日本血吸虫精氨酸编码基因CDNA的分离和序列分析

【目的】分离和鉴定日本血吸虫(Schistosomajaponicum,S.j)新基因。【方法】应用免疫学方法筛选S.jcDNA文库获得阳性克隆;通过PCR直接序列测定技术,表达序列标签（EST）同源性检索对阳性克隆插入片段进行鉴定;采用亚克隆技术及生物信息学等技术,对获得的日本血吸虫精氨酸酶编码基因序列进行结构与功能的分析。【结果】获得了一个含1061bp外源插入片段的阳性克隆,其cDNA序列含有一个840bp的阅读框,编码279个氨基酸,属精氨酸酶家族,与国际蛋白质库中的酵母、蛙、人和大鼠精氨酸酶序列的同源性分别为44%,50%,51%,53%和54%。【结论】获得了编码日本血吸虫精氨酸酶编码基因全长cDNA,为日本血吸虫精氨酸酶基因功能的研究奠定了基础。     

日本血吸虫精氨酸酶编码基因cDNA的分离和序列分析

目的：分离和鉴定日本血吸虫（Schistosoma japonicum,S.j)新基因。方法：应用免疫学方法筛选S.j.cDNA库获得阳性克隆;通过PCR直接序列测定技术,表达序列标答（EST）同源性检索对阳性克隆插入片段进行鉴定／采用亚克隆技术及生物信息学等技术,对获的日本血吸虫精氨酸酶编码基因序列进行结构与功能的分析。结果：获得了一个含1061bp外源插入片段的阳性克隆,其cDNA序列含有一个804bp的阅读框,编码279个氨基酸,属精氨酶家族,与国际蛋白质库中的酵母、蛙、人和大鼠精氨酸酶序列的同源性分别为44％,50％,51％,53％和54％,结论获得了编码日本血吸虫精氨酸酶编码基因全长cDNA,为日本血吸虫精氨酸酶基因功能的研究奠定了.     

两个日本血吸虫成虫新基因的克隆和分析

目的 克隆并分析日本血吸虫（Sj)新的抗原基因，为血吸虫病疫苗研制提供有效的侯选抗原分析。方法 用日本吸虫肝门型童虫表膜抗原（SjHmAg)免疫兔血清筛选日本血吸虫成虫cDNA库，并对阳性克隆的插入片段进行PCR扩增及测序，通过互联网对测序获得的核苷酸序列进行同源性比较分析，并预测蛋白质结构与功能。结果 经3轮筛选，获10个阳性克隆，其插入的Sj成虫cDNA片段大小自0.6-3.0kb不等。对其中3个阳性克隆测序的结果显示，两个为新基因片段，SjHm1、SjHm2的Genbank登录号分别为AY118085，AY124772，分别编码79、66个氨基酸。SjHm1蛋白为跨膜蛋白，含1个跨膜区，1个N－肉豆蔻酰化位点。SjHm2蛋白为一个非跨膜蛋白，含1个酪蛋白激酶Ⅱ磷酸化位点。结论 获得的阳性克隆插入基因片段可能为编码具有保护性作用的日本血吸虫抗原的基因片段。     

日本血吸虫文库筛选、Tetraspanin 2的抗体检测及其单抗的制备

目的筛选新的可能具有诊断意义的血吸虫蛋白分子,并对其进行生物信息学分析。方法以血吸虫病人血清筛选日本血吸虫15 d肝期童虫cDNA文库,对阳性克隆插入片段的核苷酸进行序列分析,将结果通过Internet送入NCBI GenBank进行同源性比较并利用蛋白质分析软件进行生物信息学分析。结果经3轮筛选,共获15个阳性克隆,对其进行测序,获得11个基因,其中5个为编码日本血吸虫线粒体的基因,1个为编码日本血吸虫肌球蛋白的基因,其他5个分别为编码SjCHGC05315、SjCHGC01371、SjCH-GC04782、SjCHGC05166、SjCHGC09769的基因。结论从日本血吸虫肝期童虫cDNA文库中筛选到一批日本血吸虫基因,为血吸虫病新的诊断候选分子的研究提供了材料。     

紫外线致弱尾蚴免疫兔血清筛选血吸虫成虫cDNA文库

目的 :寻找照射致弱尾蚴中起免疫保护作用的抗原分子 ,为血吸虫病疫苗研究提供新的候选抗原。方法 :用紫外线照射致弱尾蚴免疫兔血清筛选日本血吸虫 (Schistosomajaponicum ,Sj)成虫cDNA文库 ,并对阳性克隆的插入基因片段进行PCR扩增及测序分析。结果 :经三轮筛选 ,获 10个阳性克隆 ,其插入的SjcDNA片段大小为 1 5～1.8kb。初步测序获 5个部分序列 ,其中 2个序列分别与Sj动力蛋白轻链 5 (DLC 5 )及Sj线粒体基因明显同源 ,其余 3个序列为未知的新基因片段。结论 :获得的阳性克隆插入基因片段可能为编码抗日本血吸虫感染的抗原基因     

