端粒酶抑制剂筛选模型诱饵载体的构建与转化

目的构建含不同人端粒酶RNA基因片段的诱饵融合表达载体并转化酵母,用于酵母三杂交方法筛选端粒酶抑制剂。方法采用RT—PCR方法从肿瘤细胞中扩增人端粒酶RNA（hTR）基因后,分别扩增hTR基因假结合体区CR4-CR5及H／ACA和CR7区hTR基因片段,纯化分离定向克隆到pRH3诱饵质粒,构建包含不同hTR目的基因片段的重组质粒,转化大肠杆菌,经PCR、酶切、测序鉴定后,将阳性重组质粒转化酵母菌,进行毒性自激活检验。结果测序结果证实,含不同hTR基因片段诱饵重组质粒pRH3-hTR构建成功,转化酵母后无毒性和自激活性。结论成功构建了酵母三杂交系统诱饵载体,可于酵母三杂交系统筛选端粒酶抑制剂及在酵母中研究hTERT和hTR间的相互作用．     

高通量端粒酶抑制剂筛选模型融合表达载体的构建及转化

目的 构建含不同结构域的人端粒酶逆转录酶(hTERT)酵母三杂交系统融合蛋白表达载体并转化酵母.方法 采用PCR法从pCLXSN-hTERT质粒中提取不同的hTERT cDNA片段,纯化分离定向克隆到pYESTrp3质粒,构建包含不同hTERT蛋白结构域的编码序列的重组质粒,转化大肠杆菌,经PCR、酶切、测序鉴定,将阳性重组质粒转化酵母菌,进行毒性和自激活检验.结果 经过测序验证,重组融合蛋白表达载体pYESTrp3-hTERT构建成功,转化酵母无毒性和自激活性.结论 成功构建能用于酵母三杂交系统的融合蛋白表达载体,可于酵母三杂交系统筛选端粒酶抑制剂及hTERT和hTR间的相互作用.     

人可溶性增殖诱导配体cDNA突变体的设计与构建

目的构建6种人可溶性增殖诱导配体(soluble a proliferation-inducing ligand, sAPRIL)cDNA突变体.方法采用PCR将可溶性增殖诱导配体(sAPRIL)cDNA进行突变,并分别在其N端和C端连接TEL Th表位序列,插入到pQE80表达载体上,最后转化至DH5α感受态细胞.重组质粒经酶切鉴定后进行测序.结果经PCR扩增分别得到了对应于6种突变体的sAPRIL序列,酶切和测序证实成功构建了含HEL Th表位的sAPRIL cDNA突变体表达质粒.结论成功构建了6种sAPRIL cDNA突变体的表达质粒,为进一步进行sAPRIL 突变体cDNA的表达和活性鉴定奠定了基础.     

硫氧还蛋白过氧化物酶Ⅱ诱饵质粒的构建和检测

目的 构建硫氧还蛋白过氧化物酶Ⅱ(PrxⅡ)诱饵质粒,同时检测其在酵母菌内的表达和对酵母细胞的毒性.方法 提取人前列腺癌细胞系PC-3M-1E8细胞的RNA,利用RT-PCR获得其cDNA, PCR方法扩增PrxⅡ基因片段,纯化后以内切酶双酶切PrxⅡ和酵母表达载体pGBKT7,回收产物并连接,重组质粒命名为pGBKT7-PrxⅡ, 将重组质粒导入酵母菌AH109,检测其表达产物对酵母细胞有无毒性作用.结果 构建的诱饵载体pGBKT7-PrxⅡ测序结果表明序列完全正确,融合区域的读码框正确.将pGBKT7-PrxⅡ成功转化入酵母感受态AH109,并证明PrxⅡ蛋白对酵母无毒性作用.结论 证实了PrxⅡ蛋白无酵母毒性,诱饵质粒pGBKT7-PrxⅡ可以用于酵母双杂交研究前列腺癌发病机制.     

分化抑制因子1相互作用蛋白AGGF1的筛选

目的 利用酵母双杂交方法,筛选成人肺组织文库中与分化抑制因子1(Id1)相互作用的蛋白.方法 PCR方法扩增Id1的编码序列并定向克隆入诱饵质粒pHybLex/Zeo,构建重组诱饵质粒pHybLex/Zeo-Id1,酶切鉴定后转化人酵母株EGY48/pSH18-34,检测重组诱饵质粒有无非特异激活报告基因Leu2、LacZ作用;扩增并提取成人肺组织文库质粒,将文库质粒及重组诱饵质粒转化入酵母细胞,依次筛选Leu2 ,Leu2 LacZ 克隆;设置阴性及阳性对照,重复筛选Leu2 LacZ 克隆并排除假阳性克隆,获取真阳性文库质粒,进行测序和同源性比对.结果 酶切证实,重组诱饵质粒构建成功.重组诱饵质粒转化入酵母细胞后.经检测无非特异激活报告基因作用.扩增并筛选成人肺组织文库.获得198个Leu2 及19个Leu2 LacZ 克隆.经排除假阳性克隆,最终获得1个Leu2 LacZ 真阳性克隆,经测序和同源性比较证实该克隆与蛋白AGGF1高度同源(556/559,99.5%),Westem blotting证实其在酵母细胞中有表达.结论 利用酵母双杂交方法,通过筛选成人肺组织文库,发现Id1与AGGF1存在相互作用.     

