舌鳞癌干细胞相关差异表达cDNA文库的鉴定与分析

目的对构建的舌鳞癌干细胞相关差异表达cDNA文库进行初步鉴定及分析。方法将差异表达cDNA文库中的cDNA片段转化大肠杆菌进行文库扩增和蓝白斑筛选,正反向文库各随机挑取120个白色克隆进行PCR鉴定并测序。将测序所得序列提交GenBank进行Blast同源性分析。利用PubMed数据库检索已报道的与干细胞相关的差异基因并进行生物信息学分析。结果 PCR鉴定显示挑取的克隆均为阳性克隆,测序获得224个EST片段。经同源性分析得到62个已知基因,其中9个已有相关文献报道与干细胞生物特性相关,可归类于细胞分化调节、低氧应答、细胞凋亡调节等。结论对前期构建的干细胞相关差异表达cDNA文库进行克隆分析得到一些干细胞相关基因。     

Balb／c小鼠胚胎皮肤无瘢痕愈合相关基因cDNA文库构建

目的筛选、克隆Balb／c成年小鼠和小鼠胚胎皮肤的特异表达基因,构建成年小鼠和小鼠胚胎皮肤差异表达的cDNA文库。方法应用Trizol法抽提样本的总RNA,进一步获得mRNA,结合PCRcDNA合成法将合成的双链cD-NA产物与载体连接构建cDNA文库,并转染大肠杆菌进行文库扩增,菌液进行PCR扩增,筛选有插入片段cDNA克隆。结果成功地构建了Balb／c小鼠皮肤无瘢痕愈合相关基因差异表达的cDNA文库,从中挑取85个克隆进行菌液PCR分析,结果显示57个克隆得到300—9Kp插入片段。结论通过RT—PCR方法并合成双链cDNA是一种自微量总RNA中构建高特异性的差异表达cDNA文库的有效方法。cDNA文库的构建有助于筛选、克隆小鼠胚胎皮肤无瘢痕愈合相关的特异表达基因。     

应用抑制消减杂交技术构建哮喘病人嗜酸细胞差异表达基因消减cDNA文库

目的构建哮喘病人治疗前后嗜酸细胞差异表达基因的消减cDNA文库。方法采用新近建立的抑制消减杂交方法,哮喘治疗前后的嗜酸细胞为试验和对比材料,分离哮喘病人嗜酸细胞中差异表达基因的cDNA片段,将其与T载体进行T／A连接构建文库,将连接产物用氯化钙转化法转化大肠杆菌进行文库扩增和蓝白斑筛选,随机挑取100个白色克隆用菌落PCR进行鉴定。结果扩增消减cDNA文库获得3000余个白色阳性克隆,随机挑取100个白色克隆用PCR进行扩增,90％的克隆中均有200～600bp的插入片段,这些片段可能是哮喘嗜酸细胞差异表达基因的cDNA片段。结论用SSH法及T／A克隆技术成功构建了哮喘病人治疗前后嗜酸细胞差异表达基因消减cDNA文库,该消减cDNA文库的建立为进一步筛选、克隆哮喘病人嗜酸细胞差异表达的新基因奠定了基础。     

应用抑制消减杂交技术构建肝癌差异表达基因消减cDNA文库

目的 构建肝癌及癌旁肝细胞差异表达基因的消减cDNA文库。方法 采用新近建立的抑制消减杂交方法,以肝癌组织及癌旁肝组织作为对比材料,分离肝癌组织中差异表达基因的cDNA片段,将其与T载体进行T／A连接构建文库,将连接产物用电穿孔法转化大肠杆菌进行文库扩半后,随机挑取100个白色克隆及菌落PCR进行鉴定。结果 扩增消减cDNA文库获得3000余个白色阳性克隆,随机挑取100个白色克隆用PCR进行扩增,95％的克隆中均有100－600bp的插入片段,这些片段可能是肝癌差异表达基因的cDNA片段。结论 用SSH法及T／A克隆技术成功构建了肝癌与癌旁肝组织差异表达基因消减cDNA文库,该消减cDNA文库的建立为进一步筛选,克隆肝癌差异表达的新基因奠定了基础。     

高凝状态大鼠肝脏差异表达基因正向消减cDNA文库的构建与初筛

目的构建高凝状态(prothrom botic states,PTS)大鼠肝脏差异表达基因正向消减cDNA文库并进行初步筛选。方法从PTS模型大鼠和对照大鼠肝脏提取po ly A mRNA,分别以po ly A mRNA为模板,依次合成单链和双链cDNA,经酶切成400~600 bp大小的片段。以PTS大鼠cDNA作为T ester,对照大鼠cDNA为D river,进行抑制性消减杂交。将消减杂交第二次PCR产物cDNA克隆至pM D 18-T载体上,然后转化细菌,获得PTS大鼠肝脏差异表达基因正向消减cDNA文库。用巢式PCR扩增法制备“正向”和“反向”消减cDNA探针;采用差异筛选方法,用这两种探针对PTS大鼠肝脏差异表达基因正向消减cDNA文库进行筛选;将获得的阳性克隆cDNA进行序列分析;并与G enB ank DNA数据库中的DNA序列进行同源性分析。结果成功构建了PTS大鼠肝脏差异表达基因正向消减cDNA文库,初步筛选发现两条PTS差异表达cDNA片段。结论成功构建了PTS大鼠肝脏差异表达基因正向消减cDNA文库。     

