变性高效液相色谱法在FALS突变位点检测中的应用

目的检测一个肌萎缩侧索硬化症(amyotrophic lateral sclerosis,ALS)家系的铜、锌超氧化物歧化酶(Cu/Zn su-peroxide dismutase,SOD1)基因的突变位点,同时观察变性高效液相色谱法(denaturing high performance liquid chromatography,DHPLC)的实用价值.方法对PCR-SSCP检测阴性的外显子,应用DHPLC法及DNA直接测序技术,再次进行SOD1基因的突变位点检测.结果经DHPLC检测,家系成员Ⅲ1SOD1基因的第4外显子有突变峰,DNA直接测序证实存在杂合子,发生了GAA→GGA错义突变,使编码的氨基酸由谷氨酸变为甘氨酸.结论DHPLC技术与PCR-SSCP相比有更高的敏感性,可作为大样本筛查突变位点的一种便捷可靠手段.     

家族性阿尔茨海默病早老素-1基因突变位点的检测

目的：探讨早老素－1基因突变在家族性阿尔茨海默病（FAD）发病中的作用。方法：应用聚合酶链反应－单链构象多态性（PCR－SSCP）、变性高效液相（DHPLC）及DNA直接测序技术检测技术,对130人的阿尔茨海默病（AD）家系和50例正常对照组的早老素－1（PS－1）基因第4、5外显子进行了检测。结果：在早老素基因的第5外显子上发现除5例AD患者外,还有4例家系内正常人（Ⅳ－30、37、41、43）PCR－SSCP出现泳动异常。DHPLC检测进一步验证以上9例均表现双峰,提示可能有突变存在。DNA序列分析表明,这9例的早老素－1基因第5外显子的136号密码子发生了GCT→GGT错义突变,使氨基酸由丙氨酸变为甘氨酸（Ala136Gly);第4外显子未发现突变。家系内其他人及正常对照组的PS1基因第4、5外显子经以上检测均未发现异常。结论：PS1基因第5外显子的突变点可能为中国FAD患者早老素基因突变位点之一。     

遗传性先天性白内障与αA-晶体蛋白基因相关性研究

目的 分析遗传性先天性核性和绕核性白内障与定位于21q22．3的αA-晶体蛋白（CRYAA）基因之间的关系。同时对比单链构象多态性（SSCP）和变性高效液相色谱法（DHPLC）两种方法检测单核苷酸多态性（SNP）。方法 用PCR方法扩增CRYAA基因的3个外显子,分别用SSCP和DHPLC两种方法分析扩增产物,对异常者进行DNA测序,寻找基因变异情况。结果用SSCP分析15个样本,未见异常条带;同时用DHPLC检测15个样本,其中1个样本出现双峰,将该样本扩增产物测序鉴定,发现在5’端第6个核苷酸为G／A杂合,二者是同一氨基酸的2个密码子,该核苷酸为CRYAA基因的多态性位点。未发现该基因突变。结论 实验中未发现15例先天性白内障患者与CRYAA基因之间有关联。应用SSCP与DHPLC同时分析,DHPLC检测出1个多态性位点。DHPLC对杂合子检出的敏感性要高于SSCP。     

具有独特表型的LHON家系及其线粒体基因突变的初步分析

目的探讨一个5代遗传的具有独特表型的Leher遗传性视神经病（LHON）家系的线粒体基因突变。方法对LHON的常见致病突变位点（3460、11778、14484、14459等）、线粒体遗传病Leigh病常见突变位点8893和线粒体脑肌病伴Leigh病表型的常见突变位点13513进行聚合酶链反应（PCR）、单链构象多态性（SSCP）和DNA测序分析。结果SSCP和测序结果均为阴性。结论该家系是新的LHON突变亚型。为明确该家系的诊断,需要进行线粒体DNA的全长序列分析。     

新疆3个家系HCM基因突变检测分析

目的：研究家族性肥厚型心肌病（HCM）的主要致病基因β肌球蛋白重链（MYH7）突变位点。方法：对3个HCM家系成员的MYH7基因3~23外显子及附近上下游序列采用DHPLC及直接测序分析。结果：P、L、Y三个家系的先证者具有MYH7基因错义突变,测序结果显示：发现三个家系患者MYH7基因均有一定的突变。P家系21号患者MYH7基因测序结果分析发现外显子3存在密码子Thr63第三位密码子c〉t转换及19号内含子第90位的a〉g的转换。L家系发现外显子3存在Thr63第三位密码子c〉t转换,并且,第14外显子有单碱基突变,造成Thr 441M et,在中国人中是首次发现。Y家系其他成员测序结果发现携带有第8外显子最后一位碱基存在t〉c突变。结论：MYH7基因在HCM家系中具有较高的突变率,不同突变基因型以及基因突变携带个体在临床表型上有所差异。采取基因突变检测和分析,有利于对HCM家族成员的诊断、患病风险预测及疾病早期预防和治疗。     

