VVC患者临床分离白念珠菌Cap1、Mrr1和MDR1在唑类药物交叉耐药中的作用

目的探讨白念珠菌锌簇转录因子Cap1、Mrr1基因突变／高表达及外排泵基因MDR1的mRNA表达水平在唑类抗真菌药物氟康唑（FCA）、伊曲康唑（ITR）、伏立康唑（VRC）交叉耐药中的作用。方法收集山西医科大学第二医院皮肤性病科门诊疑似外阴阴道念珠菌病（VVC）患者阴道分泌物标本,进行真菌直接镜检、培养、分离鉴定和体外药物敏感性试验,获得试验用白念珠菌耐药和敏感菌株共68株。采用PCR方法扩增68株白念珠菌临床分离菌株Cap1、Mrr1基因,并进行双向测序,应用Blast软件,将测序结果与GenBank中已发表的序列进行比对,分析基因突变情况,实时荧光定量PCR检测锌簇转录因子Cap1、Mrr1和外排泵基因MDR1的mRNA表达水平。结果Cap1测序中1株ITR、VRC交叉耐药菌株出现P311S错义突变。Mrr1测序中1株FCA、ITR、VRC交叉耐药菌株出现5个错义突变（T917M,I＇923I,E1020Q,F1032L,S1037L）;FCA、ITR、VRC交叉耐药菌株组中Cap1／Mrr1／MDR1的mRNA表达量分别高于敏感菌株组;CA、ITR、VRC交叉耐药菌株组Cap1和MDR1（r=0．173,P=0．571）,Mrr1和MDR1（r=-0．091,P=0．766）,Cap1和Mrr1基因（r=0．025,P=0．936）的mRNA表达量不相关（均P〉0．05）。结论Cap1和Mrr1的突变可能和唑类药物交叉耐药有关。     

白念珠菌AP-PCR基因分型与耐药性关系研究

目前白念珠菌的耐药表型与基因型的关系仍不清楚，寻找一种恰当的基因分型方法，探讨其与耐药表型的关系，对监测耐药株的分子流行病学以及深化耐药分子机制的研究有重要的临床意义。以我院近3年出现的8株耐药白念珠菌（MIC〉64μg/ml），及用体外氟康唑诱导的方法，诱导了2株耐氟康唑白念珠菌，同期选择了48株临床敏感白念珠菌，共58株采用AP-PCR（Arbitrary primod-PCR, AP-PCR）技术对其进行了基因分型。将其电泳图谱扫描入计算机后采用Lab-work 4．0软件转化为数值图表后进行聚类分析，以探讨耐氟康唑白念珠菌基因多态性是否与其耐药性相关。     

体内外光滑假丝酵母菌氟康唑耐药机制的比较研究

目的 研究比较临床分离和体外诱导的耐氟康唑光滑假丝酵母菌的耐药机制.方法 选取临床分离的4对氟康唑敏感-耐药配对的光滑假丝酵母菌,其中4株敏感株在体外氟康唑的作用下均被诱导为耐药株.利用罗丹明6G试验比较敏感株与两种耐药株的外排泵作用,实时荧光定量RT-PCR检测外排相关基因CDR1、CDR2、SNQ2和ERG11的表达.同时,对PDR1基因进行PCR扩增和测序,对比分析临床耐药株和体外诱导耐药株的突变位点.结果 临床耐药株和体外诱导耐药株外排泵的功能均明显强于敏感株；两者CDR1表达均显著升高,而CDR2和SNQ2则无明显变化；ERG11在敏感与耐药菌株之间的表达水平也无显著差别；两种耐药株的PDR1均发现错义突变位点,其中P927S、L543P及S947L突变尚未被报道过.结论 光滑假丝酵母菌在体内外氟康唑作用下PDR1均产生突变,引起外排相关基因,尤其是CDR1的表达增加从而增强了外排泵的作用导致耐药.     

