两性多肽结构影响鲍曼不动杆菌外膜通透性的实验研究

目的明确两性多肽结构对鲍曼不动杆菌(AB)外膜通透性的影响。方法 PCR扩增LysAB3基因及删除两性多肽结构的LysAB3-D基因,以pET28a(+)为载体构建重组质粒,在大肠杆菌BL21(DE3)中表达LysAB3及LysAB3-D,金属离子螯合亲和层析法纯化重组蛋白。将AB分别经LysAB3及LysAB3-D处理后,用扫描电子显微镜观察菌体形态,荧光显微镜观察菌体中是否有绿色荧光的聚集。结果经LysAB3处理后的AB,在扫描电子显微镜下可见菌体表面粗糙、皱缩,部分裂解为碎片;在荧光显微镜下可见菌体内有绿色荧光聚集。而删除两性多肽结构的LysAB3-D作用AB后,却没有上述现象的发生。结论两性多肽结构可增加AB外膜通透性,有助于裂解酶进入其中,达到抗菌目的。     

两性多肽结构影响鲍曼不动杆菌外膜通透性的实验研究*两性多肽结构影响鲍曼不动杆菌外膜通透性的实验研究

目的：明确两性多肽结构对鲍曼不动杆菌（AB）外膜通透性的影响。方法 PCR扩增LysAB3基因及删除两性多肽结构的LysAB3-D基因，以pET28a（＋）为载体构建重组质粒，在大肠杆菌BL21（DE3）中表达LysAB3及LysAB3-D ，金属离子螯合亲和层析法纯化重组蛋白。将AB分别经LysAB3及LysAB3-D处理后，用扫描电子显微镜观察菌体形态，荧光显微镜观察菌体中是否有绿色荧光的聚集。结果经LysAB3处理后的AB ，在扫描电子显微镜下可见菌体表面粗糙、皱缩，部分裂解为碎片；在荧光显微镜下可见菌体内有绿色荧光聚集。而删除两性多肽结构的LysAB3-D作用AB后，却没有上述现象的发生。结论两性多肽结构可增加AB外膜通透性，有助于裂解酶进入其中，达到抗菌目的。     

铜绿假单胞菌噬菌体PaP1内溶素基因克隆、表达及活性分析

目的 构建铜绿假单胞菌噬菌体内溶素重组原核表达载体,检测重组蛋白的酶学活性及抑菌活性.方法 利用重组技术将铜绿假单胞菌裂解性噬菌体PaP1内溶素基因构建到表达载体pQE31中,并在大肠杆菌M15(pREP4)中诱导表达,通过非变性方法纯化重组融合蛋白,利用酶潜电泳、抑菌实验等技术检测酶学活性及抑菌活性.结果 酶谱电泳方法结果显示重组融合蛋白对铜绿假单胞菌细胞壁肽聚糖有降解作用,抑菌活性检测结果显示重组融合蛋白对金黄色葡萄球菌有一定的抑菌效果.结论 该研究从实验方面证实了噬菌体PaP1内溶素基因的生物学功能.     

