粉尘螨主要变应原基因Der f1和Der f3改组的研究

目的:探索粉尘螨变应原基因Der f1和Der f3改组条件,以期获得粉尘螨变应原融合突变基因。方法:RT-PCR方法扩增粉尘螨变应原Der f 1和Der f3基因,用DnaseⅠ分别酶解Der f 1和Der f3基因10、15、20、25、30 min;酶解相同时间的Der f1和Der f3基因片段两两混合,采用DNAshuffling技术对粉尘螨变应原Der f1和Der f3基因进行重组,琼脂糖凝胶电泳检测重组子。结果:以Der f 2 F和Der f2 R为引物在酶切10、15、20、25、30min均可扩增出清晰条带;以Der f1 F和Der f1 R、Der f1 F和Der f2 R、Der f2 F和Der f1 R为引物在酶切10、15、20、25、30min的模板中均无条带出现;而其他引物组合则在不同酶切时间的模板中均出现条带。结论:通过多种条件的组合,可获得多个粉尘螨变应原基因Der f1和Der f3间的融合基因,为大规模制备高效、低价的哮喘疫苗奠定了基础。     

尘螨Ⅱ类改组变应原对哮喘小鼠免疫治疗的效果

目的 探讨以改组尘螨Ⅱ类变应原基因Der f2和Der p2后表达的蛋白为变应原,对小鼠哮喘模型的特异性免疫治疗效果。方法 随机将160只清洁级BALB/c小鼠分为PBS组（阴性对照组）,Der f2和Der p2免疫治疗组（阳性对照组）,哮喘组,改组变应原免疫治疗M01、M03、M08、M10蛋白治疗组。用尘螨提取液于0、7、14d腹腔注射致敏激发BALB/c小鼠,第21天雾化吸入激发,连续7d,其中各免疫治疗组于第25～27天雾化前30min分别用Der f2、Der p2和改组变应原进行特异性免疫治疗,PBS组则用PBS进行腹腔注射和雾化吸入。最后1次雾化24h后,引颈处死。分别观察肺组织病理变化、支气管肺泡灌洗液（BLAF）白细胞计数,酶联免疫吸附试验（ELISA）测定BLAF和脾细胞培养上清液细胞因子IL-4、IL-17和IFN γ的含量及血清中特异性IgG1、IgE抗体水平变化。结果 与哮喘组比较,改组变应原免疫治疗组和阳性治疗组肺部炎症显著减轻, BALF中的总细胞数及嗜酸性粒细胞数,血清中抗原特异性IgG1、IgE抗体均显著降低;免疫治疗组（包括阳性对照）的BALF和脾细胞培养上清液中的细胞因子IL-4、IL-17均明显降低,IFN-γ含量显著升高。结论 通过基因改组获得的尘螨Ⅱ类变应原改组疫苗免疫治疗小鼠哮喘,可有效降低尘螨引起的小鼠肺部炎症。     

重组Der f1变应原诱导小鼠哮喘模型的建立

目的建立重组粉尘螨Ⅰ类变应原（Der f1）诱导BALB/c小鼠哮喘模型。方法 30只BALB/c小鼠随机分成3组：PBS阴性对照组、卵清蛋白（OVA）阳性对照组、重组Der f1模型组。用酶联免疫吸附试验（ELISA）检测小鼠血清中IgG1、IgG2α和IgE含量,ELISA法检测脾细胞培养上清液和支气管肺泡灌洗液（BALF）IL-2、IL-4、IL-5、IL-17和IFN-γ含量,计数BALF中细胞总数,同时切取小鼠未灌洗侧肺组织进行病理学观察。结果 OVA阳性对照组、重组Der f1模型组小鼠血清中IgG1、IgG2a和IgE含量,脾细胞上清液和BALF中IL-2、IL-4、IL-5、IL-17和IFN-γ含量与PBS阴性对照组相比差异均具有统计学意义（P〈0.05或P〈0.01）;OVA阳性对照组、重组Der f1模型组小鼠BALF中细胞总数和嗜酸性粒细胞（EOS）计数显著高于PBS组（P〈0.05或P〈0.01）。OVA阳性对照组和重组Der f1模型组小鼠肺部病理改变呈明显的变态反应性炎症。结论用重组Der f1能成功诱导BALB/c小鼠哮喘模型。     

