重组柞蚕抗菌肽AD对E.coli抗菌机制研究

目的利用透射电子显微镜和流式细胞仪等技术手段,对重组柞蚕抗菌肽AD抗菌作用机制进行初步研究。方法用微量肉汤法鉴定重组柞蚕抗菌肽AD对E.coli敏感质控标准菌株ATCC25922和产β-内酰胺酶菌株ATCC35218的抑菌作用后,利用透射电子显微镜观察经重组柞蚕抗菌肽AD处理前后E.coli超微结构变化,再用流式细胞Ca2+荧光探针技术检测经重组柞蚕抗菌肽AD处理前后E.coli细胞膜内游离钙离子浓度的变化。结果微量肉汤法证明重组柞蚕抗菌肽AD对E.coli敏感株ATCC25922和耐药株ATCC35218的MIC值均为8μg/m L;透射电镜观察到重组柞蚕抗菌肽AD对E.coli的作用主要表现为破坏细菌胞膜的完整性;流式细胞Ca2+荧光探针检测发现,经重组柞蚕抗菌肽AD作用后,细菌胞内表面上的二价阳离子Ca2+浓度增高。结论重组柞蚕抗菌肽AD对E.col i的抗菌机制可表现为膜完整性的破坏,并伴随胞内Ca2+浓度的增高。     

蛇毒抗菌肽OH-CATH在血浆环境中对大肠杆菌的抗菌作用

目的 研究蛇毒抗菌肽OH-CATH在血浆环境中是否会失去抑菌活性.方法 向含血浆LB(B组)和不含血浆LB (A组)中加入不同剂量的蛇毒抗菌肽OH-CATH,使抗菌肽的终浓度为0μg/mL、1 μg/mL、2μg/mL、4μg/mL、8μg/mL、16μg/mL和32μg/mL,再加入普通大肠埃希菌(E.coli ATCC 25922)培养后分别测定两组的O.D值.结果 A和B组O.D值对应点间比较无显著性差异(P＞0.05),蛇毒抗菌肽OH-CATH的最小抑菌浓度(MIC)在4～8 g/mL之间.结论 蛇毒抗菌肽OH-CATH在血浆环境中不会迅速降解而失活并且对大肠杆菌具有良好的抗菌效果.     

氟喹诺酮类药物体外诱导大肠埃希菌耐药性变化

目的:近年来,由于E.coli对氟喹诺酮药物耐药现象日趋严重.文中研究3种氟喹诺酮药物体外诱导大肠埃希菌(E.coli)耐药性,为临床合理使用抗菌药和控制E.coli感染提供指导. 方法:采用环丙沙星(CIP)、左氧氟沙星(LEV)和加替沙星(CAT)分别对10株E.coli进行体外分步诱导,采用琼脂平板二倍稀释法测定诱导前后CIP、LEV及GAT对E.coli的最低抑菌浓度(MIC). 结果:E.coli经体外诱导产生获得高水平耐药株(MIC≥128μg/ml).经1种药物诱导耐药后也不同程度对另外2种药物耐药. 结论:大肠埃希菌经氟喹诺酮类药物诱导后MIC变化显著(比诱导前增加8～8205倍),从体外证实大肠埃希菌对氟喹诺酮类药物逐步耐药的进程.     

抗生素持续作用下细菌传代次数对接合频率的影响

目的探究抗生素作用下细菌传代次数对接合频率的影响及其可能机制。方法将本实验室构建的携带庆大霉素(GM)抗性基因的质粒p UCP24T转入E.coli SM10λpir获得重组菌E.coli SM10λpir(p UCP24T)。将重组菌在含30μg/m L GM的琼脂平板上不断传代,每次挑取单个菌落传代,获得第50代和100代的E.coli SM10λpir(p UCP24T)。分别以铜绿假单胞菌PAO1、大肠埃希菌EC600和肺炎克雷伯菌A10为受体菌,第1、50、100代E.coli SM10λpir(p UCP24T)为供体菌进行接合实验,计算接合频率。采用微量肉汤稀释法检测第1、50、100代E.coli SM10λpir(p UCP24T)对GM的最低抑菌浓度(MIC)值。分别提取第1、50、100代E.coli SM10λpir(p UCP24T)的全基因组DNA,构建DNA文库并进行高通量测序,对测序结果进行生物信息学分析。结果成功获得重组菌E.coli SM10λpir(p UCP24T)。第50、100代E.coli SM10λpir(p UCP24T)与三种受体菌的接合频率较第1代均显著增高(P0.05),第100代菌株对GM的MIC值大于2 048μg/m L,较第1和第50代菌株对GM的MIC值1 024μg/m L升高。第1、50、100代E.coli SM10λpir(p UCP24T)重组菌中携带GM抗性基因的p UCP24T质粒的拷贝数分别为601.3、1 808.2及9 190.5,呈现增高的趋势。结论在抗生素持续作用下,E.coli SM10λpir(p UCP24T)通过增加耐药质粒拷贝数使接合频率及MIC值升高,以适应抗生素的持续作用。     

