大鼠全长OBRb cDNA克隆及真核表达重组体的构建

目的克隆SD大鼠全长长胞内段瘦素受体基因cDNA(OBRb cDNA)及构建其真核表达重组体,为进一步研究该基因打下基础.方法应用RT-PCR方法分段扩增全长OBRb cDNA,再将它们连接起来,并获得载有全长OBRb cDNA的阳性克隆质粒.将上述质粒中全长OBRb cDNA定向插入真核表达质粒pcDNA3中,筛选出阳性克隆.通过限制性内切酶酶切和核苷酸序列测定进行鉴定.结果重组的克隆质粒和真核表达质粒经限制性内切酶酶切后获得的片段大小均与理论值一致.结论成功地克隆了SD大鼠全长OBRb cDNA,并构建了真核表达重组体.     

炎症信号受体TLR-4基因的克隆及真核表达

为探讨TLR 4基因在真核细胞的表达情况 ,用RT PCR技术从人脐静脉血管内皮细胞总RNA扩增TLR 4全密码子cDNA序列。限制性内切酶HindⅢ和BamHⅠ双酶切真核表达质粒pcDNA3和TLR 4cDNA片段 ,构建重组质粒pcDNA3 TLR4。用Dosper阳离子转染试剂包裹质粒 ,转染人胚肾 2 93细胞 (HEK 2 93细胞 )。用免疫荧光细胞化学染色及流式细胞术分析TLR 4基因表达。结果 :从人脐静脉血管内皮细胞扩增出 2 72 6bp的TLR 4cDNA片段。经PCR、酶切及序列测定分析证实质粒 pcDNA3内插入了TLR 4cDNA片段 ,免疫荧光细胞化学染色和流式细胞术分析均检测到TLR 4基因在人胚肾 2 93细胞表达。本研究显示重组真核表达质粒 pcDNA3 TLR4在HEK 2 93细胞表达TLR 4蛋白 ,有利于进一步研究其相应配体和信号传导通路     

EBV-LMP1 cDNA的克隆测序与原核表达质粒构建

目的：克隆EBV－LMP1 cDNA，构建EBV－LMP1基因的原核表达重组质粒。方法：通过RT－PCR技术从B95－8细胞中扩增EBV－LMP1 cDNA，克隆至pGEM-T easy载体上并测序；利用引物上的BamH I与HindⅢ酶切位点将LMP1基因插入载体pET-32a，转化JM109菌。提取质粒，限制性内切酶消化，琼脂糖凝胶电泳鉴定。结果：扩增的LMP1 cDNA长度为1158bp，测序结果与已知序列吻合，重组原核表达质粒经酶切鉴定表明获得正确重组子。结论：成功克隆了EBV－LMP1 cDNA，并构建了pET-32a-LMP1原核表达载体，为研究LMP1在EBV致瘤中的作用及肿瘤疫苗奠定了基础。     

人Fn14基因真核重组质粒的构建及其表达

目的构建人成纤维细胞生长因子诱导早期反应蛋白14（fibroblast growth factor—inducible 14，Fn14）基因真核重组质粒及其表达。方法从人肝细胞癌组织抽取总RNA，RT—PCR扩增出Fn14基因全长cDNA，经测序正确后，核酸内切酶EcoR I、Xba I酶切PCR产物，亚克隆人pcDNA3．1-Myc载体，重组质粒pcDNA3．1-Myc—Fn14经酶切及测序鉴定正确后转染COS-7细胞，通过免疫组织化学DAB法检测Myc—Fn14融合蛋白的表达。结果重组质粒pcDNA3．1-Myc—Fn14经EcoR I、Xba I酶切及测序鉴定正确，重组质粒包含人Fn14基因完整编码区。免疫组织化学检测结果显示，重组质粒转染COS-7细胞后成功表达Myc—Fn14融合蛋白，转染空载体pcD—NA3．1-Myc的COS-7细胞未见Myc—Fn14融合蛋白表达。结论构建了人Fn14基因真核表达载体，该重组质粒可表达含Myc-Fn14的融合蛋白，为研究Fn14功能奠定了基础。     

人自然杀伤细胞抑制性受体P58.2基因克隆与鉴定

目的：P58．2基因克隆与鉴定。方法：采用RT—PCR技术，从正常人外周血单个核细胞中扩增自然杀伤细胞抑制性受体P58．2基因全长cDNA，经酶切后构建重组克隆载体，双酶切和测序鉴定。结果：RT—PCR获得了预期的扩增产物P58．2全长cDNA，成功构建了pSPORT1-P58．2重组克隆载体，酶切、酶谱分析与预期结果相符。DNA测序结果与GenBank登记的人P58．2cDNA全长碱基序列一致。结论：pSPORT1-P58．2重组克隆载体构建成功；P58．2基因序列与文献报道一致，为下一步构建表达载体和基因转染等工作打下良好的基础。     

