胃癌组织中DNA聚合酶β基因突变特点分析

目的:探讨人胃癌DNA聚合酶β(polβ)基因突变的特点. 方法:对32例人胃癌组织及32例正常对照组织中提取总RNA,进行RT-PCR,SSCP,对PCR产物克隆后以Sanger's法测序,并采用Omiga及Dansis软件分析结果. 结果:人胃癌标本的polβ基因突变率21.9%. 测序分析了7例SSCP异常中的3例,其突变分别为196位A→G,268位A→G,790位A→T;经OMIGA软件分析,这3个polβ的点突变均导致了氨基酸的改变,具体形式为:核酸196位A→G导致氨基酸28位Asn→Ser;核酸268位A→G导致氨基酸52位Lys→Arg;核酸790位A→T导致氨基酸226位Asp→Val. DNASIS软件Chou-fasman算法推测,polβ的196位A→G突变可以导致polβ酶蛋白的二级结构图明显改变. 结论:人胃癌组织polβ基因突变率达21.9%;这3个polβ的点突变都导致了氨基酸的改变,196位A→G突变可以导致polβ酶蛋白的二级结构明显改变.     

红细胞丙酮酸激酶缺陷症基因变异型的研究

目的：了解中国人红细胞丙酮酸激酶（pyruvate kinase,PK）缺陷症的基因变异类型。方法：应用聚合酶链反应－单链构象多态性（polymerase chain reaction-single strand conformation polymorphism,PCR-SSCP)和限制性内切酶酶切方法，分析上海地区8个PK缺陷症家系红细胞PK基因变异的情况。结果：在8例PK缺陷症家系中发现3个泳动变位片段，DNA直接测序证实3例均为错义点突变（cDNA 1330G→T、1339C→A、809G→C）。结论：3个突变点分别位于PKLR基因第7和第10外显子，导致相应的氨基酸发生置换（443Ala→Ser、446Leu→lle、270Ag→Pro），造成酶活力下降，红细胞糖酵解途径受阻，临床出现慢性溶血性贫血。     

3个遗传性对称性色素异常症家系中DSRAD基因的突变

目的:检测国内3个遗传性对称性色素异常症家系中DSRAD基因的突变.方法:PCR扩增3个家系中成员DSRAD基因的全部外显子,并行DNA测序.以100例无关正常人作对照.结果:PCR结合DNA测序发现3个家系中患者均存在DSRAD基因的异常:家系A中第3220位碱基发生了C→T的杂合突变,对应1074位的精氨酸被半胱氨酸替代;家系B与家系C中发现的突变相同,为第3325位碱基发生了G→T的杂合突变,对应1109位的天冬氨酸被酪氨酸替代.家系中未患病者及无关正常人未发现相应突变.结论:此3个遗传性对称性色素异常症家系中存在DSRAD基因的特异性突变,突变可能使蛋白功能缺陷,导致临床上出现皮肤色素异常.     

中国人群MEF2A基因突变与冠心病易感性的关系

目的 研究冠心病相关基因MEF2A在中国人群的突变和多态性位点.方法 利用聚合酶链反应-单链构象多态性(polymerase chain reaction-single strand conformation polymorphism,PCR-SSCP)和DNA测序技术检测210例冠心病(CAD)患者及190名健康人MEF2A基因第11外显子.结果 冠心病患者SSCP电泳条带异常样本测序分析发现突变:①密码子451G/T(147191位点G→T)杂合或纯合同义突变;②147108～147131位点之间分别发生1个氨基酸(Q)、2个氨基酸(QQ)、3个氨基酸(QQP)、6个氨基酸(425QQQQQQ430)、7个氨基酸(424QQQQQQQ430)缺失;③密码子435G/A(147143位点)杂合突变.密码子451G/T杂合或纯合同义突变以及氨基酸的缺失是基因多态性,密码子435G/A考虑为新的突变.结论 中国人群在MEF2A基因第11外显子也存在多种基因多态性,其中6或7个氨基酸的缺失和1个147143位点突变可能与冠心病易感性有关.     

糖原累积病za型分子遗传学家系研究

目的 研究1例糖原累积病Ⅰa型患者发病的分子遗传学机制。方法 从全血中抽提患者及其父母、姐姐和正常人基因组DNA,通过多聚酶链反应扩增葡萄糖-6-磷酸酶(G6Pase)基因的5个外显子,用DNA直接测序法确定其突变位点;测定患者父母亲及其姐姐的相应序列,确定突变的遗传来源。结果 患者G6Pase基因外显子5发生单个碱基纯合突变,突变碱基位于序列第727位,由G转变为T,造成剪切位点突变。患者父母亲和姐姐的G6Pase基因外显子5均发生727G→T杂合突变。结论 糖原累积病Ⅰa型可直接进行DNA分析以明确诊断,葡萄糖-6-磷酸酶外显子5的727G→T突变的发生在中国人中的发生率有待进一步研究。     

糖原累积病za型分子遗传学家系研究

目的研究1例糖原累积病Ⅰa型患者发病的分子遗传学机制.方法从全血中抽提患者及其父母、姐姐和正常人基因组DNA,通过多聚酶链反应扩增葡萄糖-6-磷酸酶(G6Pase)基因的5个外显子,用DNA直接测序法确定其突变位点;测定患者父母亲及其姐姐的相应序列,确定突变的遗传来源.结果患者G6Pase基因外显子5发生单个碱基纯合突变,突变碱基位于序列第727位,由G转变为T,造成剪切位点突变.患者父母亲和姐姐的G6Pase基因外显子5均发生727G→T杂合突变.结论糖原累积病Ⅰa型可直接进行DNA分析以明确诊断, 葡萄糖-6-磷酸酶外显子5的727G→T突变的发生在中国人中的发生率有待进一步研究.     

