葡萄糖激酶基因逆转录病毒表达载体及包装细胞系的构建及鉴定

目的构建葡萄糖激酶(glucokinase,GK)基因逆转录病毒表达载体及稳定的产毒细胞系,为将GK基因应用于糖尿病的基因治疗打下基础.方法质粒pCMV4-GKZ1经EcoRⅠ/BamHⅠ双酶切后亚克隆至逆转录病毒载体PLXSN,构建成重组逆转录病毒表达载体PLX-GK,采用酶切及测序对重组体进行鉴定.而后脂质体转染PLX-GK至包装细胞PA317,检测培养上清病毒滴度,挑选滴度较高的稳定产毒细胞系并对其行PCR及免疫组化鉴定.结果构建了GK基因逆转录病毒表达载体PLX-GK,经酶切及序列分析证实目的基因插入位点和读码框架正确、无突变;建立了稳定产毒细胞系PA317/GK,其培养上清平均病毒滴度为6.8×105cfu/ml,最高为1.8×106cfu/ml,PCR及免疫组化证实GK基因整合入PA317/GK细胞基因组并有GK的表达.结论成功构建了携GK基因的逆转录病毒表达载体及高滴度的稳定产毒细胞系.     

葡萄糖转运蛋白-2逆转录病毒表达载体构建/包装及其鉴定

目的 构建葡萄糖转运蛋白-2(glucose transporter-2,GLUT-2)基因逆转录病毒表达载体及其稳定产毒细胞系,为新型"人工胰岛β细胞"的构建奠定基础.方法 质粒pCB7/GLUT2经EcoRⅠ、BamHⅠ双酶切,克隆至逆转录病毒载体PLXSN,构建成重组逆转录病毒表达载体pL-GLUT2-SN,经酶切及测序鉴定;转pL-GLUT2-SN至包装细胞系PA317,G418抗性筛选,检测上清病毒滴度,挑选滴度较高的稳定产毒细胞克隆行PCR及RT-PCR鉴定.结果 建立GLUT-2基因逆转录病毒表达载体pL-GLUT2-SN,经酶切及测序证实目的基因插入位点及读码框架正确;所建稳定产毒细胞克隆PA317/GLUT2,其最高病毒滴度达7.1×105 CFU/ml,PCR及RT-PCR证实GLUT-2基因整合入其中并稳定表达.结论 成功构建GLUT-2基因逆转录病毒表达载体及其稳定产毒细胞系,为"人工胰岛β细胞"的构建奠定了基础.     

NIH3T3细胞中前脑啡肽原基因的稳定表达

目的:利用重组逆转录病毒载体pLNCX2-pENK,研究pENK基因在NIH3T3细胞中是否能够稳定表达.方法:用pLNCX2-pENK转染病毒包装细胞PT67,其病毒上清液感染NIH3T3细胞,检测pENK基因是否整合人NIH3T3细胞基因组中并通过RT-PCR及放射免疫法检测基因表达情况.结果:病毒基因组整合到NIH3T3细胞基因组中,并检测到目的基因转录及蛋白表达.结论:用逆转录病毒载体能将pENK基因整合到靶细胞NIH3T3基因组中,且该基因能长期稳定表达,为慢性疼痛的进一步治疗奠定了基础.     

