Objective:To investigate the physiological role of F1Fo-ATP synthase α-subunit encoding gene (ATP1) in promoting Candida albicans ( C. albicans) to escape from macrophage killing through eliminating intracellular reactive oxygen species (ROS) by using a reverse genetics approach. Methods:An ATP1 deletion strain and a parental strain of C. albicans were cultured on the YPD media, and the number of formed colonies on the plates was counted to evaluate in vitro viability of C. albicans. To evaluate their in vivo viability, the ATP1 deletion strain and parental strain of C. albicans were inoculated into mice through the caudal vein, kidney tissues were taken out from the mice 1-7 days after the infection, and inoculated onto the YPD medium followed by numeration of colonies after 48 hours of culture. After co-culture of overnight-cultured C. albicans suspensions with macrophages, some of the C. albicans suspensions were inoculated onto the YPD solid medium followed by numeration of colonies and determination of survival rate, and some culture supernatants were inoculated into the 96-well plate for detection of the level of lactate dehydrogenase (LDH) released by macrophages by LDH release assay. A model mimicking oxidative stress in macrophages was established by using hydrogen peroxide. After treatment with hydrogen peroxide, the number of colonies was counted to compare the viability of the C. albicans strains. DCFH-DA staining was conducted to detect the intracellular ROS level in C. albicans after co-culture with macrophages or treatment with hydrogen peroxide, and real-time fluorescence-based quantitative PCR to measure mRNA expression of catalase 1 (CAT1) , superoxide dismutase 4 (SOD4) and SOD5 genes in C. albicans after treatment with hydrogen peroxide. Statistical analysis was carried out by using two-way analysis of variance or Student t test. Results:In vitro, the colony number in both the parental strain group and ATP1 deletion strain group gradually increased over time; after 24 hours, the colony number of the ATP1 deletion strain group was only 10% of that in the parental strain group ( F = 481.84, P < 0.001) . The number of colony formed by the parental strain-infected mouse kidney tissues gradually increased over time, but that by the ATP1 deletion strain-infected mouse kidney tissues gradually decreased, and there was a significant difference between the two groups ( F = 78.27, P = 0.001) . After in vitro co-culture of C. albicans with macrophages, the survival rate in the ATP1 deletion strain group (62.67% ± 3.51%) was significantly lower than that in the parental strain group (82.33% ± 2.52%, t = 7.88, P = 0.001) , and the percentage of LDH released by macrophages was also significantly lower in the ATP1 deletion strain group (27.80% ± 3.54%) than in the parental strain group (87.78% ± 0.17%, t = 33.89, P < 0.001) , which were consistent with the in vivo results. In the model mimicking oxidative stress, the viability of the ATP1 deletion strain group was significantly lower than that of the parental strain group ( F = 3 440.65, P < 0.001) . Both in the co-culture model with macrophages and in the model mimicking oxidative stress in macrophages, the intracellular ROS levels were significantly higher in the ATP1 deletion strain group than in the parental strain group (both P < 0.001) . Furthermore, the mRNA expression of CAT1, SOD4 and SOD5 genes was significantly lower in the ATP1 deletion strain group than in the parental strain group after treatment with hydrogen peroxide (all P < 0.001) . Conclusion:ATP1 deletion may reduce the capabilities of C. albicans to counteract oxidative stress and eliminate ROS, likely by down-regulating the expression of oxidative stress- and ROS clearance-related genes respectively, which may prevent C. albicans from escaping from the macrophage killing and lead it to be eliminated by the host ultimately.

To investigate the effect of CDR1/CDR2 or MDR1 genes overexpression on oxidative stress in Candida albicans,we evaluated the effect of H2O2 on cell viability in C.albicans overexpressing genes CDR1 /CDR2 or MDR1 and their parent strains.After establishing an oxidative stress model with H2O2,we detected reactive oxygen species (ROS),mitochondrial membrane potential (Δψm) and the expression of oxidative stress response-related genes (CAP1 and GRP2) and ROS clearance related-genes (SOD2 and SOD5).The results showed that C.albicans growth were inhibited by 100％ after the treatment of 5 mmol/L H2O2.HeO2 caused more ROS accumulation and Δψm reduction in parent strains than in CDR1/CDR2 or MDR1 genes overexpressed strains (P＜0.05).Compared to parent strains,the up-regulated expression of CAP1 and GRP2 were relatively less in CDR1/CDR2 or MDR1 genes overexpressed strains,moreover,the down-regulated expression of SOD2 and SOD5 were also relatively less in CDR1/CDR2 or MDR1 genes overexpressed strains (P＜0.05).In conclusion,the overexpression of CDR1/CDR2 and MDR1 genes could reduce the oxidative stress response and enhance the adaptability of C.albicans to oxidative stress.