广州地区质粒和染色体介导的淋球菌对青霉素和四环素耐药株趋势分析

目的监测2000—2005年间广州地区质粒和染色体介导的淋球菌对青霉素和四环素的耐药性及耐药株流行趋势。方法采用琼脂稀释法检测淋球菌对青霉素和四环素抗菌药物的最低抑菌浓度（MIC），用纸片酸度法测定质粒介导的产青霉素酶淋球菌（PPNG）。结果6年来共测定631株临床分离的淋球菌，检出PPNG132株（20．9％），阳性率从2000年的17．1％上升到2005年的23．7％（Χ^2=0．955，P〉0．05）；质粒介导的高度耐四环素淋球菌（TRNC）222株（35．2％），阳性率从2000年的20％上升到2005年的46．1％（Χ^2=11．94，P〈0．05）。染色体介导的青霉素耐药率从2000年的76％上升到2005年的98．3％（Χ^2=12．94，P〈0．05），染色体介导的四环素的耐药率介于70．7％-85．7％（X2=3．246，P〉0．05）。在2000—2005年期间，青霉素的MIC50和MIC90由1mg／L和2mg／t,上升到4mg／L和〉32mg／L，四环素的MIC50和MIC90由1mg／L和2mg／L上升到2mg／L和4mg／L，且都超过了耐药标准。结论广州地区近6年来质粒介导的淋球菌耐药株PPNG和TRNG增长速度较快。呈逐年上升趋势；染色体介导的淋球菌对青霉素和四环素耐药比率很高。     

粉防己碱对缺血-再灌注损伤大鼠心肌细胞凋亡及Bcl-2/Bax蛋白表达的影响

目的探讨粉防己碱对缺血-再灌注损伤大鼠心肌细胞凋亡及Bcl-2/Bax蛋白表达的影响。方法结扎大鼠左冠状动脉前降支（LAD）30min后松开,再灌注24h建立心肌缺血-再灌注损伤模型。将48只雄性SD大鼠随机分为：假手术组（Sham组）,缺血-再灌注损伤组（IRI组）,粉防己碱预处理组（Tet组）。再灌注结束后检测血清肌酸激酶同工酶MB（CK-MB）和心肌梗死范围（IS/AAR,%）。应用原位末端标记法（TUNEL法）检测各组凋亡细胞,计算凋亡指数（AI）,应用免疫组化法检测心肌Bcl-2、Bax蛋白表达,计算Bcl-2/Bax值,进行组间比较。结果与IRI组相比,Tet可明显降低CK-MB值（976.57±160.69）vs（1910.38±221.10）U/L,P〈0.01,减小心肌IS/AAR%,（23.28±4.38）%vs（43.76±6.30）%,P〈0.01。与IRI组比较,粉防己碱预处理可显著减少心肌细胞凋亡,AI显著降低（8.62±2.45%vs19.36±5.28%,P〈0.01）。粉防己碱预处理使Bcl-2表达增加,Bax表达下降,Bcl-2/Bax值显著升高（P〈0.01）。结论粉防己碱能明显抑制缺血-再灌注损伤引起的心肌细胞凋亡,其作用机制可能与促进Bcl-2蛋白表达,减少Bax蛋白表达、升高Bcl-2/Bax比值有关。     

粉防己碱与表阿霉素联用对表阿霉素在荷瘤裸鼠血、心与肿瘤组织浓度的影响

目的:研究多药耐药(MDR)裸鼠移植瘤模型中,粉防己碱以不同频率和时间间隔给药对抗癌药物表阿霉素进入肿瘤组织细胞能力的影响。方法:裸鼠背部皮下接种人口腔上皮癌(耐药株KBV和非耐药株KB)细胞,按粉防己碱给药次数和提前表阿霉素给药的时间,试验分组为:组1(单次,提前48h)、组2(单次,提前24h)、组3(单次,提前2h)、组4(两次,提前72h和24h)、组5(两次,提前48h和24h)、组6(两次,提前24h和2h)、耐药株对照、敏感株对照。粉防己碱(50mg.kg-1)腹腔注射给药,表阿霉素(8mg.kg-1)尾静脉注射给药。分别于表阿霉素给药后3小时取血、解剖取心脏和肿瘤组织,HPLC法测定血液和心脏组织中表阿霉素的含量,质谱法测定肿瘤组织样品的表阿霉素的含量。结果:耐药组裸鼠表阿霉素血液含量为7.2±1.1 ng.ml-1,心脏组织中为60.1±20.2ng.g-1,与敏感组的测定值一致,但肿瘤中的含量只有8.4±3.9ng.g-1,与耐药组相比有统计学差异(P<0.05),组1、2、3、5肿瘤组织中表阿霉素含量同耐药对照组相比无统计学意义,相互之间也无显著差异。组4(-48,-24h)的肿瘤组织浓度高于耐药对照组,同时心脏组织中的含量比耐药对照组的低,有统计学意义。结论:粉防己碱提前48h或24h给药似有较好的抗MDR效果,且既能提高耐药肿瘤中表阿霉素的浓度,又减少表阿霉素对心肌的药源性损伤,具有潜在的临床意义。     

