PDZ1/2结构域与腺病毒穿梭载体重组体的构建

目的构建PSD-95结构域PDZ1/2腺病毒重组体。方法以异硫酸氰胍-酚-氯仿一步法提取的总RNA为模板,采用RT-PCR法获得PDZ1/2的cDNA;与腺病毒穿梭载体用T4 DNA连接酶连接;连接产物转化大肠杆菌JM109进行筛选、序列测定。结果①经RT-PCR得到了PDZ1/2的cDNA;②酶切鉴定能观察到PDZ1/2和pAdTrack-CMV两条带;③PDZ1/2测序图谱与文献报道完全一致。结论成功构建了含目的基因PDZ1/2的腺病毒穿梭载体重组体。     

反义BTEB2腺病毒载体的构建及其在血管平滑肌细胞中的表达

目的 :构建反义BTEB2重组腺病毒载体 ,以研究BTEB2反义RNA对血管平滑肌细胞 (VSMC)增殖及动脉再狭窄的影响。　方法 :从培养的大鼠VSMC中提取总RNA ,通过RT PCR制备BTEB2cDNA ,将BTEB2cDNA反向连接至腺病毒穿梭质粒pDC31 5中 ,构建重组穿梭质粒pDC31 5ASBTEB2 ,将其与腺病毒基因组质粒pBHGloxΔE1 ,3Cre共转染 2 93细胞获得重组腺病毒 ,采用PCR方法对其进行鉴定 ;用重组腺病毒感染VSMC ,通过RT PCR检测BTEB2反义RNA在VSMC中的表达。　结果 :在病变 2 93细胞的冻融上清中含有反义BTEB2重组腺病毒 ,其滴度为 5×1 0 9ml。VSMC被重组腺病毒感染后 ,可检测到BTEB2反义RNA的表达。　结论 :成功构建了反义BTEB2重组腺病毒载体 ;重组腺病毒可在VSMC中表达BTEB2反义RNA ,为进一步研究BTEB2反义RNA对VSMC增殖及动脉再狭窄的影响奠定了基础     

重组复制缺陷型腺病毒AdIL-10构建及其在HSC中的表达

目的利用AdEasy系统构建重组复制缺陷型腺病毒AdIL-10,并观察其在肝星状细胞(HSC)中的表达.方法从SD大鼠脾脏单核细胞中抽提总RNA,通过RT-PCR获得鼠IL-10 cDNA 片段, 插入穿梭质粒pAdTrack巨细胞病毒(CMV)启动子下游,构建重组穿梭载体pAdTrack-CMV-IL-10,线性化后与AdEasy-1电穿孔共转化BJ5183,获得重组腺病毒质粒pAdIL-10.pAdIL-10用Lipofectamin包装转染HEK 293细胞,获得重组腺病毒AdIL-10.将AdIL-10体外感染HSC,3 d后抽提HSC总RNA,以RT-PCR检测IL-10的表达.结果通过酶切和测序法筛选出pAdIL-10;经293细胞包装,3 d后观察到绿色荧光蛋白(GFP)表达;AdIL-10感染HSC 3 d后观察到GFP表达,通过RT-PCR可检测到IL-10的表达.结论利用AdEasy系统可制备重组腺病毒AdIL-10;AdIL-10感染HSC后可表达IL-10.     

低氧诱导因子基因重组腺病毒载体的构建及其在内皮细胞中的表达

目的：构建携带低氧诱导因子-1α（HIF-1α）基因的腺病毒载体（pAdxsi-GFP-HIF）,观察其在内皮细胞中的表达。 方法：低氧处理A549细胞后提取总RNA并逆转录为cDNA,以之作为模板,依据基因库公布的HIF-1α cDNA 设计引物,分别引入KpnI和BamHI酶切位点,PCR扩增后将目的基因HIF-1α连接到载体pShuttle-CMV-EGFP上,构建穿梭质粒pShuttle-GFP-HIF。采用细菌内重组方法将目的序列重组到pAdxsi病毒骨架载体上构建携带HIF-1α基因的重组腺病毒载体。检测重组腺病毒效价后,转染人脐静脉内皮细胞ECV304,检测目的基因的转染表达。 结果：通过对构建质粒克隆进行测序及酶切,证实携带HIF-lα基因的重组腺病毒载体pAdxsi-GFP-HIF构建成功,且构建的重组腺病毒纯度好、效价高。以100 MOI转染ECV304细胞24 h后在荧光显微镜下可观察到细胞有较强的绿色荧光表达,转染48 h时荧光表达更强,且培养上清液中HIF-1蛋白表达水平为（48.93±3.86）ng/mL。 结论：本实验构建的携带HIF-1α基因的腺病毒载体pAdxsi-GFP-HIF转染效率及目的基因的蛋白表达水平均较高,有望应用于缺血缺氧组织局部。     