Objective To investigate the differences in the expression of Candida albicans SRB1 in Candida albicans fluconazol resistant strain and sensitive strain,and to explore the relationship between SRB1 and drug resistance in Candida albicans.Methods Two Candida albicans strains were from a same parent.We used real-time RT-PCR to observe the expression of the target genes of SRB1,CDR1,ERG11 in Candida albicans fluconazole resistant strain CA-16 and sensitive strain CA-3.Result Compared with CA-3,the expression of the mRNA of SRB1 and drug resistance gene CDR1,ERG11 in CA-16 increased.There was significant difference in the expression levels of SRR1 、CDR1 and ERG11 (P ＜0.05).Conclusion The increased expression of SRB1 and the Candida albicans resistance to flueonazole was closely related,and SRB1 might be a new candidate genes associated with drug resistance.

目的 探讨汉防己甲素(tetrandrine,TET)对伊曲康唑(itraconazole,ITR)、伏立康唑(voriconazole,VRC)和泊沙康唑(posaconazole,PCZ)体外抗烟曲霉有无增效活性.方法 参照MS8-A2方案的微量稀释法,以23株烟曲霉临床株为研究对象,测定TET对其的最大非细胞毒性剂量和ITC、VRC、PCZ单独及联合TET对其的最小抑菌浓度(minimal inhibitoryconcentrations,MICs).结果 TET抗烟曲霉的最大非细胞毒性剂量为80 μg/mL.ITC单独及联合TET对烟曲霉的MIC值范围分别为0.5 μg/mL～32μg/mL和0.0625 μg/mL～4 μg/mL,差异有统计学意义(P＜0.05);VRC单独及联合TET的MIC值范围分别为0.25 μg/mL～2 μg/mL和0.03125 μg/mL～0.25 μg/mL,差异有统计学意义(P＜0.05); PCZ单独及联合TET的MIC值范围分别为0.125 μg/mL～1 μg/mL和0.03125 μg/mL～0.25 μg/mL,差异有统计学意义(P＜0.05).结论 汉防己甲素在体外对伊曲康唑、伏立康唑和泊沙康唑抗烟曲霉有增效活性.

Objective To evaluate the synergistic effect of tetrandrine (TET) with voriconazole (VRC) against Aspergillus fumigatus.Methods A checkerboard microdilution method was used to estimate the activity of TET combined with VRC against 21 clinical isolates of AspergilIus fumigatus according to the Clinical and Laboratory Standards Institute (CLSI) M38-A2 document.The minimal inhibitory concentration (MIC) was determined using spectrophotometry.Fractional inhibitory concentration index (FICI) was calculated to evaluate the interaction between the two antifungal agents.The isobologram method was used to verify the test results with the Aspergillus fumigatus strain CMCC (F) A.la as the representative strain.Results The MICs of TET and VRC against these clinical isolates were 512 μg/ml and 0.125-2 μg/rnl,respectively when used alone,8-128 μg/ml and 0.03-0.5 μg/ml respectively with clear end points accompanied by disappearance of the so-called trailing phenomenon when used in combination.The FICI of these agents ranged from 0.13 to 0.63.The interaction between TET and VRC was synergistic in 16 clinical isolates,but indifferent in the remaining strains.Moreover,the isobologram analysis revealed concaves with a shape characteristic of synergy.Conclusion TET exerts a synergistic effect with VRC against Aspergillus fumigatus. Key words: Tetrandrine; Voriconazoles; Aspergillus fumigates; Synergistic effect; Checkerboard microdilution method

国内外学者将改良CLSI M38-A方案应用于红色毛癣菌时进行了改进。这些改进包括红色毛癣菌培养基为燕麦培养基、马铃薯葡萄糖琼脂(PDA)、沙堡弱葡萄糖琼脂(SDA),接种物仅含小分生孢子,培养基为RPMI1640,接种物量为CFU/ml,培养温度为28℃,培养时间为7 d,MICs终点确定标准为MIC-0,质控菌株选择须癣毛癣菌MRL1957和红色毛癣菌MRL666。