鲍曼不动杆菌噬菌体SWH-Ab-1分离鉴定及其重要功能基因的生物信息学分析

目的 分离鲍曼不动杆菌噬菌体,测定其基因组DNA序列,分析预测穿孔素和裂解酶等重要功能基因及其编码蛋白的性质和功能.方法 利用双层琼脂平板法从医院污水中分离鲍曼不动杆菌噬菌体,透射电子显微镜观察噬菌体的形态特征;提取噬菌体基因组DNA,采用Illumina Hiseq2000测序仪进行全基因组测序;利用生物信息学方法对噬菌体基因组DNA序列进行功能编码基因定位、同源性分析以及对噬菌体重要功能基因编码蛋白的结构和生物功能等进行预测.结果 成功从医院污水中分离获得1株鲍曼不动杆菌噬菌体,命名为噬菌体SWH-Ab-1.噬菌体SWH-Ab-1属长尾噬菌体,对鲍曼不动杆菌具有显著裂解性.噬菌体SWH-Ab-1基因组为全长41 567 bp的双链DNA,G+C％含量为39.42％,包含51个预测功能基因,穿孔素基因和裂解酶基因分别位于噬菌体基因组的ORF02(320 ～655 bp)和ORF03(642～1 199 bp)开放阅读框,穿孔素基因序列全长336 bp,共编码111个氨基酸,其编码的HolSA1为疏水性Ⅰ型跨膜蛋白,相对分子质量为11.957 96×103,理论等电点为4.87,可能在脂肪酸代谢、转运载体及信号转导中发挥重要作用.裂解酶基因序列全长558 bp,共编码185个氨基酸,其编码的LysSA1为亲水性蛋白,相对分子质量为21.010 4×103,理论等电点为9.56,无跨膜区,可能参与氨基酸生物合成、脂肪酸代谢、辅因子生物合成等重要生理过程.两种功能蛋白均不存在信号肽序列,二级结构主要以α-螺旋和无规则卷曲为主.结论 噬菌体SWH-Ab-1是一种新分离的长尾裂解性噬菌体,其基因组的ORF02和ORF03开放阅读框是穿孔素和裂解酶的编码基因,编码的HolSA1和LysSA1可能组成“二元裂解系统”,协同参与噬菌体对宿主菌的裂解过程.     

不动杆菌的分离与鉴定

【目的】 鲍曼不动杆菌是一类非发酵、专性需氧的革兰阴性球杆菌,是临床常见的条件致病菌之一。近年来鲍曼不动杆菌感染率不断上升,其耐药率也逐年增强。鲍曼不动杆菌几乎对所有抗生素呈现高度耐药,给临床治疗带来极大困难。噬菌体是感染细菌、真菌、放线菌或螺旋体等微生物的病毒,具有严格的宿主特异性,只能在活的微生物细胞内复制、增殖。毒性噬菌体在宿主菌内复制增殖,并最终裂解细菌,其特殊的生物学特性有望成为临床上治疗细菌感染新的有效手段。温和噬菌体能将基因组整合于宿主菌染色体中,不产生子代噬菌体,不裂解细菌,使菌体处于溶原状态。本课题通过分离鉴定鲍曼不动杆菌噬菌体,研究噬菌体对耐药性的鲍曼不动杆菌的影响。 【方法】 采集污水,人及动物上呼吸道内和医院ICU环境的标本,经CaCl₂处理后,离心取上清液,细菌滤器滤过,将滞留在滤膜上的细菌进行分离传代培养,纯化;染色镜检,观察细菌形态、排列,初步生化试验筛选、ATB生化鉴定菌种;药敏试验选择耐药菌株;经噬斑试验,分离或诱导毒性噬菌体;负染色法制备电镜标本,观察其超微结构。 【结果】 ICU环境中,特别是呼吸机导管可分离到鲍曼不动杆菌,鲍曼不动杆菌表面存在温和噬菌体,呈多边形,细菌耐药性明显,为多重耐药。 【结论】 耐药的鲍曼不动杆菌菌体表面存在温和噬菌体,未裂解细菌,可能将基因整合于宿主菌体,研究噬菌体与不动杆菌耐药性的相关性,通过诱导毒性噬菌体,裂解细菌,可治疗和控制细菌性感染,将为防治鲍曼不动杆菌引起的医院感染带来希望。     

2006～2009年鲍曼不动杆菌感染分布特征及耐药性变迁

目的:研究2006～2009年临床分离鲍曼不动杆菌(Acinetobacter baumannii,A.b)的分布特征及对抗菌药物的耐药性变迁,比较多粘菌素B与亚胺培南对多重耐药鲍曼不动杆菌的体外抗菌活性.方法:监测近4年来临床分离鲍曼不动杆菌感染分布特征.采用纸片琼脂扩散法药敏试验,分析鲍曼不动杆菌对抗菌药物敏感性,比较近4年来鲍曼不动杆菌对抗菌药物的耐药性变迁.采用常量肉汤稀释法测定多粘菌素B及亚胺培南对多重耐药鲍曼不动杆菌的最低抑菌浓度(Minimal inhibitory concentration,MIC)值,比较其体外抗菌活性.结果:近4年来,鲍曼不动杆菌感染占临床分离革兰阴性杆菌的构成比逐年增加,以ICU病房患者呼吸道标本检出率最高.临床分离鲍曼不动杆菌对亚胺培南的耐药率从2008年起明显上升,多重耐药鲍曼不动杆菌比例也逐渐增加.比较多粘菌素B及亚胺培南对多重耐药鲍曼不动杆菌的体外抗菌活性显示,多粘菌素B对多重耐药鲍曼不动菌株的敏感率为93.8%,多粘菌素B对多重耐药鲍曼不动杆菌的体外抗菌活性明显高于亚胺培南.结论:鲍曼不动杆菌在医院感染所占的比例明显上升.鲍曼不动杆菌对临床常用抗菌药物的耐药性也呈逐年上升趋势,多重耐药菌比例明显增加.应加强鲍曼不动杆菌细菌耐药监测.多粘菌素B对多重耐药鲍曼不动杆菌仍呈现出较强的体外抗菌活性,对于多重耐药鲍曼不动杆菌引起的严重感染可以考虑使用多粘菌素B治疗.     