大鼠DNA多聚酶β基因克隆及其腺病毒重组体构建

目的 构建大鼠DNA多聚酶β基因克隆及其腺病毒介导的表达载体。方法 提取大鼠脑组织总RNA．用逆转录PCR扩增DNA多聚酶β（POLB）的编码区，与T载体连接，作DNA测序后，酶切POLB基因并连入腺病毒穿梭质粒pAdTraek-CMV，电转至含腺病毒pAdeasy骨架质粒的大肠杆菌BJ5183。酶切鉴定，重组腺病毒质粒（pAd-polb）Lipofectamine2000转染293细胞，用293细胞扩增重组腺病毒。结果 8个TA克隆中有2个克隆的序列与GENE BANK中大鼠POLB cDNA序列完全一致。DNA序列正确的POLB重组腺病毒质粒Ad-polb转染293细胞后，荧光显微镜下可见绿色荧光。结论 用TA克隆和AdEasy系统，成功构建了大鼠DNA多聚酶β的腺病毒重组体。     

MafA基因重组腺病毒的构建及其对小鼠肝癌细胞的影响

目的利用AdEasy-1系统构建胰岛素转录因子之一的MafA基因重组腺病毒（Ad-MafA）,并观察其对小鼠肝癌细胞的影响。方法将质粒cDNA3/MafA扩增、酶切获得MafADNA片段插入腺病毒穿梭载体质粒pAdTrack-CMV的巨细胞病毒（CMV）启动子下游,构建重组穿梭载体pAdTrack-CMV-MafA,线性化后与骨架载体AdEasy-1在细菌BJ5183内同源重组得到腺病毒质粒pAd-MafA,经293细胞包装后得到含MafA全长DNA片段的重组腺病毒AdMafA;将Ad-MafA体外感染小鼠肝癌细胞（Hepa1-6）,以RT-PCR和Westernblot检测MafA在hepa1-6细胞的表达。结果连接、重组后通过酶切法筛选出pAd-MafA,经293细胞包装,5d后观察到绿色荧光蛋白（GFP）明显表达,通过反复感染HEK细胞扩增得到高滴度的重组腺病毒,Ad-MafA体外感染肝癌细胞1d后,MafA基因表达和蛋白表达明显增加。结论利用新型腺病毒载体AdEasy-1系统可在短期内制备同时表达GFP和MafA的重组腺病毒（Ad-MafA）,Ad-MafA体外感染小鼠肝癌细胞可显著提高MafA的表达,这将为基因治疗糖尿病提供新的手段。     

人骨形成蛋白-2基因的克隆及其重组腺病毒载体的构建

目的：克隆人骨形成蛋白-2（BMP-2）基因cDNA，构建含BMP-2基因的重组腺病毒载体。方法：采用RT-PCR方法克隆BMP-2基因cDNA，并插入克隆载体pGEM-T中进行全序列分析。将BMP-2基因cDNA克隆到穿梭载体pShuttle-CMV中，构建pShuttle-BMP2重组质粒，再将其中的表达盒克隆入Adeno-X腺病毒DNA中，获得重组腺病毒DNA-pAd-BMP。 结果：成功地克隆长约1 200 bp的BMP-2 cDNA，并成功地构建其腺病毒载体，经线性化的pAd-BMP2 DNA转染HEK293细胞，包装、扩增后得到人骨形成蛋白-2重组腺病毒，其滴度约为1×10¹¹nfu•L^-1，该滴度可满足进一步的BMP-2成骨作用的研究。结论:成功地构建BMP-2腺病毒载体。     

人神经营养素-3受体基因的扩增及其重组腺病毒载体的构建

【目的】构建人神经营养素-3（neurotrophin-3，NT-3）受体（即酪氨酸蛋白激酶受体C，tyrosine protein kinase C，TrkC）基因重组的腺病毒表达载体。【方法】从人脑组织mRNA中扩增Trkc基因全长cDNA，定向克隆于穿梭质粒pShuttle中，获得一个带有CMV启动子的表达盒。再将表达盒与腺病毒骨架DNA（Adeno-X viral DNA）体外连接，形成重组腺病毒质粒（pAd-TrkC）。用pAd-TrkC转染人胚肾293细胞后包装成有感染能力的重组腺病毒颗粒（Adeno-Trkc）。【结果】TrkC基因RT-PCR扩增产物为2478bp。Adeno-TrkC经PCR鉴定为正确重组子。【结论】应用体外连接法已构建了人TrkC重组腺病毒表达载体，这为进一步应用NT-3进行基因治疗中枢神经损伤奠定了基础。     

重组穿梭质粒pAdTrack-cmv-rHSG载体的构建及鉴定

目的构建大鼠增殖抑制基因（rat hyperplasia suppressor gene,rHSG）腺病毒穿梭质粒,为进一步研究体内诱导rHSG在恶性胶质瘤的特异性表达奠定基础。方法提取SD大鼠心肌总RNA,利用RT-PCR方法扩增大鼠增殖抑制基因（rHSG）的全开放读码框区片段,将其亚克隆入T载体,双酶切后与经同样处理的腺病毒穿梭质粒pAdTrack-cmv相连,用酶切和测序进行鉴定。结果扩增rHSG克隆出rHSG基因并成功重组于入腺病毒基因并成功的穿梭质粒。结论构建成功重组大鼠增殖抑制基因腺病毒穿梭质粒载体pAdTrack-cmv-rHSG。     