To investigate the synergistic effect of tetrandrine (TET) to fluconazole (FLC) in vivo, animal model for vaginal candidiasis was established in mice by intravaginal inoculation of C. albicans. The infected mice were separated into 10 groups randomly. The mice of the groups given drugs were treated with FLC and TET alone or simultaneously at different doses once daily for 7 consecutive days, started at day 3 after inoculation. while the control groups were not given any drugs. Both of TET and FLC were administered intravaginally at 26 mg/(kg·d) as high dose or 13 mg/(kg·d) as low dose. The synergistic efficacy of TET was assessed by colony forming units(CFU) counts, microscopic examination of the vaginal lavage fluid and histopathological examination of vagina at day 2, 6, 11 after inoculation. On the day 11 after inoculation, compared with the group given FLC at 26 mg/(kg·d) alone, in the group treated with TET at 26 mg/kg and FLC at 26 mg/kg simultaneously (FLC26+ TET26 group), the number of the CFU decreased significantly (2.11±0.36×104 vs 0.19±0.04×104 CFU/ml, P=0.039). Compared with the other 9 groups, the microscopic examination of the vaginal lavage fluid indicated none of the pseudohyphae was shown in the FLC26+ TET26 group, the pseudohypha grades in the 10 groups had statistic difference (χ2=33.084,P=0.000). The histopathologic findings illustrated vaginal mucosal edema was slighter, the infiltrated inflammatory cells were less and none of pseudohyphae was seen in vaginal canal and vaginal mucosa in this group. However, in the other 9 groups, the microscopic examination showed some pseudohyphae or pseudohypha clumps, the histopathologic findings illustrated vaginal mucosal edema was severe with abundant inflammatory cells infiltrated into vaginal mucosa and submucosa. A number of pseudohyphae exsited in vaginal canal and vaginal mucosa. It is evident that TET given to mice at 26mg/(kg·d) intravaginally can improve the anti-C. albicans activity of FLC in vivo.

Objective To explore the feasibility and superiority of using Proseal laryngeal mask airway(PLMA)in gynecological emergencies.Methods Twenty ASAI-II patients with emergencies of ectopic pregnancy aged 22~32 years undergoing laparoscopy were divided into two groups of ten each:Proseal laryngeal mask airway group(L)and endotracheal tube group(E).The patients in group L were conducted of PLMA insertion,while those in group E were performed endotracheal intubation with tracheal tube after induction of anesthesia.All the patients were performed controlled-ventilation.SBP,DBP,HR,SpO2,Ppeak,PETCO2,VT,PaCO2 were monitored at seven time-points:before anesthesia(T0),before insertion of tracheal tube and PLMA(T1),at once after insertion of tracheal tube and PLMA(T2),5min(T3),20min(T4)after insertion,extubation(T5)and 5min after extubation(T6).The parameters measured included cases of difficult insertion or intubation,time of analepsia,cases of celostomia and cough.Results Ppeak、VT,SpO2,PETCO2 and PaCO2 remained unchanged before,during and after surgery in both groups(p0.05).BP and HR at T0 and T1 time points ware similar.BP and HR at T2 and T5 time points increased significantly than that of T0 and T1 time points in group E(p0.05).BP,HR in group E increased significantly than that in group L at T2 and T5 time points(p0.05).Bp,HR,at each time point ware similar in group L(p0.05).SpO2,PETCO2 at the same time point ware similar between the two groups.The retaining time of PLMA was(43.3±9.6)min.The complications after operation included cough(60%),sore-throat(28%)in group E,and there is nothing in group L.Conclusion PLMA can provide the same safe and effective ventilation as tracheal intubation in general anesthesia for gynecological emergencies.Compared with endotracheal intubation,PLMA was easy to use.the stress of patients was low.PLMA avoid the occurrence of complications of pharynxand laryngeal.

白念珠菌是念珠菌属中最常见的致病菌,常引起黏膜及系统感染.近年来,随着耐药菌株的出现,系统性真菌病发病率及病死率显著增加而引起人们的普遍关注.因此研究其致病机制并寻找新的更有效的治疗途径已成为重要课题.钙调神经磷酸酶(calcineurin,CaN)在白念珠菌感受环境刺激、信号转导及其毒力方面均起关键作用[1],实验证明其抑制剂与氟康唑等联合应用有显著的协同作用并最终达到杀菌目的[2].

脂质体两性霉素B是在两性霉素B分子的外面包裹了生物性脂质体,通过脂质体的携带可改善药物在体内的分子代谢,较多地分布在肝、脾、肺,而在肾组织中分布减少,从而显著地减少了其不良反应,使患者的耐受性提高,临床疗效也获得了提高,适用于系统性真菌病患者的治疗。现就脂质体两性霉素B的临床应用作一综述。

【目的】研制适合于普通医学实验室使用的耗氧剂。【方法】采用正交设计法,得出一种耗氧粉的最佳配方。以此对实验室保存菌株和临床菌株进行验证。【结果】该耗氧粉配方:每升容积需低亚硫酸钠5 g、碳酸氢钠5 g、还原性铁粉1 g和水2.2 ml。以此对3种厌氧菌做厌氧培养、并于临床标本中做厌氧菌分离的结果,证实其除氧效果与Gas Pak配方相当,同时具有价廉、方便、效果稳定可靠、培养结果无相互污染、安全无害及不需特殊设备等特点。【结论】该经济型耗氧粉可靠、实用,在基层有重要应用价值。

目的研究对人类致病的主要暗色丝孢科真菌裴氏着色霉、紧密着色霉、疣状瓶霉、皮炎瓶霉、卡氏枝孢霉、甄氏外瓶霉间的系谱格局.方法采用薄层扫描方法对上述6种30株重要致病性暗丝孢科真菌可溶性全细胞蛋白单向SDS-PAGE图谱结合基型趋异方法进行分析.结果得出其系谱格局为 {[P. verrucosa (F. pedrosoi F. compacta) ]W. dermatitidis[E. jeanselmei C. carrionii]}.结论显示(F. pedrosoi 和F. compacta,E. jeanselmei和C. carrionii,以及Fonsecaea 和Phialophora之间有密切关系.

