultured normal human epidermal keratinocytes. Alteration of intracellular free calcium (Ca²⁺) in single living keratinocytes was studied using an inverted fluorescence microscope and Ca²⁺ -sensitive dye, Fura 2-AM. Treatment of normal human epidermal kertinocytes with substance P resulted in an increase in inositol 1,4,5-trisphosphate and in intracellular Ca²⁺. Substance P inhibited DNA synthesis of the keratinocytes in a dose-dependent manner. These results are consistent with the view that substance P stimulates phosphatidylinositol-4,5-bisphosphate hydrolysis of human keratinocytes, resulting in inositol 1,4,5-trisphosphate-Ca²⁺ signal.     

Selective incorporation and specific cytocidal effect as the cellular basis for the antimelanoma action of sulphur containing tyrosine analogs

Tyrosine analogs are good candidates for developing melanoma chemotherapy because melanogenesis is inherently toxic and uniquely expressed in melanocytic cells. Sulphur containing substrate (tyrosine) analogs, N-acetyl-4-S-cysteaminylphenol (NAcCAP) and N-propionyl-4-S-cysteaminylphenol (NPrCAP), have been shown to have potent antimelanoma activity in mice bearing melanoma. Both NAcCAP and NPrCAP show selective cytotoxicity towards melanoma cell lines. But the mechanism leading to selectivity is not clear as these drugs are also toxic to other cell lines to a lesser extent. Here we show that these drugs have both cytostatic and cytocidal effects, which could account for this. Cytostatic effect is suggested by DNA flow cytometry. The drug causes cell cycle changes in four human cell lines (normal skin fibroblasts, HeLa cells, and melanoma cell lines, C32 and SK-MEL-23) in a dose-dependent manner blocking cells in S phase with concomitant decrease in the number of cells in G1 phase. There is also a gradual decrease in cells in G2 + M phases. The dose-concentration curves give IC50 values in the range of 50-400 microM and the melanotic melanoma cell line SK-MEL-23 has the lowest IC50 value consistent with our hypothesis that these drugs are selective towards melanoma cells. The concentration-dependent accumulation of cells in S phase suggest a cytostatic effect as a consequence of inhibition of DNA synthesis in agreement with [3H] thymidine incorporation assay. There is a highly specific uptake of [14C]NAcCAP and irreversible damage to DNA synthesis machinery in SK-MEL-23 cells, indicating a melanotic-specific cytocidal effect as well. Trypan blue exclusion study and competitive inhibition assay indicated that visible cytocidal effect occurs slowly and oxidative stress resulting from tyrosinase mediated oxidation of the drug appears to be the underlying mechanism. The primary antimelanoma effect of cysteaminylphenols derives from a selective cytostatic effect, but is followed by a specific cytocidal action rendering the drugs useful for targeted melanoma chemotherapy.     

C/EBPα expression is downregulated in human nonmelanoma skin cancers and inactivation of C/EBPα confers susceptibility to UVB-induced skin squamous cell carcinomas

Human epidermis is routinely subjected to DNA damage induced by UVB solar radiation. Cell culture studies have revealed an unexpected role for C/EBPα (CCAAT/enhancer-binding protein-α) in the DNA damage response network, where C/EBPα is induced following UVB DNA damage, regulates the G(1) checkpoint, and diminished or ablated expression of C/EBPα results in G(1) checkpoint failure. In the current study we observed that C/EBPα is induced in normal human epidermal keratinocytes and in the epidermis of human subjects exposed to UVB radiation. The analysis of human skin precancerous and cancerous lesions (47 cases) for C/EBPα expression was conducted. Actinic keratoses, a precancerous benign skin growth and precursor to squamous cell carcinoma (SCC), expressed levels of C/EBPα similar to normal epidermis. Strikingly, all invasive SCCs no longer expressed detectable levels of C/EBPα. To determine the significance of C/EBPα in UVB-induced skin cancer, SKH-1 mice lacking epidermal C/EBPα (CKOα) were exposed to UVB. CKOα mice were highly susceptible to UVB-induced SCCs and exhibited accelerated tumor progression. CKOα mice displayed keratinocyte cell cycle checkpoint failure in vivo in response to UVB that was characterized by abnormal entry of keratinocytes into S phase. Our results demonstrate that C/EBPα is silenced in human SCC and loss of C/EBPα confers susceptibility to UVB-induced skin SCCs involving defective cell cycle arrest in response to UVB.