儿童肺炎支原体感染检测MBL和CRP的意义

探讨肺炎支原体（MP）感染患儿血清甘露糖结合凝集素（MBL）和C-反应蛋白（CRP）水平变化的意义。用EL／SA和速率散射比浊法分别检测患儿血清MBL和CRP水平。结果显示,57例MP感染患儿MBL水平（3．64±1．72mg／L）和CRP（21．44±15．02mg／L）比对照组（2．18±1．05mg／L和2．76±1．81mg／L）显著增高（P〈0．001）。年龄小于8岁患儿MBL水平低于年龄大于8岁患儿,其中8例反复呼吸道感染患儿MBL水平（2．02±1．21mg／L）显著低于其它患儿（3．95±1．63mg／L,P〈0．01）。患儿恢复期CRP水平明显降低。结果提示,血清MBL和CRP水平可作为监测MP感染患儿天然免疫功能的指标,动态检测有助于评价疗效。     

早期阿托伐他汀治疗对颅内动脉支架术前后患者C-反应蛋白的影响

目的测定颅内支架术治疗前后患者C-反应蛋白（CRP）的水平,探讨阿托伐他汀对预防患者颅内支架再狭窄的可能性。方法将42例颅内支架患者随机分为对照组和治疗组（n=21）。对照组口服玻立维75mg＋拜阿司匹林100mg,1次／d;治疗组口服阿托伐他汀40mg（术前7d开始）＋玻立维75mg＋拜阿司匹林100mg,1次／d。术前7d及1d,术后7d、3个月、6个月采用免疫透射比浊法分别测血清CRP水平。结果与对照组术前1d（7．44±0．73）mg／L、术后7d（27．61±1．81）mg／L、术后3个月（36．46±1．67）mg／L、术后6个月（26．84±0．76）mg／L比较,治疗组同期的CRP明显下降（P〈0．05）;与术后7d（12．05±0．55mg／L）比较,治疗组术前1d（6．22±0．57）mg／L、术后3个月（6．03±0．46）mg／L、6个月（6．12±0．77）mg／L下降明显（P〈0．01）;术前1d、术后3个月、6个月之间差异无统计学意义（P〉0．05）。结论阿托伐他汀可有效抑制炎症因子,是预防支架内再狭窄的有效治疗方法之一。     

