石英谐振传感器液相稳定平台构建及检测HCMV的实验研究

目的 建立石英谐振压电基因传感器检测系统液相稳定平台并应用该检测系统实时检测链置换扩增(Strand displacement amplification，SDA)反应。方法 ①观察以金属夹具式和粘胶式两种检测池连接方式构成的传感器在液相中的频率稳定性；②以巨细胞病毒(Human cytomegalovirus，HCMV)为检测对象，建立SDA反应系统；③传感器检测系统对HCMVSDA反应进行实时检测。结果 ①新型金属夹具式可调节及可换式传感器检测池可实现传感器在液相中的频率稳定性；②SDA反应可引起压电基因传感器的频率持续下降；③在一定范围内核酸杂交所引起的频率下降幅度随加入的靶序列浓度提高而增大。结论 成功构建压电基因传感器液相检测稳定平台并实现对SDA反应的实时检测；该系统可直接检测基因组DNA并可广泛应用于病原微生物的临床检测。     

链置换扩增(SDA)压电DNA传感器阵列

目的　构建SDA压电DNA传感器阵列 ,解决SDA压电DNA传感器的检测通量不足问题。方法　①采用传感器集成技术将 1 0个SDA压电DNA传感器制成 2× 5的传感器阵列芯片 ;②以结核菌IS61 1 0片段为检测对象 ,研究SDA压电DNA传感器阵列的特性。结果　传感器阵列芯片的各传感器微单元能够相互独立工作 ,互不干扰。结论　SDA压电DNA传感器的阵列化不影响传感器性能 ,同时在一定程度上提高了SDA压电DNA传感器的检测通量 ,使SDA压电DNA传感技术更具临床实用性     

压电基因传感器检测巨细胞病毒PCR产物的实验研究

目的　应用自行研制的压电基因传感器检测巨细胞病毒临床标本的PCR产物 ,探讨压电传感器检测核酸反应的可行性以及各种影响因素。方法　设计特异性DNA探针巯基法固定于传感器晶振表面并调节谐振频率在液相中稳定性达到± 1Hz ,定量加入 65份巨细胞病毒临床标本的PCR产物并观察核酸反应导致的频率变化情况 ,并与PCR产物的电泳结果作对照。结果　加入HCMVPCR阳性产物的传感器谐振频率明显下降 ,3 8例阳性产物的平均频率下降值为73 66Hz ,阴性产物的频率没有变化。与PCR电泳结果的阳性符合率为 86 8%,阴性符合率为 93 10 %。结论　我们自主研发的压电基因传感器能有效地检测巨细胞病毒临床标本的PCR产物     

压电石英晶体传感器检测血浆凝血酶原时间及其初步应用

目的：建立一种压电石英晶体传感器检测血浆凝血酶原时间（PT）的方法。方法：采用AT切向，基频10MHz的银膜石英晶体作为压电元件；观察血浆凝集过程中晶体振荡频率的变化，确定血浆凝集反应的起点和终点。结果：（1）血浆标本加入反应体系后, 晶体振荡频率呈阶梯状快速下降，接着频率瞬时上升，然后再缓慢降低达到平稳，频率阶梯下降的起点为血浆凝集反应的起点，频率从快速上升变为下降的转折点为血浆凝集的终点，计算两点之间的时间即为血浆凝血酶原时间。（2）该方法的检测结果与STAGO磁珠检测法的相关性好（γ＝0．97），批内CV＝0．78％，批间CV＝2．38％。结论：压电石英晶体传感器检测血浆凝血酶原时间，血浆凝集反应起点和终点确定方便，可靠，且操作简单，快速，成本低廉，检测结果准确，是一种敏感性高，重复性好的血浆凝血酶原时间检测新方法，可用于常规检测。     

靶DNA长度对压电传感器基因杂交效应的影响

目的：探讨靶DNA长度对压电传感器基因杂交效应的影响，方法：在压电传感器阵列上固定20碱基的巯基DNA序列作为探针后分别与不同长度的含互补片段的靶序列进行杂交，计算机采集并分析传感器频率数据，比较各靶DNA长度下杂交达到平衡所需时间，杂交前后频率差，并计算杂交动力学参数。结果：随靶DNA长度增加，杂交时间有延长趋势。在靶DNA长度为20－108个碱基时，其频率变化值与碱基数呈线性关系。而225，379，654个碱基时的频率变化反应则明显降低，结合常数变化不明显，而解离常数呈增大趋势变化，使平衡常数逐渐减少，结论：在威武央晶体传感器阵列上，频率变化值随靶序列长度增长而增大但反应效率不一样。     

