正、反义hTERT基因真核表达载体的构建与鉴定

目的构建正、反义hTERT基因真核表达载体,为进一步研究端粒酶的活性调节与恶性肿瘤基因治疗作准备。方法根据已发表在Genebank的hTERT cDNA序列,设计并合成了1对两端带特定限制性酶切位点引物,提取子宫颈癌HeLa细胞株总RNA,进行RT-PCR,并将扩增产物TA首先克隆至pGEMR-T载体,然后双酶切pGEM-T载体回收目的片段再克隆至真核表达质粒pcDNA3.1(±)中,并对重组体进行酶切鉴定和测序分析。结果PCR扩增片段与预期结果相符,双酶切证实构建的正、反义hTERT基因真核表达载体克隆成功,插入片段测序结果与Genebank的hTERT cDNA的部分序列完全一致。结论成功地构建了正、反义hTERT基因真核表达载体,为下一步研究端粒酶的活性调节与恶性肿瘤基因治疗打下了实验基础。     

肝癌相关新基因BC047440反义真核表达载体构建及鉴定

目的构建肝癌相关新基因BC047440反义真核表达载体.方法提取人肝癌组织总RNA,逆转录酶-多聚酶链式反应(RT-PCR)扩增BC047440基因cDNA序列,经与pMD18-T simple载体连接,筛选、克隆、抽提质粒和双酶切后,将纯化的BC047440基因cDNA序列反向插入真核表达载体pcDNA3.1( )中,筛选、克隆、抽提质粒,从而构建BC047440基因反义真核表达载体pcDNA3.1( )BC047440AS.结果经RT-PCR获得826 bp含限制性内切酶位点的cDNA片段,T载体克隆后经PCR扩增目的片段、测序,确定该片段为BC047440基因cDNA,进而构建反义真核表达载体pcDNA3.1( )BC047440AS,并经PCR扩增目的片段,测序确证.结论成功构建了BC047440基因反义真核表达载体pcDNA3.1( )BC047440AS,为进一步研究该基因功能奠定了基础.通过抑制消减杂交法(suppression subtractive hybridization,SSH)对肝癌组织与癌旁肝组织间差异表达基因进行分离、鉴定,我们发现了1条新的肝癌相关基因cDNA片断(EST)[1],长447 bp,经GenBank检索,90%与已知基因无同源性.进一步采用RACE技术,克隆出该基因的全长cDNA序列[2].经GenBank检索,与近期登录的基因BC047440同源.但该基因是从大脑组织中获得[3],而且国内外少见该基因功能研究的报道.在此基础上,本研究从原发性肝癌组织中克隆了BC047440基因的全长cDNA序列,并构建了其反义RNA真核表达载体,为进一步研究该基因功能奠定基础.     

小鼠cyclin A2正义和反义真核表达载体的构建及鉴定

目的构建小鼠cyclin A2的正义和反义真核表达载体。方法采用小鼠胚胎组织取材,提取总RNA。设计引物,采用RT-PCR方法扩增小鼠cyclin A2 mRNA得到cyclin A2的DNA,将此DNA插入pGEM-T载体,转化入JM109感受态细菌。将鉴定得到的重组质粒进行扩增,EcoRⅠ酶切后,回收cyclin A2目的片段亚克隆入pcDNA3.1真核表达载体。酶切筛选鉴定重组的真核表达载体pcDNA3.1-cyclin A2。结果将小鼠基因的开放阅读框(ORF)重组到真核表达载体pcDNA3.1内,用EcoRⅠ酶切重组质粒后可得到一接近1660 bp的DNA条带,核酸测序证实和GenBank所公布序列相符。HindⅢ酶切鉴定重组质粒后,证明分别为正向和反向插入载体的质粒。DNA测序证明构建的真核表达载体pcDNA3.1-cyclin A2的序列是正确的。结论成功构建了小鼠cyclin A2的正义和反义真核表达载体。     

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。     

强制泛素化乙肝病毒核心抗原融合基因表达质粒的构建

目的 构建强制泛素化乙肝病毒核心抗原(HBcAg)融合基因真核表达质粒.方法 以HBV adr亚型全基因质粒pADR.为模板,PCR扩增HBcAg基因片段;无茵操作下分离Balb/C小鼠肝脏,提取mRNA,RTPCR扩增小鼠泛素基因片段.PCR产物经测序鉴定后双酶切,插入真核表达质粒pcDNA 3.1(-),构建重组真核表达质粒ub-HBcAg-pcDNA 3.1(-)酶切鉴定和基因测序.结果 扩增的泛素基因片段和HBcAg基因片段经测序证实序列正确;将上述基因双酶切后插入质粒pcDNA 3.1(-)中,构建强制泛素化rmcAg融合基因表达质粒ub-HBcAg-pcDNA3.1(-);融合基因真核表达质粒ub-HBcAG-pcDNA 3.1(-)经酶切、基因测序等鉴定证实目的基因序列和插入方向正确,与预期结果一致.结论 成功构建了强制泛素化乙肝病毒核心抗原融合基因真核表达质粒,为进一步研究强制泛素化HBcAg诱导HBV特异性CTL奠定了实验基础.     