郑州市B19病毒分离株全基因的克隆及序列测定

目的：克隆和测定在郑州市得到的B19病毒分离株全基因序列。方法：采用聚合酶链反应(PCR)技术从l例B19阳性的流产患者血清中分段扩增B19病毒全基因,获得7个互相重叠的片段,分别长814bp(1～814),849bp(753～1601),663bp(1523～2185),738bp(2124～2861),1011bp(2790～3800),1014bp(3747～4760),914bp(4681～5594)。将其分别克隆入T载体,进行测序分析。结果：郑州市B19病毒分离株全基因长5594个核苷酸,有3个开放读码框架(ORF)。第1个ORF位于615～2630位核苷酸,编码672个氨基酸,第2个ORF位于2623～4968位核苷酸,编码782个氨基酸,第3个ORF位于3304～4968位核苷酸,编码555个氨基酸。在该基因中4种碱基在基因组中所占比例为：A,29.58％(1655／5594),T,26.52％(1484／5594),G,22．88％(1280／5594),C,21．02％(1175／5594)。郑州B19病毒分离株基因序列与GenBank中NC_000883高度同源;存在10个碱基改变,只有1个引起氨基酸改变(3752位C→G导致Ser→Cys)。结论：郑州B19病毒分离株和NC_000883属同一基因型。     

犬MC1R基因多态性的研究

目的 利用PCR—RFLP和PCR—SSCP技术分别检测犬MC1R基因中两个序列T105A和R306Ter的基因多态性，为遗传育种、培育出更加优良的实验用犬奠定基础。方法 以224只犬为实验材料，对犬MC1R基因T105A片段和R306Ter片段分别进行PCR-RFLP和PCR—SSCP分析，并且分别对具有RFLP和SSCP多态性的片段进行克隆测序，找出等位基因的差异位点。结果 经对犬MClR基因的PCR-RFLP分析，发现犬MC1R基因的T105A序列具有多态性，表现为三种基因型：AA、AB和BB。通过对具有RFLP多态性的片段进行克隆测序发现MC1R基因在编码第105位氨基酸的密码子处存在由G到A的单碱基突变，该突变导致第105位氨基酸发生由精氨酸向苏氨酸的改变，此外在第207位氨基酸的密码子处还发现由A到G的单碱基突变，并产生了一个HhαI限制性内切酶位点。同时，经过犬MC1R基因的PCR—SSCP分析，发现犬MC1R基因的R306Ter序列具有多态性，表现为三种基因型：CC、CD、DD.通过对具有SSCP多态性的片段进行了克隆测序发现，MC1R基因在编码第306位氨基酸的密码子处存在一个由CGA到TGA的终止突变，而使翻译终止。结论 利用PCR—RFLP和PCR—SSCP分析技术证实犬MC1R基因具有明显的多态性。     

人原发性肝癌组织内抑癌基因PTEN的突变检测

目的 检测人原发性肝细胞性肝癌(HCC)组织中抑癌基因PTEN的突变。方法 采用PCR—SSCP技术和DNA测序技术检测60例HCC组织中PTEN的突变。结果 在60例配对HCC标本中，发现了共6个突变位点的存在。其中，出现在外显子内的突变有1个，即在31B号标本第4外显子中5’—侧翼下游13bP处检测到C→T的突变(89031C→T)，该突变导致了PTEN蛋白第84伉氨基酸由精氨酸变为赖氨酸。另外5个突变均位于内含子中，包括在34B号标本的第4外显子3’-侧翼下游106bP处的5碱基(AATAG)缺失突变、在9B号标本的第4外显子3’—侧翼下游67bP处检测到的一个G→T点突变(89125G→T)、在33B号标本的第6外显子3’—侧翼下游58bP处检测到的一个T→A点突变(110285T→A)、在44B号标本的第8外显子5’—侧翼上游29bP处检测到的一个C—T点突变(118833C→T)、在22B号标本的第8外显子5’—侧翼上游82bP处检测到一个A→C点突变(118780A→C)。在我们选择的SK—HEP—1、HePG2、Huh—7、ChangliVer和WRL68肝脏细胞中未检测到PTEN基因的突变。结论 肝癌组织中PTEN突变可能参与了原发性肝癌的形成。     

高血压患者及大鼠线粒体ATPase 6基因的克隆、表达与突变研究

目的：研究正常血压与高血压鼠以及正常血压与高血压患者的差异基因。方法：用差别杂交法（Differentialhybridization）筛选正常人心脏cDNA文库的部分克隆,得到多个在自发性高血压鼠（SHR）心肌和高血压患者外周白细胞中高表达的线粒体基因。重组质粒,PCR－SSCP和测序,进行基因突变分析。结果：SHR鼠心肌线粒体ATPase6基因构象改变,8701位碱基由G→A（G8701A）,编码的第59位氨基酸密码子由丙氨酸变成苏氨酸;A8708G,编码的第61位氨基酸由组氨酸变成精氨酸。高血压病患者外周血白细胞线粒体ATPase6基因8584位碱基突变（G8584A）,编码的第20位氨基酸由丙氨酸变成苏氨酸,这一变化与SHR线粒体ATPase6基因改变相一致。结论：高血压病患者外用血白细胞线粒体ATPase6基因G8584A突变很可能在高血压病心肌肥厚的发生、发展中具有意义,并可能是高血压病的特异性改变。     

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.