人IL-10基因重组复制缺陷型腺病毒DNA的构建

目的构建人IL-10基因的重组腺病毒并进行体外表达和检测.方法从质粒pcDNA3IL-10的CMV启动子下游完整切下IL-10 cDNA的片段,将其定向插入Gateway载体,在克隆酶的作用下将阳性克隆质粒转入目的腺病毒载体.PacI酶线性化IL-10腺病毒DNA后阳离子质脂体转染293A细胞,获得人IL-10的复制缺陷型重组腺病毒.体外感染人胰腺癌细胞株Bxpc-3和大鼠胰腺细胞株AR-42J;Western blot检测人IL-10的表达.结果成功地构建人IL-10重组腺病毒,病毒滴度达2×109PFU/mL.体外感染的细胞株均检测到IL-10的表达.结论构建复制缺陷型重组腺病毒能够介导IL-10的基因表达,为细胞因子的抗炎治疗奠定实验基础.     

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.     

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，插人穿梭质粒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，3d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，3d后观察到绿色荧光蛋白(GFP)表达；AdIL-10感染HSC3d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。     

携带人白细胞介素18基因的复制缺陷型腺病毒载体的构建、鉴定和滴度测定

目的构建人白细胞介素18（IL-18）基因复制缺陷型腺病毒表达载体。方法以质粒pJWIL—18为模板，设计并合成上、下游引物，利用多聚酶链反应（PCR）扩增获得IL-18基因片段，酶切后克隆至pCA13质粒，构建重组质粒pCA13IL—18。鉴定正确后，将pCA13 IL—18与含有腺病毒右臂的质粒pBHGE3共转染HEK293细胞，9-12天出现病毒空斑。提取重组腺病毒的DNA。进行PCR鉴定。大量扩增后，氯化铯梯度离心纯化腺病毒，测病毒滴度。结果酶切分析、序列测定、PCR验证表明，IL-18基因成功克隆到复制缺陷型腺病毒载体中。结论成功构建了表达人IL-18基因的复制缺陷性腺病毒载体。     

mIL-10重组复制缺陷型腺病毒载体的构建及制备

目的构建小鼠白介素-10(mIL-10)重组腺病毒真核表达载体,为其体外表达及动物体内试验研究提供依据。方法以pORF5-mIL-10质粒为模板通过PCR方法扩增回收mIL-10,将其定向连接到腺病毒载体pSGCMV中,构建pSGCMV-mIL10质粒。此质粒与5型腺病毒质粒pBGHE3通过Lipofectamine 2000共转染至293细胞,经同源重组产生重组复制缺陷型腺病毒Ad5-mIL-10。纯化后Ad5-mIL-10在293细胞大量扩增并通过氯化铯密度梯度离心法纯化,测定其滴度。结果扩增所得mIL-10经酶切鉴定、PCR扩增、DNA测序均证实为小鼠白介素-10,重组腺病毒Ad5-mIL-10经PCR鉴定正确,滴度达2.5×1010pfu/ml。结论成功构建了Ad5-mIL-10重组腺病毒载体系统,为研究IL-10在临床移植中的免疫学作用及基因治疗奠定基础。     

含结核分枝杆菌ESAT6和CFP10基因的重组腺病毒表达载体的构建

目的克隆结核分枝杆菌的ESAT-6和CFP-10基因,构建能同时表达两基因的重组腺病毒活载体,为研制能有效防治结核病的重组腺病毒活载体疫苗打下基础。方法首先利用PCR技术克隆结核分枝杆菌的ESAT-6和CFP-10基因,将两者通过IRES串连插入腺病毒转移载体中,构建重组穿梭质粒pShuttle-CMV-ESAT-6-IRES-CFP-10。该穿梭质粒转化含有腺病毒骨架质粒pAdeasy-1的BJ5183感受态细胞,经同源重组获得复制缺陷型重组腺病毒载体pAdeasy-1-pShuttle-CMV-ESAT-6-IRES-CFP-10。PacI酶切线性化该质粒,转染AD293细胞进行病毒包装。结果ESAT-6和CFP-10基因成功克隆,其序列与GenBank公布的一致。两者通过IRES串连插入腺病毒转移载体,获得的重组穿梭质粒经同源重组,成功获得复制缺陷型重组腺病毒质粒Adeasy-1-pShuttle-CMV-ESAT-6-IRES-CFP-10,转染该质粒的AD293细胞出现细胞病变效应。结论成功克隆了分枝杆菌的ESAT-6和CFP-10基因,构建了含IRES串连的ESAT-6和CFP-10基因的重组腺病毒,为重组腺病毒活载体疫苗的研制奠定了基础。     