2006～2009年鲍曼不动杆菌感染分布特征及耐药性变迁

目的:研究2006～2009年临床分离鲍曼不动杆菌的分布特征及对抗菌药物的耐药性变迁,比较多粘菌素B与亚胺培南对多重耐药鲍曼不动杆菌的体外抗菌活性.方法:监测近4年来临床分离鲍曼不动杆菌感染分布特征.采用纸片琼脂扩散法药敏试验,分析鲍曼不动杆菌对抗菌药物敏感性,比较近4年来鲍曼不动杆菌对抗菌药物的耐药性变迁.采用常量肉汤稀释法测定多粘菌素B及亚胺培南对多重耐药鲍曼不动杆菌的最低抑菌浓度(Minimal inhibitory concentration,MIC)值,比较其体外抗菌活性.结果:近4年来,鲍曼不动杆菌感染占临床分离革兰阴性杆菌的构成比逐年增加,以ICU病房患者呼吸道标本检出率最高.临床分离鲍曼不动杆菌对亚胺培南的耐药率从2008年起明显上升,多重耐药鲍曼不动杆菌比例也逐渐增加.比较多粘菌素B及亚胺培南对多重耐药鲍曼不动杆菌的体外抗菌活性显示,多粘菌素B对多重耐药鲍曼不动菌株的敏感率为93.8%,多粘菌素B对多重耐药鲍曼不动杆菌的体外抗菌活性明显高于亚胺培南.结论:鲍曼不动杆菌在医院感染所占的比例明显上升.鲍曼不动杆菌对临床常用抗菌药物的耐药性也呈逐年上升趋势,多重耐药菌比例明显增加.应加强鲍曼不动杆菌细菌耐药监测.多粘菌素B对多重耐药鲍曼不动杆菌仍呈现出较强的体外抗菌活性,对于多重耐药鲍曼不动杆菌引起的严重感染可以考虑使用多粘菌素B治疗.     

鲍曼不动杆菌UDP-葡萄糖4-差向异构酶Gne1的表征

异源表达鲍曼不动杆菌AB0057的UDP-葡萄糖4-差向异构酶并表征其酶学性质以及分析其结构与功能.将异构酶基因构建到pET-28a表达载体并在大肠埃希菌BL21(DE3)中异源表达,使用高效液相色谱检测酶活力及表征酶学性质.系统发育分析、序列比对、同源建模与分子对接分析其结构与关键催化位点.结果显示,重组酶Gne1获...     

诃子提取物的抗菌活性研究

目的：考察中药诃子提取物的体外抗菌活性。方法分别用水乙酸乙酯及95％乙醇对诃子粉末进行提取,获得3种提取组分。以二倍稀释法测定3种提取组分对鲍曼不动杆菌大肠埃希菌肺炎克雷伯杆菌和金黄色葡萄球菌的最低抑菌浓度和最低杀菌浓度。采用同样方法考察提取物对临床分离的泛耐药鲍曼不动杆菌的抗菌作用。结果诃子的3种提取组分中水提取组分的抗菌活性最强;3种提取组分对4种致病菌均具有抑制和杀灭作用,对泛耐药鲍曼不动杆菌的作用最强。结论中药诃子提取物具有广泛的抗菌活性,有望用于细菌感染性疾病特别是耐药菌所致感染的治疗。     