酵母双杂交筛选与分化抑制因子Id1′相互作用的蛋白

目的 筛选成人肺组织文库中与分化抑制因子Id1′相互作用的蛋白。方法 构建重组诱饵质粒pHybLex／Zeo-Id1′，转化入酵母细胞EGY48／pSH18．34，检测重组质粒有无非特异激活报告基因；通过筛选成人肺组织文库，获取真阳性文库质粒，进行测序和同源性比较，获得真阳性克隆。结果 经测序证实，重组诱饵质粒pHybLex/Zeo-Id1′构建成功；将重组质粒转化入酵母细胞：EGY48／pSH18-34，经检测无非特异激活功能。进一步通过对文库进行筛选，获得一个真阳性克隆，通过测序和同源性比较证实该阳性克隆是酪氨酸蛋白激酶Fyn。结论 分化抑制因子Id1′与Fyn在酵母细胞中存在相互作用。     

sBLyS突变体酵母表达载体的构建

目的构建人BLyS胞外区(soluble BLyS)突变体毕赤酵母分泌型表达载体,为深入探讨BLyS的功能和机制创造条件。方法以构建的人pUC19/sBLyS及其突变体重组质粒为模板,利用PCR扩增出6×his/sBLyS及其突变体的cDNA,克隆入酵母表达载体pPIC9K;行序列测定后电转化入毕赤酵母GS115,并进行表型筛选和G418抗性筛选;对高拷贝整合的重组酵母表达菌株进行PCR鉴定。结果构建得到酵母融合重组表达载体pPIC9K/6×his/sBLyS及其突变体;经G418浓度梯度筛选及PCR鉴定得到高拷贝整合的重组酵母表达菌株。结论成功构建了人sBLyS及其突变体的真核表达载体,为获得人sBLyS及其突变体蛋白,进一步研究BLyS的生物学功能奠定了基础。     

人组织型纤溶酶原激活剂cDNA糖基化位点突变体的构建

构建人组织型纤溶酶原激活剂（ｔｉｓｕｅｔｙｐｅｐｌａｓｍｉｎｏｇｅｎａｃｔｉｖａｔｏｒ，ｔ－ＰＡ）ｃＤＮＡ糖基化位点突变体，为ｔ－ＰＡｃＤＮＡ在Ｐｉｃｈｉａｐａｓｔｏｒｉｓ酵母表达系统中的表达提供条件。方法运用ＤＮＡ重组技术，构建ｐＡＨＲ、ｐＡＲＨ质粒，然后应用改良的寡核苷酸诱导定位突变技术，构建ｐＭＡＨＲ、ｐＭＡＲＨ质粒，并用限制性核酸内切酶鉴定、核酸序列分析证实。结果琼脂糖凝胶电泳显示了特异的片段，序列分析证实获得的ｐＭＡＨＲ为Ａｓｎ１１７和Ａｓｎ１８４位点突变的阳性克隆，ｐＭＡＲＨ为Ａｓｎ４４８位点突变的阳性克隆。结论成功地构建了ｔ－ＰＡｃＤＮＡ糖基化位点突变体。     

菠菜乙醇酸氧化酶编码区cDNA在酵母表达载体中的克隆

目的：克隆菠菜乙醇酸氧化酶cDNA并构建其酵母表达载体。方法：从新鲜菠菜叶子中提取总RNA，用RT－PCR法扩增菠菜乙醇酸氧化酶编码区cDNA并插入pMD-T载体中进行序列测定；将此cDNA构建入P.Pastoris酵母表达体系中的pPIC3.5k上的SnaBI/NotI位点，进行PCR筛选和限制性酶切鉴定。结果：测序表明获得的基因为乙醇酸氧化酶cDNA序列，与国外文献报道的序列相比有约98％的同源性；重组质粒的SnaBI/NotI双酶切证明构建正确。结论：菠菜乙醇酸氧化酶cDNA克隆及重组质粒pPIC3.5K-GO的获得，为酵母表达菠菜乙醇酸氧化酶提供了前题条件。     

hTERT片段真核表达质粒的构建

目的：构建人端粒酶逆转录酶(hTERT)片段的真核表达质粒,方法：从肿瘤组织中提取总RNA．采用RT—PCR法扩增hTERT基因并克隆人PGEM—T Easy载体中,然后再亚克隆于pcDNA3．1真核表达载体,用PCR扩增及限制性酶切法筛选重组真核表达质粒,并以DNA测序鉴定。结果：PCR扩增及限制性酶切法证实重组真核表达质粒的构建成功。测序表明,hTERT基因片段与GenBank公布的相应基因同源性比较,核苷酸序列的同源性为98．73％,氨基酸序列的同源性为99．68％。结论：成功构建了hTERT真核表达质粒,为进一步研究与hTERT相关的抗肿瘤免疫治疗奠定了基础。     

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.