幽门螺杆菌Ⅳ型分泌系统cagM基因的克隆、表达及抗体制备

目的:构建表达幽门螺杆菌(Helicobacter pylori, H.pylori) NCTC 11637 IV型分泌系统cagM(HP0537)全长编码基因的原核表达载体,分析其抗原性并制备抗体.方法:应用PCR技术从H.pylori基因组扩增 cagM基因片段,TA克隆后测序分析,并构建表达载体pET-28a(+)-cagM,转化 E.coli BL21,IPTG诱导表达,SDS-PAGE及鉴定表达蛋白,表达蛋白以 Ni2+-NTA 柱进行纯化,用软件分析其抗原性后,免疫家兔制备多克隆抗体,ELISA检测多克隆抗体效价.结果:测序结果表明cagM基因全长1 131 bp(基因库登录号为GU269568),编码376个氨基酸,与基因库中已知菌株J99,26695和G27的氨基酸同源性为98%～99%;诱导表达后经SDS-PAGE检测表明,在43 700处发现一条新生条带,Ni2+-NTA柱纯化后为单一条带,软件分析表明其抗原性较强,免疫家兔制备的多克隆抗体效价为1∶ 1.6×105.结论:首次成功克隆并表达了H.pylori pNCTC 11637 cagM基因,并制备了其抗体,为以后进一步研究其生物学功能以及H.pylori相关疾病的检测与治疗奠定了基础.     

幽门螺杆菌外膜蛋白cagL基因的克隆、表达及其对IL-8 mRNA表达的影响

目的:克隆表达幽门螺杆菌(Helicobacter pylori, H.pylori)NCTC11637菌株cagL基因,并探讨其对IL-8 mRNA表达的影响.方法:设计cagL引物,应用PCR扩增目的基因片段,TA克隆后测序分析, 用信息学软件分析其物理化学特性,并构建表达载体cagL-PET28a(+)转化E.coli BL-21(DE3),IPTG诱导表达, 镍亲和层析纯化,SDS- PAGE及蛋白质印迹鉴定表达蛋白.纯化蛋白与细胞GES-1共培养,提取细胞总RNA,RT-PCR检测细胞IL-8mRNA表达.结果:测序分析表明cagL编码成熟肽的碱基序列为654 bp,编码217个氨基酸,与基因库公布的其他H.pylori菌株的基因序列同源性为96%～98%,氨基酸同源性为 97%～99%,软件预测显示有多个明显的具有抗原活性的结构域;表达蛋白的相对分子质量约为32 000,蛋白质印迹检测融合蛋白具有良好的抗原活性;重组蛋白作用于GES-1细胞后,可诱导细胞因子 IL-8 在 mRNA 水平上的表达.结论:CagL蛋白可刺激GES-1细胞IL-8 mRNA的表达.     

幽门螺杆菌重组1188蛋白的表达

目的 表达幽门螺杆菌(H.pylori)重组1188蛋白,为研究幽门螺杆菌的黏附机制奠定基础.方法 用聚合酶链反应(PCR)技术从H.pylori标准株NCTC 11637的基因组DNA中扩增hp1188基因片段,插入原核表达载体pQE-30,构建重组载体pQE30-hp1188,经DNA测序分析确认后,转化大肠杆菌DH5α,异丙基硫代半乳糖苷(IPTG)诱导表达.Ni2+-NTA树脂纯化表达蛋白,十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)分析表达形式和表达量.结果 重组表达质粒pQE30-hp1188构建成功,所得序列完整,插入的基因片段全长810 bp,与基因文库中的hp1188基因同源性达98%.SDS-PAGE显示表达产物相对分子质量约为30.6 kD,与预期一致,蛋白表达量占全菌总蛋白的47%,上清液和沉淀中均有表达,重组蛋白纯化后纯度达90%以上. 结论 成功克隆hp1188基因,并在大肠杆菌DH5α中获得高效表达.     

幽门螺杆菌hp1188基因的原核表达

目的:重组表达幽门螺杆菌(Helicobacter pylori,H.pylori)hp1188基因,为研究该基因所编码蛋白在H.pylori的黏附过程中的作用奠定基础.方法:以H.pylori标准株NCTC 11637基因组DNA为模板,PCR扩增hp1188基因片段,克隆至质粒pQE-30中获得重组质粒pQE30-hp1188,经DNA测序确认后将重组质粒转化大肠杆菌DH5 α,IPTG诱导表达.Ni2+-NTA树脂亲和层析纯化表达蛋白,SDS-PAGE检测表达,Western blot鉴定相应抗原表位.结果:扩增的hp1188基因片段全长810 bp,与基因文库中的hp1188基因同源性达98%.SDS-PAGE显示表达产物相对分子量约为30 600 Dalton,与预期相符;大肠杆菌裂解液中表达产物接近可溶性蛋白总量的一半,但沉淀中仍能检测到表达蛋白.目标蛋白纯化后均一性接近90%,Western blot证实与多克隆抗体有良好结合能力.结论:成功克隆了hp1188基因,并在大肠杆菌DH5α中获得高效表达.     