粉尘螨Ⅰ类变应原成熟肽段基因的克隆和生物信息学分析

目的了解不同地区粉尘螨Ⅰ类变应原(Der fⅠ)成熟肽段基因序列的变异性及其编码蛋白的化学及免疫学特征。方法从皖南地区分离鉴定的活粉尘螨,提取总RNA,采用RT-PCR扩增Der fⅠ成熟肽段基因,克隆到T载体测序确认。应用生物信息学软件对该基因进行初步分析。结果获得皖南地区两种不同基因型的Der fⅠ成熟肽段基因,与GenBank公布的其他地区Der fⅠ基因序列比较同源性在98.73%～99.84%之间;相应编码氨基酸序列的同源性在99.04%～100%之间,存在6个氨基酸残基变异位点,并影响其抗原表位的改变。结论不同地区Der fⅠ成熟肽段基因序列及其编码氨基酸序列存在差异,并对其免疫原性产生影响。克隆并分析研究我国各地的尘螨变应原基因序列及氨基酸序列变化,对于临床变应性疾病的诊治有重要的意义。     

粉尘螨Der f5克隆表达、纯化和免疫原性鉴定

目的克隆表达粉尘螨第5组变应原(dermatophagoides farinae,Der f5)基因,并鉴定纯化蛋白免疫原性。方法根据Der f5基因已知序列,设计出相应的引物,提取粉尘螨总RNA,采用RT-PCR方法扩增出Der f5基因片段,PCR产物克隆入pMD18-T载体,转化大肠埃希菌Top10,经PCR和酶切鉴定并测序。将上述所得阳性克隆菌株扩大培养,碱裂解法提取质粒,所得重组质粒pMD18-T-Der f5和空质粒pET-32a同时用限制性内切酶Bam HⅠ与HindⅢ双酶切,经纯化后连接并转化至大肠埃希菌Top10。构建的重组质粒pET32a-Der f5,经PCR、酶切和测序鉴定后,再转化至大肠埃希菌BL21(DE3),异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达。用十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和蛋白质印迹法(Western blotting)鉴定其表达效果,用Ni+离子亲和层析柱纯化重组质粒pET32a-Der f5表达产生的组氨酸重组蛋白。结果构建了重组质粒pMD18-T-Der f5和pET32a-Der f5。SDS-PAGE结果表明Der f5基因在大肠埃希菌BL21(DE3)中获得良好的表达,经亲和层析纯化后,SDS-PAGE结果显示单一条带。该蛋白以尘螨过敏患者血清进行Western blotting,结果表明具有良好的IgE结合活性。结论克隆、表达并纯化了具有良好尘螨致敏患者IgE结合活性的Der f5,为粉尘螨变态反应性疾病的特异性诊断和治疗以及进一步的实验研究奠定了基础。     

粉尘螨I类变应原成熟肽段基因的克隆和生物信息学分析

目的 了解不同地区粉尘螨Ⅰ类变应原(Der f I)成熟肽段基因序列的变异性及其编码蛋白的化学及免疫学特征.方法 从皖南地区分离鉴定的活粉尘螨,提取总RNA,采用RT-PCR扩增Der fⅠ成熟肽段基因.克隆到T载体测序确认.应用生物信息学软件对该基因进行初步分析.结果 获得皖南地区两种不同基因型的Der fⅠ成熟肽段基因,与GenBank公布的其他地区Der fⅠ基因序列比较同源性在98.73%-99.84%之间;相应编码氨基酸序列的同源性在99.04%-100%之间,存在6个氨基酸残基变异位点,并影响其抗原表位的改变.结论 不同地区Der fⅠ成熟肽段基因序列及其编码氨基酸序列存在差异,并对其免疫原性产生影响.克隆并分析研究我国各地的尘螨变应原基因序列及氨基酸序列变化,对于临床变应性疾病的诊治有重要的意义.     