黄芪多糖抑制人乳腺癌MCF－7细胞增殖和诱导凋亡

目的探讨中药黄芪多糖的体外抗人乳腺癌MCF．7细胞活性。方法实验分为空白对照组、黄芪多糖组和阳性对照组,黄芪多糖组MCF．7细胞给予不同浓度（2．5、5、10、20mg／L）的黄芪多糖,阳性对照组给予10gmol／L顺铂,空白对照组给予等体积培养基。48h后应用四甲基偶氮唑蓝（MTT）法测黄芪多糖对MCF-7细胞增殖抑制率,计算IC_50;吖啶橙（AO）,溴化乙啶（EB）荧光染色法测定黄芪多糖对MCF-7细胞诱导凋亡作用;应用流式细胞仪分析黄芪多糖对MCF-7细胞凋亡和细胞周期的影响。结果在给予2．5、5、10、20mg／L黄芪多糖48h后,黄芪多糖呈浓度依赖性抑制MCF．7细胞的增殖（r=0．985,P〈0．05）,抑制率分别为（4．14±2．96）％、（7．14±2．10）％、（20．13±2．33）％、（64．66±5．15）％,高于空白对照组0％,但4个不同浓度组的抑制率均低于阳性对照组（90．31±4．92）％。黄芪多糖48h的IC_50=16．83mg／L。随着黄芪多糖浓度的逐渐增高,MCF-7细胞中代表凋亡的玛瑙色逐渐增多,细胞核骤缩、核分裂,细胞形态呈现典型的凋亡特征。2．5、5、10、20mg／L黄芪多糖的凋亡率分别为（2．37±0．98）％、（6．76±1．31）％、（11．65±1．46）％、（20．75±2．68）％,高于空白对照组（1．14±1．25）％（均P〈0．05）,但均低于阳性对照组（35．09±2．88）％（均P〈0．05）。与空白对照组比较,黄芪多糖以浓度依赖性诱导MCF-7细胞凋亡（r=0．991,P〈0．05）,随浓度的增加,细胞凋亡率升高,但均低于阳性对照组。黄芪多糖随浓度的增加促使s期细胞比例逐渐升高,但低于空白和阳性对照组（均P〈0．05）,使处于G_0-G_1期细胞的比例逐渐减少,仍高于空白和阳性对照组（均P〈0．05）。结论黄芪多糖抑制人乳腺癌MCF-7细胞增殖并诱导其凋亡,使MCF-7细胞生长增殖停滞在S期?     

用持续血液净化治疗急性重症胰腺炎11例分析

目的探讨持续血液净化对急性重症胰腺炎的应用疗效。方法选择2004年11月～2007年8月急性重症胰腺炎患者11例．其中男性8例，女性3例，年龄27～49岁，平均年龄36．7岁；在常规治疗基础上，加用持续血液净化治疗．每次治疗24～48h后更换滤器，持续治疗3～10d，置换液以前稀释方式输入，流量为3000～6000ml／h，血流量200～300ml／min。采用普通肝素抗凝。结果11例患者中9例好转而转入普通病房继续治疗，1例因合并严重肺部感染并发多脏器功能衰竭死亡，1例因其他原因放弃治疗；治疗后患者平均动脉压[（67．23±11．69）mmHg]、心率[（85．61±14．36）次／分1和氧合指数（259．96±29．51）均有所改善（P〈0．05）；血淀粉酶[（87．59±31．68）U/L]、脂肪酶[（190．21±66．50）U/L]、血乳酸[（1．69±0．82）mmol／L]和C-反应蛋白[（39．33±10．17）mg/L]较治疗前[（638．65±79．42）U/L、（734．79±86．91）U/L、（7．11±3．25）mmol/L、（141．21±33．63）mg／L]下降明显（P〈0．05），APACHEⅡ评分亦较治疗前降低（P〈0．05）。结论持续血液净化疗法对急性重症胰腺炎患者在治疗过程中血流动力学稳定．有改善愈后的作用．是抢救急性重症胰腺炎的有效手段之一．     

International and multicenter comparison of EUCAST and CLSI M27-A2 broth microdilution methods for testing susceptibilities of Candida spp. to fluconazole, itraconazole, posaconazole, and voriconazole

The aim of this study was to compare MICs of fluconazole, itraconazole, posaconazole, and voriconazole obtained by the European Committee on Antibiotic Susceptibility Testing (EUCAST) and CLSI (formerly NCCLS) methods in each of six centers for 15 Candida albicans (5 fluconazole-resistant and 4 susceptible-dose-dependent [S-DD] isolates), 10 C. dubliniensis, 7 C. glabrata (2 fluconazole-resistant isolates), 5 C. guilliermondii (2 fluconazole-resistant isolates), 10 C. krusei, 9 C. lusitaniae, 10 C. parapsilosis, and 5 C. tropicalis (1 fluconazole-resistant isolate) isolates. CLSI MICs were obtained visually at 24 and 48 h and spectrophotometric EUCAST MICs at 24 h. The agreement (within a 3-dilution range) between the methods was species, drug, and incubation time dependent and due to lower EUCAST than CLSI MICs: overall, 94 to 95% with fluconazole and voriconazole and 90 to 91% with posaconazole and itraconazole when EUCAST MICs were compared against 24-h CLSI results. The agreement was lower (85 to 94%) against 48-h CLSI endpoints. The overall interlaboratory reproducibility by each method was > or =92%. When the comparison was based on CLSI breakpoint categorization, the agreement was 68 to 76% for three of the four species that included fluconazole-resistant and S-DD isolates; 9% very major discrepancies (< or =8 microg/ml versus > or =64 microg/ml) were observed among fluconazole-resistant isolates and 50% with voriconazole (< or =1 microg/ml versus > or =4 microg/ml). Similar results were observed with itraconazole for seven of the eight species evaluated (28 to 77% categorical agreement). Posaconazole EUCAST MICs were also substantially lower than CLSI MIC modes (0.008 to 1 microg/ml versus 1 to > or =8 microg/ml) for some of these isolates. Therefore, the CLSI breakpoints should not be used to interpret EUCAST MIC data.     