幽门螺杆菌Cag-PAI hp0523基因的克隆及功能预测

目的:明确幽门螺杆菌Cag-PAI hp0523基因的功能,探讨其在幽门螺杆菌致病中的作用.方法:设计引物,以幽门螺杆菌NCTC11637为模板,采用聚合酶链反应(PCR)扩增获得目的片段;将其进行T-A克隆后,进行测序.将该基因序列与其他菌株做BLAST比对分析,并结合生物信息学数据库和软件,分析其序列特征和理化特性.结果:经酶切,测序分析表明,插入的基因片断为510 bp,与基因库公布的幽门螺杆菌 26695及J99序列相比较,同源性高达 94%;软件预测其编码的蛋白为169个氨基酸,其相对分子质量约为19.7 kDa,等电点约为9.53;基序分析表明,在33～165位氨基酸之间存在一个保守的SLT催化基序,可能是溶菌糖基转移酶;对其催化能力进行预测,结果表明,HP0523(33.7%)催化能力可能较T4溶菌酶(32.3%)和溶菌酶SLT70(28.6%)的活性高.结论:成功克隆了幽门螺杆菌Cag-PAI的hp0523基因,对其序列进行了深入分析,为进一步研究其功能,阐明幽门螺杆菌Cag-PAI的致病机制奠定了基础.     

线粒体途径在幽门螺杆菌诱导胃癌细胞凋亡中的作用

目的 探讨线粒体途径在幽门螺杆菌(H.pylori)诱导胃痛细胞凋亡中的作用.方法 采用流式细胞术检测H.pylori NCTC 11637诱导的SGC-7901细胞凋亡,RT-PCR和Western blot法检测H. Pylori感染对线粒体途径相关基凶和蛋白表达的影响,并观察Caspase抑制剂对H. Pylori所致SGC-7901细胞凋亡的影响.,结果 H. Pylori能够在体外直接诱导SGC-7901细胞凋亡,6、12、24 h和48 h的凋亡率分别为6.30%、11.57%、8.63%和7.22%;H. Pylori能够呈时间依赖性地上调SGC-7901细胞的Bax蛋白表达,并促进Caspase-9和3的激活;Caspase-9和3抑制剂能够显著抑制NCTC 11637所致的SGC-7901凋亡,而Caspase-8抑制剂仪能部分抑制约20%NCTC 11637所致的SGC-7901凋亡.结论 H.pylori可引起SGC-7901细胞Bax基因和蛋白表达升高,激活Caspase-9和Caspase-3,且主要通过线粒体途径诱导细胞凋亡.     

幽门螺杆菌Cag致病岛hp0540基因的克隆表达及其多克隆抗体的制备

目的：构建幽门螺杆菌（Helicobacter pylori,Hp）Cag致病岛（Cag—pathogenicity island,Cag—PAI）编码的hp0540基因的原核表达系统,并制备其多克隆抗体。方法：应用PCR技术从Hp11637基因组DNA中扩增hp0540基因片段,克隆至pMD18-2T载体后,进行序列测定,并对其序列进行生物信息学分析;构建pET-28a—hp0540原核表达载体,转化表达宿主菌BL21DE3;经IPTG诱导表达后,SDS—PAGE法鉴定目的蛋白的表达,并以Ni^2＋-NTA柱分离纯化目的蛋白;将纯化蛋白免疫新西兰大白兔制备多克隆抗体并测定抗体效价。结果：成功克隆了hp0540基因,全长1086bp,编码361个氨基酸,与基因库公布的其他坳菌株基因序列的核苷酸同源性为98％。工程菌诱导后SDS—PAGE显示新生表达蛋白带,相对分子质量约为39000,经Ni^2＋NTA柱纯化后可获得重组蛋白,重组蛋白免疫新西兰大白兔后获得效价为1：160000的多克隆抗体。结论：成功构建了hp0540基因的原核表达系统并制备了其多克隆抗体,为研究该基因的功能奠定了基础。     

幽门螺杆菌适应性定植相关基因HP0318的克隆及序列分析

目的 测定幽门螺杆菌(HP)适应性定植蒙古沙鼠前后HP0318基因序列并分析其序列差异, 以了解该基因在HP适应性定植过程中在基因水平是否发生了改变,为进一步研究HP的适应性变异机制提供实验依据.方法 对幽门螺杆菌临床分离株M0、沙鼠适应株M13及标准株NCTC11637基因组中的HP0318全长基因进行扩增,然后连接到PMD18-T载体进行测序,最后通过DNAsis软件对不同菌株来源的HP0318基因进行多重序列比对,并与Genbank公布的2个标准株(HP26695株和J99株)的HP0318序列进行对比分析.结果 PCR法成功扩增出了3个菌株的HP0318基因片段,克隆到PMD18-T载体上酶切鉴定正确.测序结果表明3株幽门螺杆菌(M0、 M13、11637)的HP0318序列大小均为756bp.序列分析显示在不同来源的HP菌株中表现出较高的保守性,最低相似性达到94%.结论 HP0318基因属于HP特异性基因,HP在沙鼠中适应性定植可能并不是由HP0318基因水平的变异引起,有必要进一步实验验证其功能和阐明其所具有的调控机制.     

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.     

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.     

Scientific principles and rigorous processes should be followed in developing clinical guidelines for therapeutic interventions of integrative medicine

A clinical guideline for the therapeutic interventions of integrative medicine may be defined as a written document which states a series of recommendations on therapeutic interventions of integrative medicine for a special disease or condition. The guideline may provide assistance to medical professionals in making clinical decisions aimed at improving the clinical outcome of patients and reducing the costs of medical care（~＇4~. Recommendations issued by a guideline should be based on the best available evidence in both Western and Chinese medicine. For fulfilling this purpose, the development of clinical guidelines for therapeutic interventions in the field of integrative medicine should follow scientific principles and undergo a rigorous processes.