人类p27kip1基因正、反义真核表达质粒的构建

目的：克隆人类p27^kip1基因，构建其正、反义真核表达质粒。方法：采用逆转录聚合酶链式反应(RT—PCR)技术，从人类伯基特淋巴瘤细胞株Raji中扩增出人类p27^kip1 cDNA全长序列，通过DNA重组技术将该基因重组于pcDNA3．1真核表达载体上，构建p27^kip1正、反义表达质粒。通过酶切电泳分析及DNA测序的方法对重组表达质粒进行鉴定。结果：通过酶切鉴定及测序分析证实，本实验构建的重组表达质粒目的基因片段为人类p27^kip1 cDNA。结论：本实验成功地克隆了人类p27kipl基因并构建了其正、反义真核表达质粒，为进一步研究p27^kip1在淋巴瘤发生中的意义奠定了基础。     

鼻咽癌相关新基因NPCEDRG真核诱导表达载体的构建及鉴定

目的构建受Tet系统调控人鼻咽癌相关新抑瘤基因NPCEDRG表达的真核诱导表达载体。方法以pcDNA3．1-NPCEDRG重组质粒为模板，采用聚合酶链反应（PCR）技术扩增NPCEDRG基因编码区，亚克隆到pRevTRE载体，PCR及双酶切鉴定，测序验证。结果以pRevTRE—NPCEDRG重组载体为模板PCR扩增以及双酶切重组载体，分别产生530bp和520bp长度的片断，测序结果证实插入片段方向正确，序列与Genebank已知序列一致，NPCEDRG基因编码区成功插入pRevTRE载体。结论成功构建了受Tet系统调控NPCEDRG基因表达的pRevTRE—NPCEDRG真核诱导表达载体，为深入研究NPCEDRG基因功能和摁示鼻咽癌发病分子机制提供实验手段。     

人C型利钠肽基因的克隆及其真核表达载体的构建

目的：为开展C型利钠肽基因治疗，克隆人C型利钠肽基因并构建其真核表达载体。方法：通过RT-PCR从人胎盘组织中克隆人C型利钠肽基因全长编码区，将该DNA片段亚克隆至克隆载体pBS-T中，用SmaⅠ单酶切筛选出负向插入片段，用HindⅢ和BamHⅠ双酶切将目的DNA片段定向克隆至真核表达质粒pcDNA3．1（＋）中，然后用双酶切、PCR鉴定和DNA序列测定三重鉴定方法对重组质粒进行鉴定。结果：酶切、PCR及测序鉴定证实，人C型利钠肽基因全长编码区被准确插入至pcDNA3．1（＋）酶切位点HindⅢ和BamHⅠ之间，并且未发现有碱基突变或移位。结论：含人C型利钠肽基因真核表达质粒的成功构建并鉴定，为开展C型利钠肽基因治疗奠定了物质基础。     

巨噬细胞移动抑制因子真核表达载体的构建

目的构建巨噬细胞移动抑制因子(macrophage migration inhibitory factor,MIF)基因真核表达载体。方法通过PCR方法从pAdTrack-CMV质粒中扩增出348 bp的靶片段,将其粘端克隆至pcDNA3.1-(+)真核表达质粒,然后应用限制性酶切、PCR扩增及序列测定等技术进行鉴定。结果成功构建了MIF真核表达载体,酶切及测序证实读码框正确。结论成功克隆MIF真核表达载体,为体内基因治疗勃起功能障碍奠定基础。     

人甲状腺刺激激素受体cDNA的克隆及真核表达载体的构建

目的克隆人甲状腺刺激激素受体(hTSHR)基因cDNA并构建其真核表达载体。方法以人甲状腺组织cDNA为模板,聚合酶链反应(PCR)扩增hTSHR基因编码区的全部序列,克隆入pGEM-T载体中,经限制性内切酶、DNA序列分析鉴定目的基因后,定向亚克隆到真核细胞表达载体pcDNA3.1中,并进行双酶切鉴定。结果PCR扩增的特异性片段长度为2 337 bp,以此构建的pGEM-T-hTSHR克隆载体,经限制性内切酶酶切及DNA序列分析证实载体中带有hTSHR基因编码区的目的片段,重组质粒pcDNA3.1-hTSHR经KpnⅠ和XbaⅠ双酶切后显示5 1000 bp和2 300 bp左右的2个条带,DNA序列分析显示与GenBank中的hTSHR基因cDNA序列一致,证明hTSHR基因已成功克隆入真核细胞表达载体pcDNA3.1中。结论成功构建了野生型pcDNA3.1-hTSHR重组真核表达载体。     

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.