桂皮醛联合万古霉素对耐甲氧西林金黄色葡萄球菌生物膜的抑制作用

目的 研究桂皮醛联合万古霉素对耐甲氧西林金黄色葡萄球菌 (methiciline-resistant Staphylococcus aureus, MRSA) 生物膜的影响.方法 96孔板法构建金黄色葡萄球菌标准菌株ATCC25923和5株临床分离MRSA菌株的体外生物膜模型, 结晶紫半定量法检测其成膜能力;选取标准菌株和强成膜临床菌株为实验菌株, 采用氯化三苯基四氮唑 (TTC) 法确定药物单用及联用的最低抑菌浓度 (minimum inhibitory concentration, MIC) ;微量棋盘稀释法测出两药联用的最小抑膜浓度 (s MIC) .结果 96孔板培养3 d可见成熟生物膜形成, 结晶紫半定量法测得编号为16187的实验菌株有较强的产生物膜能力;桂皮醛和万古霉素对标准菌株的MIC为256μg/m L和1μg/m L, 对MRSA16187菌株的MIC为128μg/m L和0.5μg/m L, 两药联用对标准菌株的MIC为16μg/m L和0.06μg/m L, 对MRSA16187菌株的MIC为16μg/m L和0.03μg/m L;MRSA16187在桂皮醛和万古霉素联用与单独用药后s MIC50相比差别有显著性 (P<0.05和P<0.05) , s MIC90相比差别有显著性 (P<0.05和P<0.05) ;ATCC25923在桂皮醛和万古霉素联用与单独用药后s MIC50相比差别有显著性 (P<0.05和P<0.05) , s MIC90相比差别有显著性 (P<0.05和P<0.01) .结论 桂皮醛能显著增强万古霉素抗MRSA生物膜的作用, 且两药具有协同抗菌作用.     

HtsA表达质粒克隆和蛋白质的纯化

目的克隆HtsA重组表达质粒以获得HtsA表达蛋白。方法通过PCR方法扩增获得A组链球菌HtsA基因完整的序列,将此片段定向克隆到表达载体pET21d质粒中,在E.coli BL21(DE3)中表达融合蛋白,并运用离子交换层析和疏水层析分离纯化HtsA蛋白。结果经琼脂糖电泳证实成功克隆了重组表达质粒pET21d-HtsA,pET21d-HtsA融合蛋白在E.coli BL21(DE3)中得到表达,SDS-PAGE分析表明纯化的HtsA纯度较高。结论成功构建了A族链球菌HtsA基因重组表达质粒,并纯化获得了目的蛋白HtsA。     

白色念珠菌对五种抗真菌药物体外敏感性研究

目的了解白色念珠菌对常用抗真菌药物的敏感性情况,为临床正确选择抗真菌药物和有效控制该菌引起的感染提供依据。方法采用美国国家临床实验标准委员会(NCCLS)推荐的微量肉汤稀释法,测定从临床标本中分离的102株白色念珠菌对五种抗真菌药的最低抑菌浓度(MIC)。结果酮康唑、氟胞嘧啶和伊曲康唑对102株白色念珠菌MIC较低,其MIC90分别为0.25μg/ml、0.5μg/ml和0.5μg/ml。两性霉素B和氟康唑MIC90分别为2μg/ml和8μg/ml。受试菌对氟胞嘧啶敏感率最高为100%,对氟康唑和伊曲康唑的敏感率分别为88.3%和75.5%。对氟康唑耐药的9株菌株也同时对伊曲康唑耐药。结论受试白色念珠菌对氟胞嘧啶敏感率最高,未发现耐药菌株;酮康唑对受试菌抗菌活性最强,对唑类药物存在耐药性,且该类药物之间存在交叉耐药。     

南宁地区解脲支原体对4种抗菌药物敏感性分析

目的:为了了解南宁地区Uu的耐药情况,对本地区支原体耐药情况进行监测,指导临床用药.方法:对南宁地区NGU患者分离出的35株Uu,采用肉汤倍比稀释法测定4种抗生素的MIC值.结果:克拉霉素的MIC≤1μg/ml有17株Uu(71%),MIC50、MIC90分别为1μg/ml、16μg/ml;阿奇霉素的MIC≤4μg/ml有17株Uu(71%),MIC50、MIC90分别为4μg/ml、16μg/ml.美满霉素的MIC≤4μg/m1有11株Uu(占45%),MIC50、MIC90分别为8μg/ml、32μg/ml.氟罗沙星的MIC≤2μg/ml仅有6株Uu(25%)MIC≥8μg/ml的有23株Uu,MIC50、MIC90分别为8μg/ml、16μg/ml.结论:南宁地区的Uu对大环内酯类药物较为敏感,其中克拉霉素优于阿奇霉素.四环素类药物的敏感性较低,喹诺酮类药的敏感性最差.对四环素类药物高度耐药者,71%菌株对其它药物的MIC值较低(17/24).提示临床应警惕三类药物均耐药的情况.     