替加环素、多粘菌素和头孢哌酮舒巴坦对多重耐药鲍曼不动杆菌体外抗菌活性的研究

目的:研究替加环素、多粘菌素B和头孢哌酮舒巴坦对多重耐药鲍曼不动杆菌体外的抗菌活性,为临床合理用药提供依据。方法:收集2013年1月-2014年6月广东省第二中医院检验科微生物室临床致病菌培养加药敏试验的标本,结果经鉴定为多重耐药鲍曼不动杆菌50株,用药敏纸片扩散法分别对替加环素、多粘菌素和头孢哌酮舒巴坦进行体外药敏试验。结果:替加环素对多重耐药鲍曼不动杆菌保持敏感性100%,对多粘菌素B中介敏感性为67%,对头孢哌酮舒巴坦的耐药率为73%。结论:替加环素、多粘菌素对多重耐药鲍曼不动杆菌有较高的抗菌活性,是临床治疗多重耐药鲍曼不动杆菌感染的最佳药物之一。     

Value of D-Dimers in patients with acute aortic dissection

Objective: To evaluatel the value of D-dimers in patients with acute aortic dissection (AAD). Methods: This study consisted of 16 patients with AAD and 27 non-AAD patients. Serum D-dimets were measured by Sta-Liatest D-DI immunoturbidimetric assay. Results: D-dimer level was higher (P ＜ 0.001) in patients with AAD(7.91 ± 5.52 μg/ml) than that in non- AAD group(1.57±1.24 μg/ml). D-dimer was positive (＞0.4 μg/ml) in all patients with AAD and in 10 control group patients (37%). Among patients with acute AAD, D-dimers tended to be higher in Stanford A than in Stanford B (8.67 ± 4.31 μg/ml vs. 3.24±1.27 μg/ml, P ＜0.01). D-dimer values tended to be higher in more extended disease(3.84 ± 1.65 μg/ml, 8.57 ± 3.58 μg/ml and 11.87 ± 5.69 μg/ml in thoracic aorta, thoracic and abdominal aorta, thoracic and abdominal aorta and iliacal arteries, respectively, P ＜ 0.05 for both 8.57 ± 3.58 and 11.87 ± 5.69 vs. 3.84 ± 1.65 ). Including the control group into the analysis, we found a sensitivity of 100%, a negative predictive value of 100%, and a specificity of 66% and a positive predictive value of 64% for D-dimer in diagnosis of AAD in our patients with suspected AAD. Conclusion: D-dimer was elevated in patients with AAD. A negative D-dimer test result could be useful in excluding AAD.     

Research of combined liver-kidney transplantation model in rats

Objective： To set up a simple and reliable rat model of combined liver-kidney transplantation. Methods： SD rats served as both donors and recipients. 4℃ sodium lactate Ringer＇s was infused from portal veins to donated livers,and from abdominal aorta to donated kidneys, respectively. Anastomosis of the portal vein and the inferior vena cava （IVC） inferior to the right kidney between the graft and the recipient was performed by a double cuff method, then the superior hepatic vena cava with suture. A patch of donated renal artery was anastomosed to the recipient abdominal aorta. The urethra and bile duct were reconstructed with a simple inside bracket. Results： Among 65 cases of combined liver-kidney transplantation, the success rate in the late 40 cases was 77.5%. The function of the grafted liver and kidney remained normal. Conclusion： This rat model of combined liver-kidney transplantation can be established in common laboratory conditions with high success rate and meet the needs of renal transplantation experiment.     

BLOOD PRESSURE CHANGE WITH AGE IN SALT-SENSITIVE TEENAGERS

Objective To observe blood pressure change with age in salt-sensitive teenagers whose salt sensitivity were determined by repeated testing.Methods Salt sensitivity was determined through intravenous infusion of normal saline combined with volume-depletion by oral diuretic furosemide in 55 teenagers. After five years, salt sensitivity was re-examined and subject blood pressure was followed up. Blood pressure changes in salt-sensitive teenagers were compared to that of non-salt sensitive teenagers over five years.Results After 5 years, the repetition rate of salt sensitivity determined by intravenous saline loading is 92.7%. In teenagers with salt sensitivity on the baseline, both the systolic blood pressure increments and increment rates were much higher than non-salt sensitive teenagers (12.7±12.1 mmHg vs. 2.8±5.2 mmHg, P＜ 0.01; 12.2%± 12.0% vs. 2.5% ±4.4%, P＜ 0.001,respectively). There was a similar trend for diastolic blood pressure (8.4 ± 6.4 mmHg vs. 3.7 ± 6.4 mmHg, P = 0.052; 13.2% ±10.6 % vs. 6.8%± 10.1%, P = 0.053, respectively).Conclusions Salt sensitivity determined by intravenous saline loading showed good reproducibility. Blood pressure increments with age were much higher in salt-sensitive teenagers than non-salt sensitive teenagers, especially in terms of systolic blood pressure.     