尘螨1类变应原Derf1和Derp1生物信息学分析

目的:分析尘螨1类变应原的理化特性。方法:Gen Bank检索尘螨1类变应原Der f 1和Der p 1的氨基酸序列,利用生物信息学软件预测分析这两个氨基酸序列的一级结构、疏水性、跨膜区、二级结构及空间结构,并对其可能的功能位点进行预测。结果:尘螨1类变应原Der f 1和Der p 1分别编码321和320个氨基酸,分子量分别为36.4和36.1 ku,理论等电点(p1)分别为5.66和5.65,两蛋白均为亲水性蛋白且可能均无跨膜区,Der f 1的二级结构以不规则卷曲为主(43.93%),而Der p1的二级结构以α螺旋为主(43.75%)。两变应原均由蛋白酶抑制子I 29和木瓜蛋白酶C-端2个结构域组成,且半胱氨酸活性位点、组氨酸活性位点和天冬酰胺活性位点的位置相似。结论:Der f 1和Der p 1的这些特性有助于为螨性过敏性疾病的变应原特异性免疫治疗提供理论支持。     

尘螨变应原Der f 2基因克隆及序列分析

目的:获得尘螨变应原Der f 2基因及其生物信息学资料.方法:提取粉尘螨总RNA,RT-PCR合成Der f 2的cDNA片段,回收PCR产物并连接至pMD19-T simple,经测序验证后亚克隆入表达载体pET-28a( ),转化大肠杆菌感受态细胞后提取质粒,双酶切鉴定.用ExPaSy,EBI,NCBI网站的在线软件对测序结果进行分析.结果:RT-PCR扩增获得了Der f 2 cDNA片段,酶切、电泳结果表明克隆和亚克隆获得成功.与参考序列相比,测序结果多了87 bp(77～163),但Blastn发现中国广州和德国Reinbek报道的序列中也含此87bp.同源性、相似性、序列比对及分子进化分析提示,该序列与中国广州和德国Reinbek报道的序列亲缘关系较近.推测该变应原由176个氨基酸组成,与附睾分泌蛋白E1具有同源性,信号肽位于1～17aa处,在6～24aa处有一跨膜螺旋.二级结构由a-螺旋(16.57%),延伸链(32.57%)和随机卷曲(50.86%)组成.结论:获得了粉尘螨变应原Der f 2编码基因及其分子特征,为进一步生产基因工程变应原用于临床诊治变态反应性疾病奠定了基础.     

粉尘螨第五组主要变应原(Der f5)的克隆表达、纯化及免疫原性鉴定

目的 克隆表达粉尘螨第五组变应原(Dermatophagoides farinae,Der f5)基因,并鉴定纯化蛋白免疫原性.方法 提取活粉尘螨总RNA,扩增Der f5片段.PCR产物与克隆载体pMD18-T连接,转化入大肠埃希菌JM109,经酶切及测序鉴定获得pMD18-Der f5阳性菌株,再提取质粒进行双酶切,与表达载体pET-30a(+)连接,转化人大肠埃希菌BL21,异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达.SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)和蛋白质印迹法(Western blotting)鉴定其表达效果.Ni-IDA亲和层析柱纯化蛋白,利用尘螨病人血清鉴定其免疫原性.结果 构建了重组质粒pMD18-Der f5和pET30a-Der f5,SDS-PAGE结果表明Der f5基因在BL21中获得良好的可溶性表达,蛋白质分子量与理论值相符.纯化的蛋白与病人血清有良好的IgE结合活性.结论 获得广州地区Der f5的原核表达载体,高效表达纯化重组蛋白.初步鉴定了该蛋白的免疫原性.     

室内空调机滤尘网尘螨、腐食酪螨变应原基因的检测

目的:检测室内空调机滤尘网中粉尘螨1类变应原(Dermatophagoides farinae 1,Der f 1)、屋尘螨1类变应原(Dermatophagoides pteronyssinus 1,Der p 1)和腐食酪螨1类变应原(Tyrophagus putrescentiae 1,Tyr p 1).方法:分...     

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.