Epidemiology of candidaemia and antifungal susceptibility patterns in an Italian tertiary-care hospital

The epidemiological and antifungal susceptibility data for 94 episodes of candidaemia in an Italian tertiary-care hospital between January 2000 and August 2003 were evaluated by prospective laboratory-based surveillance. The incidence of fungaemia was 0.90 episodes/10 000 patient-days, and the most common species isolated were Candida albicans (40.4%), Candida parapsilosis (22.3%), Candida tropicalis (16.0%) and Candida glabrata (12.8%). Among 24 patients who received antifungal prophylaxis, non-albicans Candida spp. were more prevalent than C. albicans (p 0.012). The 30-day mortality rate was high (38.2%), particularly for haematological (71.4%) and solid-organ transplant patients (50.0%), and in individuals with C. tropicalis and C. glabrata bloodstream infections (60.0% and 50.0%, respectively). In-vitro susceptibility tests demonstrated that 95% of the isolates were susceptible to amphotericin B (MIC < 2 mg/L), 98.1% to posaconazole (MIC < 1 mg/L), 95.8% to flucytosine (MIC < 32 mg/L) and fluconazole (MIC < 64 mg/L), and 94.7% to itraconazole (MIC < 1 mg/L). Posaconazole was active (MIC 0.5 mg/L) against all three isolates of Candida krusei, which had reduced susceptibility to both fluconazole and itraconazole. Overall, non-albicans Candida spp. accounted for 60% of the episodes of candidaemia, which could be related to the use of antifungal prophylaxis. Resistance is still uncommon in Candida spp. recovered from blood cultures. The in-vitro activity of posaconazole is encouraging, and this agent could play an important role in the management of invasive candidiasis, including episodes caused by inherently less susceptible species such as C. krusei.     

高通量血液透析对维持性血液透析患者细胞免疫功能的影响

目的探讨高通量血液透析对维持性血液透析（MHD）患者细胞免疫功能的影响。方法收集2012年3月至8月于本院门诊行MHD治疗的患者40例,随机数字表法分为血液透析（HD）组（n=20）和高通量血液透析（HFHD）组（n=20）,分别接受HD和HFHD治疗,均为每周透析3次,每次4h。透析前、透析后4、24、48h,流式细胞术检测两组患者外周血CD4＋．CD8＋、CD25＋,记录CD47CD8＋比值,酶联免疫吸附测定（ELISA）检测血清IL-2、可溶性IL-2受体（sIL．2R）;另设健康对照组（C组）20例,清晨空腹抽血检测上述指标。结果与C组比较,透析前HD组和HFHD组患者外周血CD4＋、CD25＋、CD4＋／CD8＋水平下降,血清IL．2水平下降,sIL．2R升高（均P〈0．05）。与透析前比较,HD组患者透析后4h外周血CD4＋、CD25＋、CD47CD8＋水平升高,血清IL-2水平升高,sIL-2R降低（均P〈0．05）,CD8＋差异无统计学意义（P〉O．05）;与透析前比较,HD组患者透析后24、48h上述各指标差异无统计学意义（均P〉0．05）。与透析前比较,HFHD组患者透析后4、24、48h外周血CD4＋、CD25＋、CD4VCD8＋水平升高,血清IL．2水平升高,slL-2R降低（均P〈0．05）,而CD8＋差异均无统计学意义（均P〉O．05）。与同时点HD组比较,HFHD组透析后4h各指标差异均无统计学意义（均P〉O．05）;透析后24、48h,HFHD组外周血CD4＋、CD25＋、CD4＋／CD8＋水平升高,血清IL．2水平升高,sIL．2R降低[CD4＋：（38．73±6．25）％比（34．92±5．84）％,（37．03±5．41）％比（32．62±5．79）％;CD25＋：（21．36±4．65）％比（15．29±4．72）％,（18．19±4．27）％比（13．94±5．05）％;CD4＋／CD8＋：1．42±0．31比1．23±0．29,1．38±0．30比1．20±0．33;IL-2：（22．03±5．18）m±L比（19．03±4．87）m#L,（20．54±5．92）mL比（18．26±4．96）mL;sIL-2R：（672．96±159．36）U／ml比（787．32±143．27）u,ml,（720．24±143．92）u,（858,42±172．13）U／ml,均P〈0．05],而CD8＋差异无统计学意义（均P〉O．05）。结论HD可短暂改善MHD患者的细胞免疫功能,HFHD可持续改善MHD患者的细胞免疫功能。     