利用Adeasy-1系统构建npcl基因非复制型腺病毒载体并鉴定

目的以复制缺陷的腺病毒为载体,细菌内同源重组法构建有npcl基因的腺病毒载体.方法利用限制性内切酶kpnl HindⅢ从带有朊病毒启动子的质粒prp-pro-mlpcl中切下目的基因npcl-1,克隆至穿梭质粒PAdtrack-CMV,再与PAdeasy-1在大肠杆菌BJ5183中进行同源重组,经抗性筛选和酶切鉴定的质粒再利用脂质体转染人293例细胞中扩增,利用Adeasy-1系统上的绿色荧光蛋白鉴定病毒,RT-PCR鉴定目的基因的表达.结果切酶切鉴定,正确的目的基因片断已克隆至穿梭质粒PAdtrack-CMV;卡那霉素进行抗性筛选及Pac Ⅰ酶切鉴定证实腺病毒重组质粒构建成功.Pac Ⅰ酶切线性化的重组质粒导入293细胞3d后见明显的绿色荧光蛋白表达.回收病毒,可重复感染293细胞,证实有感染能力的病毒颗粒包装成功.结论利用腺病毒载体系统Adeasy-1可构建能成功表达Pac Ⅰ基因的腺病毒载体,为对Niemann pick' s病C型的进一步研究提供了条件.     

携带乙肝核心抗原基因复制缺陷型重组腺病毒的高效制备和表达

目的构建表达乙肝病毒核心抗原(HBcAg)的复制缺陷型重组腺病毒载体。方法扩增乙肝病毒(HBV)C基因片段,亚克隆到穿梭质粒pAdTrack-CMV上,与5型腺病毒骨架质粒pAdeasy-1共同电转化到大肠杆菌BJ5183内进行同源重组,经卡那霉素抗性筛选和酶切鉴定筛选出携带HBVC区基因的重组腺病毒载体,用脂质体包裹PacⅠ酶切线性化的重组质粒,转染到293细胞内进行重组腺病毒的包装。体外转染真核细胞,通过示踪基因绿色荧光蛋白表达的观察、RT-PCR和ELISA检测目的基因的表达。结果成功获得重组腺病毒质粒pAd-HBc。重组质粒pAd-HBc导入293细胞,经过包装和二次扩大培养获得了具有感染能力的重组腺病毒颗粒Ad-HBc,其病毒滴度达5×109pfu/mL,并能在真核细胞中有效表达目的基因。结论成功构建表达HBcAg复制缺陷型重组腺病毒载体,为进一步开展HBV基因治疗研究提供实验基础。     

复制缺陷型人TPO重组腺病毒的构建与鉴定

目的:构建并鉴定携带人TPO基因的复制缺陷型重组腺病毒.方法:将含全部编码序列的人TPOcDNA克隆于腺病毒载体穿梭质粒pCA3的CMV启动子下游,然后与含有腺病毒基因组的质粒pBHG10共转染293细胞,经同源重组产生人TPO重组腺病毒(AdCMVTPO);体外转染A549细胞并检测其表达活性.结果:扩增后获得的病毒滴度达4.5×109pfu/ml,经体外转染A549细胞检测到TPO的表达.结论:所构建的重组腺病毒能有效介导人TPO基因的转移,可望用于造血功能重建的基因治疗研究.     

新型Smad7 重组腺病毒载体的构建

目的构建鼠Smad7重组复制缺陷型腺病毒载体(AdSmad7)。方法采用常规分子生物学方法，抽提SD大鼠肝脏总RNA，RT-PCR获得鼠Smad7 cDNA片段，插入腺病毒穿梭载体质粒pAdDTrack CMV启动子下游，构建重组穿梭载体pAdTrackCMV-Smad7，线性化后与腺病毒骨架载体质粒AdEasy-l在细菌BJ5l83内同源重组，鉴定后在HEK293细胞包装成重组腺病毒AdSmad7。RT-PCR检测AdSmad7在HEK293中的表达情况，结果该重组缺陷型腺病毒载体经测序、限制性内切酶酶切分析、PCR等鉴定，与预期结果一致；重组腺病毒质粒转染HEK293细胞后3d可观察GFP明显表达；RT-PCR检测证实Smad7表达增强。结论利用新型腺病毒载体AdEasy系统可快速构建同时表达GFP和Smad7的重组复制缺陷型腺病毒AdSmad7，为进一步研究TGFl3信号转导和肝纤维化的基因治疗奠定基础。     

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…     

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.     

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     

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.     

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.     

Acupuncture Combined with Spinal Tui Na for Treatment of Primary Dysmenorrhea in 30 Cases

Objective: To observe the therapeutic effects in acupunture treatment of primary dysmenorrhea combined with spinal Tui Na, and study its mechanism. Methods: Thirty cases of the treatment group were treated by acupuncture combined with spinal Tui Na, and thirty cases in the control group were treated by routine acupuncture. Results: The total effective rate was 93.3% in the treatment group, and 73.3% in the control group, with a significant difference between the two groups (P<0.05). Conclusions: Acupuncture combined with spinal Tui Na has good prospects for treatment of primary dysmenorrhea.     

Luo Lingjie's Experience in Treating Chronic Nephropathy

In treating chronic nephropathy,Luo Lingjie,a chief physician,pays attention to regulating the balance between yin and yang,treating infection if present,and removing pathogenic factors.He prescribes gentle drugs and uses carefully strongly warming-tonifying ones,emphasizes the importance of persuading the patient to persist in treatment with medication and nurse one's health for recuperation,and is good at combined use of TCM and western medicine therapy and brings the merits of various therapies into full play,with obvious theraoeutic effects.