人端粒重复序列结合因子1^33—277在大肠杆菌中的？…

目的：建立能高效表达人端粒重复序列结合因子１第＾３３－２７７位（ＴＲＦ１＾３３－２７７）氨基酸的菌株并制备多抗。方法：设计一对引物,分别带有Ｋｐｎ Ｉ和ＥｃｏＲＩ位点,从已经克隆了ＴＲＦ１ｃＤＮＡ全长的重组质粒ｐＣＲⅡ－ＴＯＰＯ－ＴＲＦ１扩增ＴＲＦ１＾３３－２７７ｃＤＮＡ片段,克隆入ＰＧＥＭ－Ｔ质粒,测序和酶切图谱鉴定,ＥｃｏＲＩ酶切后亚克隆入ｐＧＥＸ－３Ｘ的ＥｃｏＲＩ位点之间,筛选在向插入重组子     

EB病毒3种全基因的扩增,克隆及表达

目的：扩增克隆及表达ＥＢ病毒（ＥＢＶ）３种（ＤＮＡ酶、甙激酶及ＨＢＲＦ１）全基因片段。方法：以Ｂ９５－８细胞株ＤＮＡ为模板，ＰＣＲ扩增３种目的基因。酶切鉴定后分别与高效表达载体ＰＢＶ２２１连接，用大肠杆菌ＪＭ１０３或ＨＢ１０１作为围论的宿主菌。抗氨苄青霉素培养形成单菌落后放大培养，根据重组质粒的电泳 行为粗筛，再双酶切。结果：各种阳性菌经３０℃→４２℃变温诱导培养，超声破碎后其上清液经十二烷基磺酸     

日本血吸虫中国大陆株安徽品系基因组文库的构建

以日本血吸虫中国大陆株安徽品系为材料，采用酚－氯仿抽提，纯化基因组ＤＮＡ。经ＢａｍＨＩ酶切后，与质粒ＰＵＣ＾１８重组，转化入大肠杆菌ＪＭ１０３，转化率为１０＾４。并对重组质粒ＤＮＡ进行酶切鉴定。     

EBV－DNA酶全基因扩增克隆和鉴定

根据Ｂｅａｒ的ＥＢ病毒（ＥＢＶ）基因全序列，设计包括ＥＢＶ特异性ＤＮＡ酶全基因的一对引物，在引物上设计有一个ＳａｌⅠ切点，以便于克隆。选用蛋白酶Ｋ裂解的Ｒａｊｉ细胞ＤＮＡ为模板，扩增出一条１４６０ｂｐ的特异条带，纯比后经ＴａｑⅠ酶切形成１１９８ｂｐ和２６２ｂｐ的两个片段，证实该扩增片段即为ＥＢＶ特异性ＤＮＡ酶的全基因片段。以ＪＭ１０３为宿主菌，以高效表达质粒ＰＢＶ２２１为载体，按常规方法作酶切、连接、转化，最后在含氨中青霉素的培养基上挑取单菌落扩大培养，用快速法少量制备质粒ＤＮＡ。根据质粒电泳结果粗筛，再行ＢａｍＨⅠ酶切初步鉴定，确定质粒大小为５１１７（１４５１＋３６６６）ｂｐ的菌落为阳性克隆。先后用ＢａｍＨⅠ和ＴａｑⅠ消化阳性重组体，以形成１２００ｂｐ和３９１７ｂｐ两片段的确定为正向连接重组体。本实验首次将ＥＢＶ－ＤＮＡ酶全基因片段克隆到高效表达载体ｐＢＶ２２１上获得成功     

琼脂糖凝胶中DNA的回收

用冻结压榨法、玻璃粉吸附洗脱法和透析袋电洗脱法，分别从琼脂糖凝胶中回收日本血吸虫中国大陆株副肌球蛋白编码基因的聚合酶链反应（ＰＣＲ）扩增产物，经ＢａｍＨ Ｉ及ＥｃｏＲ Ｉ双酶切的线状质粒载体ｐＴｒｃＨｉｓ Ａ，以及碱裂解法制备的质粒载体，均取得较为满意的回收率。冻结压榨法适于回收少量的ＤＮＡ，透析袋电洗脱法适于回收较大量ＤＮＡ，玻璃粉吸附洗脱法则应用范围较宽。     