Novel Fluorescent Broth Microdilution Method for Fluconazole Susceptibility Testing of Candida albicans

A comparative evaluation of the reference National Committee for Clinical Laboratory Standards (NCCLS) broth microdilution method with a novel fluorescent carboxyfluorescein diacetate (CFDA)-modified microdilution method for the susceptibility testing of fluconazole was conducted with 68 Candida strains, including 53 Candida albicans, 5 Candida tropicalis, 5 Candida glabrata, and 5 Candida parapsilosis strains. We found trailing endpoints and discordant fluconazole MICs of < 8 microg/ml at 24 h and of > or =64 microg/ml at 48 h for 12 of the C. albicans strains. These strains satisfy the definition of the low-high MIC phenotype. All 12 low-high phenotype strains were correctly shown to be susceptible at 48 h with the CFDA-modified microdilution method. For the 41 non-low-high phenotype C. albicans strains, the CFDA-modified microdilution method yielded 97.6% (40 of 41 strains) agreement within +/-1 dilution at 24 h compared with the reference method and 92.7% (38 of 41 strains) agreement within +/-1 dilution at 48 h compared with the reference method. The five strains each from C. tropicalis, C. glabrata, and C. parapsilosis that were tested showed 100% agreement within +/-2 dilutions for the two methods being evaluated.     

Antifungal activity of ibuprofen alone and in combination with fluconazole against Candida species

Ibuprofen, a non-steroidal anti-inflammatory drug, exhibited antimicrobial activity against Candida albicans and non-albicans strains. At 10 mg/ml, ibuprofen showed a rapid cidal activity against exponential growth phase C. albicans, accompanied by rapid and extensive leakage of intracellular K+, permeation to propidium iodide, lysis of spheroplasts and severe membrane ultrastructural alterations. These results indicate that the killing of Candida cells is due to direct damage to the cytoplasmic membrane. At 5 mg/ml, ibuprofen inhibited growth; however, it did not kill the yeasts and did not directly affect the cytoplasmic membrane. Evaluation of yeast metabolic vitality with the fluorescent probe FUN-1 showed that growth inhibition induced by the fungistatic drug concentration was due to metabolic alterations. The combination of ibuprofen with fluconazole resulted in synergic activity with eight of the 12 Candida strains studied, including four of the five fluconazole-resistant strains. The MICs of fluconazole for the fluconazole-resistant strains decreased 2-128-fold when the drug was associated with ibuprofen. When in combination with fluconazole, MICs for ibuprofen decreased by up to 64-fold for all the 12 strains studied. These results point to the practicability of using ibuprofen, alone or in combination with azoles, in the treatment of candidosis, particularly when applied topically, taking advantage of the drug's antifungal and anti-inflammatory properties.     

Comparative evaluation of macrodilution and alamar colorimetric microdilution broth methods for antifungal susceptibility testing of yeast isolates.