安心颗粒对急诊经皮冠状动脉介入术后生活质量影响的研究

目的：评价使用安心颗粒对急诊经皮冠状动脉介入术（PPCI）术后生活质量的影响.方法：将160例接受PPCI的急性ST段抬高型心肌梗死患者随机分为安心颗粒组（术前顿服安心颗粒8.8g,术后安心颗粒4.4 g/次,每日2次）和对照组（仅接受基础药物治疗）.所有患者均服用阿司匹林、氯吡格雷和阿托伐他汀.分别在入院时、出院前1d、出院后180 d时,应用心肌梗死多维度量表（MIDAS）、中文版SF-36评价量表对患者生活质量评分.并观察术后30 d以内的出血并发症、血小板减少症发生情况.结果：入院时和出院前1d,两组患者的心肌梗死MIDAS、SF-36量表评分比较无差异（P＞0.05）;出院后180 d时,与对照组比较,安心颗粒组MIDAS、SF-36评分明显减低（P＜0.05）;组内与入院时比较,两组出院前1d、出院后180 d时,MIDAS、SF-36评分均降低（P＜0.05）.两组患者在随访期间均无大量出血、少量出血、重度和极重度血小板减少症发生,安心颗粒组有4例、对照组有7例发生不明显出血（P＞0.05）.两组发生轻度血小板减少症的患者数比较无差异（P＞0.05）.结论：PPCI使用安心颗粒,能改善急性ST段抬高型心肌梗死患者的生活质量,且不增加出血风险.     

The comparative studies of the influences of Urapidil and Nicardipine on sino-atrial node function, atrio-ventricular node function and hemodynamics

Objective:To investigate the influences of urapidil and nicardipine on rabbit sinus function,atrio-ventricular node function and hemodynamics.Methods:Thirty-two Angora's rabbits were selected and randomly divided into four groups.U1 group:urapidil 0.25 mg/kg;U2 group:urapidil 0.5 mg/kg;N1 group:nicardipine 10 μg/kg;N2 group:nicardipine 20 μg/kg.All these medicine were administrated within 30 seconds.Measurements were taken before and after the administration of urapidil or nicardipine for the following data:mean blood pressure(MAP),heart rate(HR),sino-atrial conduction time(SACT),maximal sinoatrial recovery time(SNRTmax)corrected sinus node recovery time(CSNRT),index of sinus node recovery time(SNRTI),Wenckebach A-V conduction frequency (WB),and P-R interval.Results:Significant MAP and HR changes were identified in all of the four groups before and after administration of both urapidil and nicardipine.No significant changes could be found in the rest of the parameters.Intergroup analysis showed that SACT and CSNRT of N1 and N2 groups were shorter than those of the U2 group(P＜0.01);the MAP decreased(P＜0.01)and the HR increased drastically(P＜0.01).Conclusions:Neither urapidil(0.25 mg/kg,0.5 mg/kg)nor nicardipine(10μg/kg,20μg/kg)has any significant influence on rabbit sinus function or rabbit atrio-ventricular node function.Nicardipine could be a better choice than urapidil for parafunctional sinus node patients.     

Expression of OPGmRNA and ODFmRNA in the patients of hip fracture due to osteoporosis

Objective：To investigate the gene expression of osteoprotegerin（OPG） and osteoclast differentiation factor（ODF） in the bone tissue of patients with hip fracture due to osteoporosis. Methods：OPGmRNA and ODFmRNA in the bone tissue in 50 cases of osteoporosis sufferers（over 50 years old） with hip fracture（Observer Group） and 30 cases of hip facture sufferers with no osteoporosis（Control group） were analyzed with the Semi-Quantitative RT-PCR method. Results：The mRNA expressed of ODF, OPG were both high in the patients with hip fracture. In the control group, the expression of OPG mRNA was observed, while the expression of ODF mRNA was very slight. Conclusion：Aged patients contained all signals including OPG, ODF that are essential for inducing osteoclastogenesis and promoting bone resorption.     