酒渣鼻病因复杂,许多因素参与了其发生与发展过程.该病患者的幽门螺杆菌感染率较正常人高,幽门螺杆菌分泌毒素和机体所产生的各种炎症介质可能导致酒渣鼻的发生和发展.综述幽门螺杆菌的生物学特性、与酒渣鼻发生和发展的关系、治疗的影响以及可能的作用机制.

Randomly amplified polymorphic DNA (RAPD) finger prints in combination with numerical phonetic analysis was used to investigate the molecular phylogeny of 6 species of 30 strains of the medically important Dematiaceae, i.e. Fonsecaea pedrosoi, Fonsecaea compacta, Phialophora vrrrucosa, Wangiella dermatitidis, Cladosporium carrionii and Exophiala jeanselmei. The cladistic pattern was described conveniently as that:{P.verrucosa [(F.pedrosoi F.compacta) W.dermatitidis] [E.jeanselnei C.carrionnii]}. W.dermatitidis was monophyletic, while E.jeanselmei was polyphyletic. These results indicate that very close relationships exist between isolates of F.pedrosoi and F.compacta, E.jeanselmei and C.carrionii as well as Fonsecaea and Wangiella.

Objective To investigate the anti-Candida albicans activity of fluconzaole combinated with ethanol in vitro.Methods Using minimum concentration of ethanol being not fungistatic,the minimum inhibitory concentration(MICs) of fluconazole alone and in combination with ethanol were determined in 10 C.albicans strains by NCCLS-M27-A microdilution method.Results The MICs of fluconazole alone to C.albicans above were 4-64 μg/ml,while those of fluconazole in combination with 3.75%(v:v) ethanol were 0.125-0.500 μg/ml.Conclusion Ethanol can increase the susceptility of C. albicans to fluconazole.

白念珠菌生物膜是相对于单个分散游离状态的白念珠菌而言的另一种白念珠菌独特的生存形式,其最具特征性的表型是对多种传统抗真菌药物高度耐药.对白念珠菌生物膜的结构、耐药机制及应对其耐药的实验研究作一综述.

Objective To investigate the differences on gene sequence between yeast-form and hyphal-form Candida albicans,a comparative study on partial ERG11 gene sequence was conducted. Methods 5 strains C. albicans from a single HIV-infected patient were grown to form yeast cells and hyphal cells respectively, and the genomic DNA was extracted. Then ERG11 was amplified by PCR and sequenced. Results Differences on ERG11 gene sequence between yeast-form and hyphal-form C. albicans were identified in strain CA-7,which were observed respectively at position 1 547, 1 587 and 1 617 of ERG11. Conclusion There were some differences on DNA sequence between yeast-form and hyphal-form C. albicans. Hyphal-form C. albicans was thought to be the appropriate object of medical mycology.

由白念珠菌引起的口咽念珠菌病是HIV感染者常见的机会感染.自从应用高效抗逆转录病毒治疗以来,HIV感染者口咽念珠菌病的发生率明显下降.HAART降低口咽念珠菌病发病率不仅是由于改善了患者的免疫力,而且还由于HAART中的蛋白酶抑制剂抑制了白念珠菌分泌型天冬氨酸蛋白酶的活性、形态转换及其生长过程而发挥其抗念珠菌的作用.本文综述了PI抗白念珠菌的机制.

Objective: To explore the differences in gene sequences between yeast -form and hyphal -form ofCandida albicans, the sequences of ERG11 gene encoding the target enzyme of fluconazole were compared. Methods:Seven C. albicans isolates from the same strain ( CA - 10、CA - 11 、CA - 13 ～ CA - 17 ) were induced to form pseudohyphae. The genomic DNA of both the yeast - form and hyphal - form of the seven isolates were extracted. DNA fragments ( position 438 to position 701 ) of ERG1 1 were amplified by PCR and DNA differences between both forms of each isolate were compared by DN.A sequencing. Results:No isolate was found to have sequence difference between yeast form and hyphal-form in this fragment of ERG11 gene of C. albicans. Conclusion:The hyphal- form of C. albicans has no difference in parts of DNA sequence with the yeast-form in this fragment of ERG11 gene of C. albicans.