大肠杆菌poly（A）化mRNA基因片段的RD—PCR克隆

目的 研究大肠杆菌（E.coli）中哪些基因mRNA3′端存在poly(A）化。方法 利用E.colimRNA中可能存在的poly（A）尾，以oligo(dT）－cellulose进行mRNSA的纯化，并以oligo(dT）18为引物逆转录合成cDNA，应用限制性显示－聚合酶链反应（RD－PCR）分离不同组别的基因片段并进行克隆。结果 成功克隆100多个基因片段，并对其中30个进行了测序分析。结论 细菌mRNA的poly（A）化可能不是偶然和个别的现象。     

大肠杆菌poly(A)化mRNA cDNA文库的构建与鉴定

目的构建和鉴定大肠杆菌poly(A)化mRNA的cDNA文库。方法应用限制性显示PCR技术构建大肠杆菌poly(A)化mRNA的cDNA文库,筛选、收集cDNA片段,并进行测序。根据测序结果进行初步的生物信息学分析和结果验证。结果构建了大肠杆菌poly(A)化mRNA的限制性cDNA文库,并对其中66个基因片段进行了分析。结论所构建的大肠杆菌poly(A)化mRNA的cDNA文库片段重复性低,质量高。     

大肠杆菌poly（A）化mRNA cDNA文库的构建与鉴定

目的：构建和鉴定大肠杆菌poly(A)化mRNA的cDNA文库。方法：应用限制性显示PCR技术构建大肠杆菌poly(A)化mRNA的cDNA文库，筛选，收集cDNA片段，并进行测序。根据测序结果进行初步的生物信息学分析和结果验证。结果：构建了大肠杆菌poly(A)化mRNA的限制性cDNA文库，并对其中66个基因片段进行了分析，结论：所构建的大肠杆菌poly(A)化mRNA的cDNA文库片段重复性低，质量高。     

骨肉瘤cDNA文库的构建和筛选

目的 研究骨形态发生蛋白-2(BMP-2)在骨肉瘤发生、发展中的作用,构建SAOS-2细胞cDNA文库,寻找与BMP-2相关作用蛋白.方法 从骨肉瘤细胞系SAOS-2中提取总RNA,进而分离poly(A) RNA,用poly(A) RNA进行反转录并以SMARTⅢTM和CDSⅢoligo(dT)为引物进行PCR扩增,得到两端具有同源臂的PCR片段,以此同源臂为基础在酵母中实现同源重组.通过文库片段,线形化的pGADT7-Rec和诱饵质粒pGBKT7/HA-BMP-2共转化酵母AH109菌株在文库构建的同时进行与BMP-2相互作用蛋白的筛选;或先将文库片段,线形化的pGADT7-Rec转化AH109,再利用AH109和Y187两种酵母菌株的接合生殖进行筛选.最后用Far-Western blotting法进一步从体外论证BMP-2相互作用蛋白.结果 构建了具有基因多样性和库容量足够大的骨肉瘤cDNA文库,双链cDNA片段的长度大小范围为250～5000 bp.共转化的效率为4.3×105,重组效率为1.9×106,筛选的克隆数为4.3×105.接合法筛选时的接合效率为32%,筛选的克隆数为1.0×106.筛选到四个与BMP-2相互作用的阳性克隆.结论 此文库的多样性和库容量均符合筛选需求.可用于骨肉瘤相关基因的进一步筛选.     

human NF-κB miRNA干扰载体的构建

目的:设计并构建针对胰腺癌基因治疗中最关键的靶基因人细胞核因子κB(human NF-κB)的miRNA干扰载体。方法:根据靶基因设计并合成miRNA oligo(引物设计软件),将oligo退火成双链,然后用载体构建试剂盒进行重组克隆,将双链的miRNA oligo插入到miRNA表达载体中,构建miRNA质粒,转化入感受态细胞DH5α,从转化平板分别挑取克隆,用载体通用引物进行菌落PCR筛选,筛选得到的阳性克隆进行测序,以验证重组克隆中插入片段序列是否与设计的oligo序列一致。结果:经过比对,重组克隆中插入片段序列与设计的oligo序列完全一致。结论:成功构建了针对靶基因human NF-κB的miRNA干扰载体,为进一步研究奠定了基础。     

B6-Co小鼠Ankrd55和Ddx4突变候选基因克隆及测序

目的 对B6-Co小鼠Ankrd55和Ddx4基因进行克隆测序分析,寻找是否存在突变位点.方法 以小鼠Ankrd55和Ddx4基因的mRNA序列设计引物,以mRNA为模板,采用RT-PCR和PCR技术分段进行目的基因扩增,将目的片段连接在T载体上,转化至感受态细胞,筛选阳性克隆,提取质粒DNA分子,电泳检测,EooR(E)酶切释放目的片段,测序、分析、比对.结果 Ddx4基因位于小鼠13号染色体第113412349的碱基由C转换成A,导致编码氨基酸的密码子改变,产物脯氨酸(P)变成谷氨酰胺(Q).结论 Ddx4基因发生单碱基突变,表明该突变可能与B6-Co小鼠的EOB表型相关,但是有待于进一步研究.     

中华眼镜蛇毒腺cDNA表达文库的建立和分析

目的: 为研究开发眼镜蛇毒药用基因工程产品,筛选克隆及表达相关药用功能基因,构建中华眼镜蛇毒腺cDNA表达文库. 方法:一步法提取总RNA,Oligo(dT)纤维素层析柱纯化mRNA,逆转录PCR合成双链cDNA,分级分离除去小片段后,收集大于500 bp的cDNA片段,取10 ng cDNA与质粒载体pSPORT1连接、转化. 结果:获得克隆总数为2×105的眼镜蛇毒腺cDNA表达文库,重组率97%. 利用PCR技术从该文库扩增了心脏毒素-3(CTX-3)和磷脂酶A2(PLA2)基因的cDNA. 通过对文库克隆的序列测定和初步生物信息学分析,获得24个中华眼镜蛇毒腺EST序列. 结论:所建立的毒腺cDNA表达文库质量较高,可用于进一步筛选、克隆蛇毒药用功能新基因.     

TNF-α致脂肪细胞胰岛素抵抗相关基因的克隆和鉴定

目的分离和克隆肿瘤坏死因子α(TNF-α)致3T3-L1脂肪细胞胰岛素抵抗(IR)的相关基因.方法分化成熟的3T3-L1脂肪细胞用TNF-α处理48 h后抽提总RNA,采用mRNA差异显示技术分离和克隆TNF-α引起脂肪细胞IR的基因,再通过半定量RT-PCR证实.结果经mRNA差异显示技术分离和克隆到10个已知基因的cDNA,3个已知表达序列标签(ESTs)片段和1个新的EST片段.半定量RT-PCR证实TNF-α上调3T3-L1脂肪细胞Synip基因和血浆淀粉样蛋白A3(SAA3)基因的表达.结论Synip基因和SAA3基因的表达升高可能与TNF-α致3T3-L1脂肪细胞IR和相关的心血管并发症有关.     

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.