链延伸反应提高压电基因传感器的灵敏度的实验研究

目的　利用链延伸反应的原理延长引物链以提高压电基因传感器表面的质量负载 ,从而提高传感器的灵敏度。方法　基因传感器阵列表面分别固定上金黄色葡萄球菌mecA及femA两种探针片段 ,待杂交上相应的靶序列片段后 ,加入Klenow酶 ,室温下 (2 0℃左右 )进行链延伸反应 8h。结果　 50nmol L单链mecA在杂交反应及链延伸反应的过程中 ,频率的下降值分别为 (99.2± 8.5)Hz、(1 92 .9± 1 3 .3 )Hz。而femA则分别下降了 (1 42 .4± 1 1 .2 )Hz、(2 2 2 .6± 1 6.6)Hz。mecA、femA在链延伸反应前的检测灵敏度为 0 .5nmol L ,但经链延伸反应后检测灵敏度提高到了 0 .0 5nmol L。结论　链延伸反应有效地提高了压电基因传感器的灵敏度 ,可用于微量DNA的检测、核酸同源性分析等方面     

2种不同类型的核酸探针对压电基因传感器阵列检测特异性的影响

目的探讨用PNA作为探针与普通的DNA探针相比对压电传感器基因杂交的优越性.方法用生物素-亲和素法分别将PNA、DNA两种探针固定在基因传感器的金膜表面,分别与完全匹配、1个碱基错配、2个碱基错配的互补靶序列进行杂交,观察2种不同的探针与靶序列反应所引起的频率变化及所用的时间.结果相对于DNA探针,PNA探针识别碱基错配的能力明显高,且杂交时间更短.结论 PNA与靶序列DNA结合有高度的特异性,选用PNA作探针,可提高压电基因传感器检测的特异性.     

链置换扩增技术的影响因素研究

目的：探讨限制酶，聚合酶，缓冲体系和DTT等对链置换扩增（SDA）实验的影响，以期在商品化试剂条件下组建SDA系统。方法：以结核菌IS6110序列为扩增对象设计SDA引物，利用商品化的BsoBI和exo-Bst为实验用酶建立SDA实验系统，对比在不同BsoBI,exo-Bst浓度条件下SDA产物的电泳条带亮度来观察限制酶，聚合酶对SDA效率影响；对比SDA在pH7.6的磷酸盐缓冲液和以Tris-HCl为缓冲体系的NF Buffer2,ThermoPol Buffer和EcoPol Buffer中的产物电泳条带的方法来观察不同缓冲体系对SDA的效率影响。对比加入DTT和不同DTT时SDA产物的电泳条带亮度来观察DTT对SDA效率的影响。结果：（1）在0．1－0．4U／ul浓度范围内，SDA的扩增率与BsoBI含量正相关，而0．8U/ul BsoBI反而使SDA受到抑制。（2）在0．08－0．32U／ul浓度范围内exo-Bst用量对SDA的扩增能力没有显著影响；（3）SDA可以在pH7.6的磷酸盐缓冲液和ThermoPol Buffer中进行，而不能在NE Buffer2和EcoPol Buffer中进行；（4）DTT对SDA的扩增率和特异性都没有显著影响。结论：以上实验结果表明在商品化试剂条件下可以构建SDA系统，并得到良好的扩增效果。     

纳米金颗粒质量放大压电DNA传感器频移信号的研究

目的探讨纳米金颗粒质量放大压电DNA传感器频移信号的可行性,以进一步提高检测灵敏度。方法将巯基化的单链DNA探针固定于压电DNA传感器石英晶体金膜表面,封闭后加入合成的生物素化的互补靶DNA与之进行杂交反应,最后用5nm粒径的链亲和素标记的胶体金追加标记于杂交后的生物素化的靶DNA上,以增加石英晶振金膜表面的质量负载,从而降低石英晶振的响应频率,进一步放大频移信号。结果检测终浓度为10-8mol/L的阳性靶序列时杂交反应前后频移为(12.7±5.5)Hz,加入终浓度为9%的纳米金颗粒放大后总频移为(126±2.6)Hz,后者显著高于前者(P<0.01)。另外,对不同终浓度的阳性靶序列检测后发现,频移与其终浓度的lg值之间具有显著的线性关系,相关系数为0.974,而且最低检出限达到了10-12mol/L。结论实验结果表明该方法可以显著放大压电DNA传感器的频移信号,从而进一步提高压电DNA传感器检测的灵敏度。     

核酸外切酶-压电石英DNA传感器检测金黄色葡萄球菌的实验研究

目的 构建一种用于检测金黄色葡萄球菌的核酸外切酶-压电石英DNA传感器新方法,同时对其重要影响因素进行探讨.方法 采用λ核酸外切酶处理金黄色葡萄球菌PCR产物,EB染色、电泳后于紫外灯下进行观察,对杂交温度、杂交时间和杂交响应性能等因素进行实验研究,进一步将构建的核酸外切酶-压电石英DNA传感器法用于金黄色葡萄球菌检测,并对其灵敏度和特异性进行研究.结果 λ核酸外切酶-压电石英DNA传感器法最适杂交温度为35℃,最适杂交时间为60 min,响应性能显著大于普通压电DNA传感器(P＜0.01),可以检测到1.0×104CFU/ml的金黄色葡萄球菌,特异性好.结论 利用核酸外切酶-压电石英DNA传感器检测金黄色葡萄球菌,具有杂交效率高、技术难度低、频率响应性能高等优点,有较好的应用前景.     

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.