A comparative evaluation of the macrodilution method and the Alamar colorimetric method for the susceptibility testing of amphotericin B, fluconazole, and flucytosine was conducted with 134 pathogenic yeasts. The clinical isolates included 28 Candida albicans, 17 Candida tropicalis, 15 Candida parapsilosis, 12 Candida krusei, 10 Candida lusitaniae, 9 Candida guilliermondii, 18 Torulopsis glabrata, and 25 Cryptococcus neoformans isolates. The macrodilution method was performed and interpreted according to the recommendations of the National Committee for Clinical Laboratory Standards (document M27-P), and the Alamar colorimetric method was performed according to the manufacturer's instructions. For the Alamar colorimetric method, MICs were determined at 24 and 48 h of incubation for Candida species and T. glabrata and at 48 and 72 h of incubation for C. neoformans. The overall agreement within +/- 1 dilution for Candida species and T. glabrata against the three antifungal agents was generally good, with the values for amphotericin B, fluconazole, and flucytosine being 85.3, 77.9, and 86.2%, respectively, at the 24-h readings and 69.3, 65.2, and 97.2%, respectively, at the 48-h readings. Most disagreement was noted with fluconazole against C. tropicalis and T. glabrata. Our studies indicate that determination of MICs at 24 h by the Alamar colorimetric method is a valid alternate method for testing amphotericin B, fluconazole, and flucytosine against Candida species but not for testing fluconazole against C. tropicalis and T. glabrata. For flucytosine, much better agreement can be demonstrated against Candida species and T. glabrata at the 48-h readings by the Alamar method.(ABSTRACT TRUNCATED AT 250 WORDS)     

HIV-1gp120蛋白对人血-视网膜屏障细胞的毒性作用

目的探索HIV-1gp120蛋白侵犯人血-视网膜屏障的机制。方法原代培养人血-视网膜屏障细胞（HBRBC），包括人视网膜微血管内皮细胞（HRCEC）、人视网膜微血管周细胞（HRCPC）、人视网膜色素上皮细胞（HRPE），以培养基作为对照。用MTT法观察7种不同浓度（0．01～0．15mg／L）的HIV-1gp120蛋白作用24h和0．08mg／LHIV-1gp120蛋白作用不同时间（4～72h）对3种细胞的生长抑制作用。0．08、0．1、0．12、0．15mg／L的HIV-1lgp120蛋白作用24h后，用流式细胞仪检测其对3种细胞凋亡率和线粒体膜电位（△ψm）的影响；用Western免疫印迹检测Cleavedcaspase-9蛋白的活化情况。用透射电镜观察经0．08mg／LHIV-1gp120蛋白处理24h前后3种细胞超微结构的变化。结果HIV-1gp120蛋白作用24h，低浓度（〈0．08mg／L）对3种细胞的活性均没有明显影响，而当浓度超过0．08mg／L时，对细胞的增殖活性有明显的抑制作用，呈浓度依赖性（HRCEC：r=-0．763，P〈0．01；HRCPC：r=-0．804，P〈0．01；HRPE：r=-0．698，P〈0．01）。HIV-1gp120蛋白（0．08mg／L）作用12h即可显著抑制细胞的增殖活性，24、48和72h抑制效应更明显，相对增殖率分别为HRCEC：84％、70％、41％、22％，HRCPC：80％、69％、38％、18％，HRPE：86％、73％、45％、26％，抑制效应呈时间依赖性（HRCEC：r=-0．833，P〈0．01；HRCPC：r=-0．784，P〈0．01；HRPE：r=-0．701，P〈0．01）。HIV-1gp120蛋白作用24h后与对照组相比，各浓度组3种细胞凋亡率增加、△ψm明显降低以及Cleavedeaspase-9蛋白表达增强，均呈浓度依赖性。透射电镜示0．08mg／LHIV-1gp120蛋白处理24h后，3种细胞均出现了线粒体肿胀、溶酶体增多等早期凋亡的微观改变。结论HIV-1gp120蛋白能够抑制人血-视网膜屏障细胞的增殖并具有诱导凋亡的作用，破坏线粒体的结构和功能是其可能的机制。