PSD-95及其突变体的重组腺病毒对皮质神经元的感染

目的构建PSD-95（postsynaptic density-95）及其突变体PSD-95Y523F和PSD-95Y533F（523和533位酪氨酸分别突变成苯丙氨酸）的复制缺陷型重组腺病毒载体,并检测腺病毒颗粒对体外原代培养皮质神经元的感染能力。方法双酶切将PSD-95及其突变体的基因分别从载体peDNA3．1（＋）亚克隆至穿梭载体pAdTrack—CMV中;电穿孔法将穿梭重组体转化到电转感受态细胞BJ5183中,使其与腺病毒骨架载体pAdEasy-1同源重组;同源重组体经PacI酶切线性化,脂质体法转入HEK293H细胞中进行病毒包装;病毒颗粒分别感染HEK293T细胞和原代皮质神经元,荧光检测或免疫印迹方法检测目的蛋白的表达水平。结果经酶切电泳鉴定和序列测定证明同源重组体中含序列正确的目的基因;重组腺病毒颗粒感染HEK293T细胞后,免疫印迹显示有较高水平的目的蛋白表达;重组腺病毒具有感染原代皮质神经元的能力,在未成熟神经元（体外培养5—9天）中感染率低,而在成熟神经元（体外培养13天）中感染率较高。结论成功构建了PSD-95、PSD-95Y523F、PSD-95Y533F的重组腺病毒载体;获得具有感染能力的重组腺病毒颗粒,易于感染成熟的神经元。     

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.     

Development on Adjustable Calibration Marker for Shock Wave Focus

Shock wave lithotripsy （SWL） is a treatment of choice for upper urinary stones. However, this procedure is inappropriate for obese patients because the focus is often unable to reach the target owing to the limited focal distance in shock wave source. Although treating such patients in a blast path may increase the application length of shock wave source, it＇s difficult to find this path on the lithotripter monitor. For this reason, we invented an adjustable calibration marker in order to set an effective focus in the shock wave hath.     

Study on the TCM Pathogenesis of Angiopathic Complications of Type II Diabetes

Excess production of reactive oxygen species(ROS)of mitochondrion mediated by hyperglycemia is the common pathogenesis of angiopathic complications of diabetes.TCM holds that the damp from the dysfunction of spleen.kidney and liver is the causative factor of complications of diabetes.This is similar to the mechanism of Ros resulting in angiopathic complications of diabetes.When the angiopathic complications of type II diabetes mellitus(T2DM)are difierentiated as caused by turbid damp in TCM can be explained as ROS.Since the obstruction of pathogenic damp in channels and collaterals is said to be the main pathogenesis,the treating principle should be dissolving the damp to remove the obstruction.     

Leptin, Ghrelin and TNF-α before and after Hypo-caloric Traditional Chinese Diet and Auricular Acupuncture

INTRODUCTION Obesity is a complex emergent problem, which can be possibly solved not only by the diet but also by the life style and promotion of a constant physical exercise. 1, 2 No doubt careful attentions must be given to the nutritional condition of obese people, the dietary habits, the somatic build (i.e. distribution of fat mass) and the organic functions linked to formation of the fat mass. All the parameters should be constantly monitored before, during and after a diet treatment. 3, 4, 5     

Relationship between Dysglycemia and Carotid Atherosclerosis in Tibetan Population

People with dysglycemia are at high risk for atherosclerotic diseases. This study aims at investigating the atherosclerotic vascular damage in dysglycemia and its metabolic origin in Tibetan population.     

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.     

CLINICAL SIGNIFICANCE OF SERUM CYTOKINES IL-1β,sIL-2R,IL-6,TNF-α,AND IFN-v IN ACUTE CORONARY SYNDROME

Objectives To explore serum cytokines levels (including IL-1 β, sIL-2R, IL-6, TNF-α, and IFN-v) and their significance in patients with acute coronary syndrome (ACS) and the subsequent follow-ups, with attempt to estimate the role of various serum inflammatory markers in the diagnosis and assessment of ACS.Methods The study population include 40 patients with acute myocardial infarction (AMI), 40 patients with unstable angina pectoris (UAP), and 40 controls. Among the 80 patients, 60 patients attended a follow up 4 months later. Serum inflammatory markers including IL-1 β, sIL-2R, IL-6, TNF-α, and IFN-v were measured by enzyme linked immunosorbent assay.Results Serum IL- 1 β, sIL-2R, IL-6, TNF-α were significantly higher in AMI group or UAP group compared to the control group and became significantly lower 4 months later in the follow-up patients. Serum levels of IFN-v shows no significant difference between AMI group or UAP group and controls, also showing no significant change when measured in follow up patients. There was no correlation between serum creatine kinase-MB isoenzyme levels and serum inflammatory markers either in UAP or AMI group. Furthermore, when divided into two subgroups using Wagner's QRS scoring system in the AMI group, there is no difference of each serum inflammatory marker between ≤ 6 scores group and ＞ 6 scores group.Conclusion Serum levels of certain inflammatory markers may have some diagnostic value for ACS, and can be a useful marker reflecting disease stability.