人骨骼肌TnI-fast基因克隆和体外表达

目的：构建人骨骼肌TnI-fast基因分泌型真核表达载体，为利用TnI-fast基因进行抗肿瘤血管生成基因治疗奠定基础。方法：采用PCR和RT－PCR制备TnI-fast cDNA，构建TnI-fast基因克隆质粒，测序证实后将TnI-fast cDNA插入到pSecTag2B构建分泌型重组真核表达质粒。然后用TnI-fast基因重组表达质粒转染COS－1细胞，检测TnI-fast基因体外表达。结果：DNA测序证实，我们从人骨骼肌总RNA和cDNA文库中扩增的TnI-fas cDNA序列与NCBIGenebank TnI-fast cDNA序列完全一致。ELISA检测证实，TnI-fast/pSec Tag2 B转染后目的基因可在真核细胞中分泌表达。结论：本研究成功地构建了TnI-fast基因重组表达质粒，可进一步用于抗肿瘤血管生成基因治疗动物实验。     

人截断型转化生长因子β受体Ⅱ真核表达载体的构建和表达

目的构建无细胞内段基因的人转化生长因子βⅡ型受体（△TβRⅡ）的真核表达载体pEGFP／△TβRⅡ，转染L02细胞以表达融合蛋白EGFP／△TβRⅡ，并检测其生物学活性。方法以质粒H2-3FF为模板，用PCR方法扩增得到人转化生长因子βⅡ型受体（TpRⅡ）胞外段和跨膜段的cDNA，将该cDNA克隆到真核表达载体pEGFP-N2上，构建成pEGFP／△TβRⅡ重组质粒。经双酶切和核酸测序鉴定，将该重组质粒转染L02细胞，用倒置荧光显微镜观察融合蛋白EGFP的表达，并检测其生物学活性。结果L02细胞转染pEGFP／△TβRⅡ重组质粒后，在荧光显微镜下可以观察到融合蛋白EGFP的表达，所表达的蛋白能显著减轻TGF-β1对L02细胞G1～S期的阻滞作用。结论成功构建了pEGFP／△TβRⅡ重组质粒，并表达了有活性的融合蛋白EGFP／△TβRⅡ，为进一步探讨其生物学效应奠定了基础。     

神经元细胞信号传导蛋白质14-3-3ζ编码基因的扩增和克隆

目的 克隆神经元信号传导蛋白质14－3－3ζ编码基因，以研究其在人神经退行性病变中的诊断价值、细胞信号传导过程中的作用及对细胞分裂、细胞凋亡的影响。方法 提取人脑胶质瘤细胞总mRNA，以其为模板逆转录合成cDNA第一链，设计合成引物，用PCR扩增14－3－3ζ基因编码序列，将其插入pGEM-T载体，并用双酶切和以质粒为模板的PCR进行鉴定。结果 RT－PCR扩增出一条约750bp大小的特异性条带，重组质粒的双酶切和以质粒为模板的PCR获得了一条与RT－PCR大小相同的条带。经DNA测序证明所克隆的DNA片段与GenBank收录序列一致。结论 信号传导蛋白质14－3－3ζ重组pGEM-T克隆载体的成功构建，为进一步研究提供了条件。     

大鼠微管相关蛋白4重组腺病毒载体的构建及在细胞中的表达鉴定

目的构建大鼠微管相关蛋白4(m icrotubu le-ossoc iated prote in 4,MAP4)重组腺病毒载体,并转染新生大鼠体外培养的心肌细胞进行表达和鉴定,为真核表达及有关实验研究提供基础。方法设计1对引物,应用逆转录多聚酶链式反应(RT-PCR)技术,从大鼠心肌细胞总mRNA中扩增出MAP4 cDNA,并将MAP4 cDNA克隆到腺病毒穿梭质粒pShuttle2中,构建pShuttle2-MAP4表达质粒。将此表达质粒扩增纯化酶切获得含有目的片段MAP4 cDNA的表达盒与Ad-eno-X V iru l DNA重组并线性化后转染HEK293T细胞,包装产生大鼠MAP4重组腺病毒。并且转染新生大鼠原代心肌细胞,应用免疫组化的方法进行表达鉴定。结果扩增所得的MAP4 cDNA片段经酶切鉴定、PCR扩增、DNA测序最终确定为大鼠MAP4基因,所得大鼠MAP4重组腺病毒滴度为2.3×108pfu/m l。转染原代心肌细胞48 h后MAP4表达显著增强。结论为研究MAP4的生物学活性及功能的基因治疗提供了基础。     

尿激酶受体反义RNA表达质粒的构建

目的 构建能在哺乳动物细胞中表达尿激酶受体（ｕＰＡＲ）反义ＲＮＡ的表达质粒ｐＵＲＡＳ。方法 采用ＲＴ－ＰＣＲ方法扩增并克隆尿激酶受体（ｕＰＡＲ）ｃＤＮＡ５’端－４６￣４５４ｂｐ一段为５００ｂｐ的顺序，利用ＤＮＡ重组技术将该片段反向插入表达载体ｐｃＤＮＡ３的ＣＭＶ启动子下游。结果 限制性内切酶分析和ＤＮＡ顺序分析证实ＲＴ－ＰＣＲ获得ｕＰＡＲ ｃＤＮＡ片段与文献报道相吻合；重组质粒ｐＵＲＡＳ转染肺癌细     

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.