多粘菌素B等对多重耐药铜绿假单胞菌的体外抗菌活性研究

目的测定多粘菌素B等抗菌药物对铜绿假单胞菌的体外抗菌活性,为临床治疗药物选择提供实验室依据。方法对127株临床分离的多重耐药铜绿假单胞菌,采用琼脂对倍稀释法测定多粘菌素B、米诺环素等12种抗菌药物的MIC值。结果亚胺培南对多重耐药的铜绿假单胞菌有较强的抗菌活性,MIC50为2μg/ml,MIC90为16μg/ml,多粘菌素与米诺环素也有很好的抗菌活性,MIC50分别为0.5μg/ml与2μg/ml,MIC90分别为4μg/ml与8μg/ml。结论碳青霉烯类、多粘菌素B及米诺环素对临床常见多重耐药铜绿假单胞菌具有良好的抗菌活性。     

罗红霉素和阿奇霉素对金黄色葡萄球菌的防耐药突变浓度的研究

目的:测定罗红霉素和阿奇霉素对金黄色葡萄球菌的防耐药突变浓度(MPC),扩增突变选择窗内筛选的耐药突变体的耐药基因.以了解金黄色葡萄球菌对大环内酯类药物耐药的发生机制.方法:肉汤法富集1013CFU/L ATCC29213,采用平板二倍稀释法测定罗红霉素和阿奇霉素对ATCC29213的MIC,MIC99、初测MPC(Provisional MPC,MPCpr)以及MPC.PCR法扩增不同药物筛选出的耐药突变株的耐药基因ermA,ermB和ermC并测序.结果:罗红霉素和阿奇霉素对ATCC29213的MPC分别是0.24 mg/L和0.18 mg/L,细菌耐药选择指数(MPC/MIC99)是106和114.不同药物筛选出的耐药突变株中扩增出ermA和ermC基因.结论:单独使用罗红霉素和阿奇霉素,金黄色葡萄球菌易产生耐药突变株,应与其他抗菌药物联合.     

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.     

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.     

Electro-Acupuncture Combined with the Trigger Point Needle-Embedding for Treatment of Primary Trigeminal Neuralgia in 31 Cases

In recent years, the author of this essay has applied electro-acupuncture combined with the trigger point needle-embedding for treatment of primary trigeminal neuralgia in 31 cases, yielding satis- factory results as reported in the following.     

Investigation of Matrix Metalloproteinase -1, 2 and tissue inhibitor of matrix metalloproteinases-1 in Endometriosis

Objective： To explore the role of matrix metalloproteinase-1,2 （MMP-1, MMP-2） and tissue inhibitor of matrix metalloproteinases-1 （TIMP-1） in endometriosis. Methods： The eutopic and ectopic endometria from 40 subjects suffering from endometriosis and regular.endometria from 40 subjects （excluding endometriosis） were collected and examined by in situ hybridization technology and western blot assay. Results： Both expressions of MMP-1 and -2 were stronger in ectopic endometrium and eutopic endometrium than in normal endometrium. On the contrary, the expression of TIMP-1 in ectopic endometrium and eutopic endometrium was lower. The differences were significant （P 〈 0.01 ）. Moreover, there was no relationship among the expressions of MMP-1, 2 and TIMP-1 in ectopic endometrium. Conclusion： The expressions of MMP-1, 2 and TIMP-1 lose balance and lack of periodic changes in ectopic endometrium , which explains the biological invasive behavior of endometriosis. It was suggested-that regulating the balance between the MMPs and TIMP-1 should be an ideal therapeutic target to endometriosis.     

Shi Da-zhuo (史大卓) —An outstanding cardiologist of integrative medicine

Prof. SHI Da-zhuo, Ph.D., male, was born on March 20, 1960. Prof. SHI entered the Ph.D. program in 1990 at the China Academy of Chinese Medical Sciences under the supervision of Prof. CHEN Ke-ji, majoring in the treatment of cardiovascular diseases. After receiving his Ph.D. degree in 1993, Prof. SHI started working at the Cardiovascular Center in Xiyuan Hospital affiliated to China Academy of Chinese Medical sciences.     

Persist in and Carry Forward the Integrative Medical Research——Speech at the 6th National General Congress of Chinese Association of Integrative Medicine by Academician CHEN Zhu,Minister of Ministry of Health

The 6th National General Congress of Chinese Association of Integrative Medicine （CALM） was convened at 19-20, April 2008 in Beijing. Academician CHEN Zhu, the minister of Ministry of Health indicated at the congress that the integration of Chinese and Western medicine is very well in keeping with the situation of our country and the general rule of development in medical science; and as a good integration of Chinese medicine and Western medicine, it is mutually beneficial and advantageous to both of them. Seeing the creativity shown in integrative medical investigation in theoretic and methodological sides, we should and must persist in and develop it.