传代培养前后内皮细胞差异表达基因消减cDNA文库的构建

目的:构建传代培养前后牛主动脉内皮细胞差异表达基因的消减cDNA文库,筛选动脉粥样硬化相关新基因构建文库. 方法:采用抑制消减杂交技术,以新鲜分离和传代培养的牛主动脉内皮细胞作为对比材料,分离传代培养主动脉内皮细胞中不表达或低表达基因的cDNA片段,将其与T载体进行T/A连接构建文库,将连接产物转化大肠杆菌进行文库扩增后,随机挑取64个白色克隆进行酶切鉴定. 结果:扩增消减cDNA文库获得760个克隆,随机挑取的64个阳性克隆经酶切后均有200～600 bp的插入片段. 结论:构建的传代培养前后内皮细胞差异表达基因消减cDNA文库,为进一步筛选早期发生改变的动脉粥样硬化相关基因奠定了工作基础.     

高胆固醇血症小鼠相关基因消减文库的建立

目的 构建高胆固醇血症小鼠cDNA消减文库.方法 以高胆固醇血症小鼠肝组织的eDNA为Teste,以正常小鼠肝组织的cDNA为Driver,应用抑制消减杂交技术(Suppression subtractive hybridization).构建正向消减文库,反之构建反向消减文库.结果 成功构建高胆固醇血症cDNA正.反向消减文库,利用葛落PCR技术鉴定文库的阳性克隆.结论 用SSH技术成功构建了高胆固醇血症小鼠差异表达基因消减cDNA文库,该消减cDNA文库的建立为进一步筛选.克隆高胆固醇血症小鼠差异表达的基因奠定了基础.     

视神经慢性损伤相关差异表达cDNA文库的鉴定与分析

目的:对构建的猫视神经慢性损伤相关差异表达cDNA文库进行初步克隆、鉴定和分析.方法:用氯化钙转化法转化大肠杆菌进行cDNA文库扩增和蓝白斑筛选,每个文库随机挑取300个白色克隆用菌落PCR进行鉴定.对阳性克隆菌落进行DNA测序以获得差异表达基因片段序列.将测序所得的序列通过互联网用Blast程序查找日本国家DNA数据库进行同源性分析.用实时定量PCR验证差异克隆.结果:PCR鉴定获得1000个阳性菌落,测序获得674个EST片段,序列长度在200～800 bp之间.同源性分析结果提示4周正向文库得到14个同源基因,4周反向文库得到20个同源基因,8周正向文库得到23个同源基因,8周反向文库得到19个同源基因.这些基因可归类于能量代谢、物质转运、信号转导、基因转录、细胞损伤与修复、MHC分子等.实时定量PCR检测的4个克隆证实是差异表达序列.结论:本研究构建的视神经慢性损伤相关差异表达cDNA文库为进一步鉴定和研究慢性视神经损伤相关基因提供了实验基础.     

人肝脏高凝状态相关基因的筛选

目的 筛选人肝脏高凝状态（PTS）相关基因的全长cDNA序列。方法 采用差异筛选法对本室建立的PTS大鼠肝脏差异表达基因消减cDNA文库进行筛选；以获得的差异cDNA克隆为模板，采用PCR方法扩增目的片段；以PCR产物作探针，用噬菌斑原位杂交法筛选人肝脏cDNA文库，经初筛、二筛和三筛后，将获得的阳性克隆转入E．coli BM 25．8，使λTripIEx环化成质粒pTripIEx，经酶切鉴定后进行DNA测序。结果 从人肝脏cDNA文库中筛选获得4条全长cDNA序列，经序列测定及生物信息学分析发现，其中3条的表达产物分别为纤维蛋白原、纤维蛋白原相关蛋白、10号染色体开放阅读框架104mRNA；另1条与氨基甲酰磷酸合成酶1同源。结论 以PTS大鼠肝脏差异表达基因cDNA为探针，从人肝脏cDNA文库中筛选获得4条PTS相关cDNA全长序列。     

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.     

APPLICATION OF LORNOXICAM TO PATIENT-CONTROLLED ANALGESIA IN PATIENTS UNDERGOING ABDOMINAL SURGERIES

FOR anesthesiologis s ,treatingpostoperativepainhas alwaysbeen a problem.Althoughopioidshave been provedtobe effective,theirsideeffectscouldnotbeignored.With thedevelopmentofscienceand pharmacology,many drugs with aspectsof satisfactoryanalgesicefficacyand couldbe welltoleratedby patientshave been developed.And lornoxicamisone of them, which isa non-steroidalanti-inflammatorydrug (NSAID ), with analgesic, anti-infl-ammatory,andantipyreticproperties.Itseliminationhalf-time(3 to 5 hours) isle…     

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.     

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.