中国妇女宫颈癌组织中HPV_(16)L_1晚期基因克隆及重组质粒的构建

本实验采用ＰＣＲ方法，以中国妇女宫颈癌组织ＤＮＡ为模板扩增出了３株ＨＰＶ１６Ｌ１晚期基因ＤＮＡ片段，扩增参数为９４℃１ｍｉｎ，５５℃３０ｓ，７２℃２ｍｉｎ，共３５个循环。将扩增产物ＨＰＶ１６Ｌ１ＤＮＡ片段与克隆载体ｐＣＲ２．１连接构建了ＨＰＶ１６Ｌ１－ｐＣＲ２．１重组质粒。经酶切ＨＰＶ１６Ｌ１－ｐＣＲ２．１重组质粒回收ＨＰＶ１６Ｌ１基因片段，将其与表达载体ｐＰＩＣ３．５连接，构建了ＨＰＶ１６Ｌ１－ｐＰＩＣ３．５重组体，并经酶切鉴定得到确认。为进一步对感染型ＨＰＶ１６Ｌ１晚期基因的测序及表达打下了基础。     

快速高效提取白色念珠菌DNA

目的建立两种简便的白色念珠菌ＤＮＡ提取方法，即纯ＤＮＡ提取法和快速ＤＮＡ提取法。方法以原生质体提取法为基础进行改良，两种方法均用Ｌｙｔｉｃａｓｅ破壁。结果与结论纯ＮＤＡ提取法提取ＤＮＡ耗时６～７ｈ，产量较高，每１０~（１０）个菌细胞可提取４μｇＤＮＡ，且纯度高，ＯＤ_（２６０）ＯＤ_（２８０）＞１．７，对核酸限制性内切酶敏感，适用于各种分子生物学方面的研究。快速ＤＮＡ提取法全过程仅需３～４ｈ，每１０~（１０）个菌细胞提取１μｇＤＮＡ，ＯＤ_（２６０）：ＯＤ_（２８０）＝１．４，不能进行核酸限制性内切酶酶切，但可用于作ＰＣＲ扩增。     

大肠杆菌耐药质粒的头孢哌酮抗性基因克隆和定位

用鸟枪法从耐药质粒ＰＦＣ上克隆到头孢哌酮抗性基因，快速小规模制备质粒ＤＮＡ进行限制性酶切分析鉴定含重组质粒的克隆株，经多次传代，克隆株抗性表达稳定。多种限制性内切酶谱分析并构建重组质粒的物理图谱与质粒ｐＥＣ物理图谱比较，定位出头孢哌酮抗性基因在ｐＥＣ物理图谱上３．２ｋｂ至４．８ｋｂ区间内，其分隔量约１．６ｋｂ。     

一种改良的质粒DNA的快速提取方法

目的：建立一种确实有效的更实用的小量质粒ＤＮＡ的提取方法。方法：将一含有目的质粒的大肠杆菌扩增后采用改良后的方法提取质粒ＤＮＡ，然后采用紫外分光光度计测定含量，并且进行酶切。结果：每毫升原细菌培养物质粒ＤＮＡ的产量明显增高，且酶切效果极好。结论：改良后的质粒ＤＮＡ提取法具有速度快、收获量多、纯度高、经济又方便等优点，适合于应用与推广。     

大肠杆菌耐药质粒的头孢哌酮抗性基因克隆和定位

用鸟枪法从耐药质粒ｐＦＣ上克隆到头孢哌酮抗性基因。快速小规模制备质粒ＤＮＡ进行限制性酶切分析鉴定含重组质粒的克隆株，经多次传代，克隆株抗性表达稳定。多种限制性内切酶谱分析并构建重组质粒的物理图谱与质粒ｐＦＣ物理图谱比较，定位出头孢哌酮抗性基因在ｐＦＣ物理图谱上３２ｋｂ至４８ｋｂ区间内，其分子量约１６ｋｂ。该基因包含有ＥｃｏＲⅠ、ＳｍａⅠ、及ＰｖｕⅡ位点。     

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.