The clinicopathological and immuohistochemical analysis of solid-pseudopapillary tumor of the pancreas： report of 9 cases

Objective：To investigate the clinical features, pathological characteristics and immunophenotype of solid-pseudopapillary tumor of the pancreas（SPTP）. Methods：Nine surgically treated cases of SPTP were retrospectively reviewed. Hematoxylin and Eosin（HE） staining and immunohistochemical staining were used to analyze all cases, and the general clinical data was collected. Results：Six patients were asymptomatic except for a palpable mass. Two patients complained of vague-epigastric pain. One patient appeared jaundice. The tumor was encapsulated and solid tissues alternately with cystic tissues. Histologically, the histological structure of solid portion was pseudopapillary with a fibrovascular core. Tumor cells were uniform and medium-sized which were arranged in sheets ets or nests or pseudopapillary patterns. Immunohistochemical studies demonstrated that SPTP proved positive in vimentin（9/9 cases）, AAT（9/9 cases）, NSE（9/9 cases）, ACT（7/9 cases）, CK20（2/9 cases）, CgA（1/9 cases）, S-100（3/gcases）, PR（4/gcases）, Syn（3/9 cases） and CD56（5/9cases）, negative in CEA and ER. Conclusion：SPTP is a tumor predominantly occurring in young women frequently without special symptoms. This tumor has various characteristical histological patterns with different immunophenotype.     

Dynamic Changes in Serum Estradiol and Progesterone levels in Patients of Premenstrual Syndrome with Adverse Flow of Liver-qi

Objective:To probe into the influence of changes of ovarian hormones on the pathogenesis of the specific sub-type premenstrual syndrome(PMS)and reveal partial microcosmic mechanisms of adverse flow of liver-qi.Methods:Estradiol(E2)and progesterone(P)levels in serum were determined at different phases of menstrual cycle by radioimmunoassay.Results:In the group of PMS with adverse flow of liver-qi.the secretive peak value Of E2 and P at the follicular phase significantly decreased,and the secretive peak value at the luteal phase did not come into being.Conclusions:Low E2 and P secretive peak at the follicular phase and absence of secretive peak at the luteal phase is one of the microcosmic mechanisms of PMS with adverse flow of liver-qi.One of the pathophysiologic mechanisms of specific sub-type PMS is probably the continuous low level of E2and P.     

Real-time Three-dimensional Echocardiography in Assessment of Left Ventricular and Right Ventricular Volumes

Real-time three-dimensional echocardiography (RT3DE)is a new ultrasound technique that enables dynamic threedimensional visualization and quantification of the heart in real time. Investigation of feasibility and methodology of RT3DE in determining left ventricular (LV) and right ventricular (RV) volumes, RT3DE was performed in 35 normal adults using Philips SONOS 7500 system with a 2-4 MHz matrix array transducer. The 60°×60° "pyramid" volume database was obtained and analyzed on a TomTec echo workstation. Both LV and RV volumes were calculated with four 3DE methods (i.e. apical 2, 4, 8, and 16-plane) through manually tracing ventricular endocardial borders in end diastole and end systole. Stroke volumes were then calculated. LV volume was also measured by 2DE Simpson's rule using GE VIVID 7 ultrasound machine.     

SCREENING FOR A 21-CHROMOSOME ABNORMALITY IN PREIMPLANTED EMBRYOS OF ELDERLY WOMEN

Increasing maternal age is the only etiological factor unequivocally linked to Down's syndrome in humans. The occurrence rate of newborns with Down's syndrome is about 1/220 in women over 35 years old. However, the occurrence rate in embryos fertilized in vitro, of the elder woman is unclear. Using FISH we screened the number of chromosome 21 in preimplanted embryos of 5 elderly women (average age, 38.4 years) to study the feasibility and necessity of screening trisomy 21 in embryos in patients over 35 years old at the in vitro fertilization (IVF) center.