青蒿鲨烯合酶基因及其cDNA的克隆与测序

【目的】克隆青蒿鲨烯合酶基因,为基因工程改造青蒿打下基础。【方法】采用聚合酶链反应(PCR)扩增、逆转录聚合酶链反应(RT-PCR)扩增、扩增片段与T-载体连接和克隆片段的序列分析等方法。【结果】通过PCR扩增和RT-PCR扩增,分别获得3590bp及1257bp片段,克隆后经PCR和酶切反应进行了鉴定。初步测序结果与GenBank上已登录的青蒿鲨烯合酶基因序列同源性为99％,氨基酸序列同源性为100％。【结论】成功克隆了青蒿鲨烯合酶基因及其cDNA。     

日本血吸虫亲免素基因的扩增及序列分析

【目的】研究日本血吸虫亲免素基因编码序列的结构及进行初步的功能预测。【方法】用5’端和3’端锚定PCR从尾蚴库中扩增，以获取日本血吸虫中国大陆株亲免素基因完整的开放阅读框(open reading frame，ORF)；用生物信息学技术确认其是否为亲免素编码基因并进行结构功能的初步分析和预测。【结果】用5’端和3’端锚定PCR从尾蚴库中获得了亲免素基因5’端和3’端所缺序列，得到的亲免素基因cDNA的总序列长为1438bp，其ORF长1296bp，编码431个氨基酸。利用生物信息学技术鉴定其为日本血吸虫亲免素基因的完整cDNA序列。并用RT-PCR从日本血吸虫尾蚴mRNA扩增出亲免素基因的ORF。【结论】成功获得了日本血吸虫亲免素编码基因的全长cDNA，为进一步的功能研究打下了基础。     

日本血吸虫真核生物翻译起始因子2α亚基全长cDNA的克隆与功能分析

目的对用表达序列标签(EST)策略从日本血吸虫(Schistosomajaponicum)尾蚴cDNA文库中筛选出的新基因进行全长cDNA克隆及功能预测。方法将插入于pTriplEx2 质粒上的cDNA进行测序,测序结果经BLASTn程序搜索,发现该插入cDNA序列与曼氏血吸虫真核生物翻译起始因子2α亚基(eukaryotic translation initiation factor 2 alpha subunit, eIF2α) 基因高度同源。根据该EST序列设计与pTriplEx2 质粒上的5'端测序引物相匹配的PCR下游引物,从该cDNA文库中扩出包含eIF2α全长ORF的cDNA片段。将PCR产物纯化后克隆到pGEM-T载体上,并对此克隆进行测序得到其全长cDNA序列。用NCBI 站点的BLASTx及BLASTn程序对所获得的新基因序列进行同源性搜索;用blast two sequence程序对同源性高的基因进行核苷酸及氨基酸水平的同源性比较。同时用网上分析软件进行基序和保守区域的搜索。结果发现一个与曼氏血吸虫eIF2α亚基mRNA高度同源的日本血吸虫新基因,核苷酸与氨基酸水平的同一性分别为87%和79%,编码由327个氨基酸组成的蛋白序列。结论用EST策略筛选到一个日本血吸虫新基因,编码eIF2α亚基,全长编码序列与曼氏血吸虫eIF2α亚基mRNA高度同源。     

日本血吸虫尾蚴66~68 kD单特异性血清对尾蚴cDNA文库的免疫学筛选

目的：获得日本血吸虫（Schistosoma japonicum，Sj）尾蚴66～68kD抗原的编码基因，为日本血吸虫疫苗和诊断研究提供材料。方法：以Sj尾蚴66～68kD抗原免疫家免获得的单特异性血清为探针，对Sj尾蚴cDNA文库进行免疫筛选，将所获阳性克隆的插入片断进行PCR扩增、琼脂糖电泳初步鉴定，并挑选出4个强阳性克隆进行测序，通过互联网NCBI／BLAST进行核苷酸序列分析。结果：共获得21个持续阳性克隆，其中10个阳性克隆初步鉴定显示为单一扩增条带，且插入的cDNA片段大小分布于0．5—3．0kb之间；4个强阳性克隆分别与日本血吸虫SJCHGC05187，SJCHGC05173，SJCHGC06989，SJCHGC01894显著同源。结论：获得了4种日本血吸虫相关的编码基因。     

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.