His348Gln杂合突变伴hrg353Gln杂合多态性导致的遗传性凝血因子Ⅶ缺陷症家系分析

目的：对1例遗传性凝血因子Ⅶ（FVH）缺陷患者进行基因分析和家系调查,初步探讨其发病的分子机制。方法：检测先证者及其家系成员凝血酶原时间（PT）、活化部分凝血活酶时间（APTT）、纤维蛋白原（FIB）、凝血酶原活性（FII：C）、凝血因子V活性（FV：C）、FVII活性（FVII：C）和凝血因子x活性（FX：C）及FVII抗原（FVII：Ag）等进行表型诊断;用DNA直接测序法分析先证者Ⅳ基因的全部外显子、侧翼、5’和3’非翻译区及家系成员相应的突变位点区域,用反向测序证实所发生的突变。结果：先证者PT延长为22．4s,FVII：C和FVII：Ag明显降低,分别为7％和10％;父亲、母亲、姐姐及儿子的PT正常或稍延长,FVII：c分别为63％、54％、57％和46％,FVH：Ag分别为67％、53％、61％和49％;先证者和所有家系成员的APTT及其他凝血指标均在正常参考范围内。测序发现先证者Ⅳ基因第8号外显子存在g．11482T〉G（His348Gln）杂合突变和g．11496G〉A（Arg353Gln）杂合多态性;其母亲、姐姐、儿子均存在F7基因g．11482T〉G杂合突变;父亲存在,7基因g．11496G〉A杂合多态性。结论：肜基因His348Gln杂合突变协同Arg353Gln杂合多态性是导致该先证者FVII缺陷症的分子机制。     

一汉族念珠状发家系Ⅱ型毛发角蛋白基因突变的检测

目的 检测一个汉族念珠状发家系Ⅱ型毛发角蛋白（hHb）基因的突变情况。方法 在取得遗传学研究知情同意书后，采集先证者及其家系成员外周血并提取基因组DNA。运用聚合酶链式反应（PCR）扩增hHb1、hHb3、hHb6外显子1和外显子7，DNA直接测序，然后与GenBank中登记序列进行比对分析。对新发现的单核苷酸多态性（SNPs）进行限制性位点酶切分析加以验证。结果 经网上比对分析，该家系患者均未发现已报道的10种hHb致病突变，但发现该家系hHb1的外显子1存在第348位的单个碱基转换（G／A），经限制性位点酶切分析法证实为一个同义cSNPs（第348位G／A，R116R）。结论 该家系念珠状发患者的致病基因不同于现已报道的10种hHb致病突变，在他们hHb1外品子1存在一个同义cSNPs。     

PCR-SSCP技术检测中国人肝豆状核变性基因第18、14外显子突变

目的 了解中国人肝豆状核变性（WD）患者基因第18、第14外显子的突变情况，为掌握该痛的突变特点并进行基因诊断提供依据。方法 聚合酶链反应-单链构象多态性（PCR—SSCP）检测中国人WD基因第18、14外显子突变，并对异常带型进行直接DNA测序。结果 45例患者和20例正常人的18外显子PCR—SSCP出现两种泳动带型，异常带型测序证实无突变存在。14号外显子PCR—SSCP出现的带型均一致，结论 14外显子和18外显子可能不是中国人WD基因突变的热区。     

常染色体显性遗传性听神经病家系全外显子组测序分析

目的：在多年围绕1个常染色体显性遗传性非综合征型听神经病家系开展系统分子遗传学研究的基础上,进一步探讨该家系耳聋的致病机制,以期发现新的听神经病致病基因和突变位点。方法：对3例耳聋患者和1例配偶进行全外显子组测序,初步筛选出与家系耳聋相关的候选致病基因。采用PCR-Sanger测序法,检测上述候选基因变异是否与家系表型共分离。最后,以50例与研究家系无关的听力正常人为对照,检测候选致病突变在正常群体中的突变频率和SNPs遗传多态性。结果：全外显子测序分析得到41个候选致病基因突变;用PCR-Sanger测序法对核心家系的9名成员和2名家系外听力正常人进行验证,仅发现1个基因突变（ALOX15B 7942797 C>T）与家系耳聋表型共分离。选取50例家系外正常对照的DNA样本对ALOX15B基因进行PCR扩增和序列分析,结果显示有2例听力正常人也检测到该基因的同一变异,提示该变异为SNPs遗传多态性。结论：对核心家系成员的全外显子组测序分析和Sanger测序法验证未发现有意义的突变位点,排除了该家系耳聋由基因编码区突变及Indels致病的可能性。     

原发性开角型青光眼一家系的基因突变和临床表型分析

目的：对中国南京地区1个原发性开角型青光眼（primary open angle glaucoma,POAG）家系进行基因突变位点的筛查和临床表型分析。方法：对该家系成员行全面的眼科检查,采集受检者的外周静脉血,提取基因组DNA,以二代测序方法进行基因组定点捕获测序分析,对发现的变异用Sanger测序技术在家系成员中进行致病基因验证。对照组为100名健康人群。结果：在先证者的小梁网诱导性糖皮质激素反应蛋白（myocilin,MYOC）基因第3外显子区域发现杂合突变c.734G>A（p.C245Y）,这一突变也出现在其他12个POAG的家系成员中,无该突变的家系成员中无POAG患者及可疑者。对照组未发现该突变位点。结论：MYOC基因C245Y突变是该POAG家系的致病突变位点,本研究结果补充了中国江苏地区MYOC基因的突变谱。     

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.     

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.     

Scientific principles and rigorous processes should be followed in developing clinical guidelines for therapeutic interventions of integrative medicine

A clinical guideline for the therapeutic interventions of integrative medicine may be defined as a written document which states a series of recommendations on therapeutic interventions of integrative medicine for a special disease or condition. The guideline may provide assistance to medical professionals in making clinical decisions aimed at improving the clinical outcome of patients and reducing the costs of medical care（~＇4~. Recommendations issued by a guideline should be based on the best available evidence in both Western and Chinese medicine. For fulfilling this purpose, the development of clinical guidelines for therapeutic interventions in the field of integrative medicine should follow scientific principles and undergo a rigorous processes.