EBV LMP2A逆转录病毒表达载体的构建及产毒细胞系的建立

目的构建含EB病毒（EBV）潜伏膜蛋白基因2A（LMP2A）的逆转录病毒表达载体，筛选建立携带该基因的高滴度产毒细胞系。方法逆转录一聚合酶链反应（RT—PCR）扩增获取目的基因LMP2A，定向插入逆转录病毒表达载体pMSCVpuro，形成重组质粒pMSCVpuro-2A，脂质体法将pMSCVpuro-2A转染逆转录病毒包装细胞PT67。嘌呤霉素筛选产毒细胞克隆，扩大培养产毒细胞克隆，收获病毒进行滴度测定，RT-PCR检测产毒PT67细胞目的基因的转录表达。结果限制性酶切、PCR及测序鉴定证实LMP2A正确插入逆转录病毒表达载体；筛选获得稳定产毒的抗性细胞克隆；收获病毒的滴度为3．2×10^7CFu／L，且重组腺病毒在PT67细胞能有效转录。结论携带LMP2A基因的重组逆转录病毒表达载体pMSCVpuro-2A构建成功，转染PT67细胞后包装出重组逆转录病毒，进而筛选获得了能转录表达LMP2A的产毒细胞系PT67-LMP2A。     

hTERT-逆转录病毒载体构建及其在脐血间质干细胞中的表达

目的:构建hTERT-逆转录病毒载体,向脐血间质干细胞(UCBMSCs)中导入hTERT基因,观察该基因的表达。方法:PCR法扩增出hTERT全部片断,定向克隆入pLNCX2载体。将病毒质粒导入包装细胞内,筛选阳性产毒细胞,测定病毒滴度。取病毒上清感染脐血MSCs,筛选转基因细胞,检测转染前后hTERT在mRNA水平的表达。结果:用BglⅡ和NotⅠ双酶切构建质粒,证明PLNCX2-hTERT构建成功,转基因细胞的hTERT基因在mRNA水平得到表达。结论:hTERT-逆转录病毒载体构建成功,在逆转录病毒介导下,外源性hTERT基因转入脐血MSCs后得到表达。     

外源hTERT基因对大鼠肝细胞增殖的影响

目的:构建含目的基因人端粒逆转录酶(hTERT)重组逆转录病毒载体pLNCX2-hTERT,体外感染原代大鼠肝细胞,观察对大鼠肝细胞增殖的影响.方法:将hTERT插入逆转录病毒载体pLNCX2获得重组逆转录病毒载体pLNCX2-hTERT,转染包装细胞293T后可获得表达hTERT逆转录病毒,感染体外培养的原代大鼠肝细胞,通过RT-PCR及western-blot检测基因在细胞中的表达情况.结果:重组载体pLNCX2-hTERT经双酶切鉴定及序列测定,证实构建正确.转染原代大鼠肝细胞后能明显促进其增殖.结论:体外转染原代大鼠肝细胞后能有效促进其增殖,这为进一步解决人工肝细胞来源问题奠定了实验基础.     

人p58.2重组逆转录病毒载体的构建及稳定包装细胞系的鉴定

目的　构建并鉴定携带人p5 8 2基因的重组逆转录病毒载体及稳定的包装细胞系。方法　用PCR技术从pSPORT1-p5 8 2扩增出编码p5 8 2N端 1～ 3 45个氨基酸的全长基因 ,定向克隆入逆转录病毒载体pMSCVneo中 ,酶切鉴定 ;脂质体法将重组逆转录病毒转入包装细胞系PT67,G418筛选建立稳定产病毒的细胞株。结果　双酶切鉴定 ,成功构建重组逆转录病毒载体 ;重组逆录病毒载体转入包装细胞 ,经G418筛选 ,形成了抗性克隆 ,并测得病毒滴度为 3×10 5cfu/ml,提示构建携带人p5 8 2基因的逆转录病毒载体及稳定的包装细胞系成功。结论　构建人p5 8 2基因的逆转录病毒载体可行 ,并且可望成为一种很好的对血液系统疾病进行基因治疗的方法     

人NT-3重组逆转录病毒载体及稳定包装细胞系的构建及鉴定

构建并鉴定携带人NT - 3基因的逆转录病毒载体以及稳定的包装细胞系。方法 :用DNA重组技术将人NT - 3定向克隆入逆转录病毒载体 pLXSN ,并用限制性内切酶进行酶切鉴定 ;磷酸钙转染方法将重组的逆转录病毒转染入包装细胞系PA317,G4 18筛选建立稳定产病毒的细胞株。结果 :构建的重组逆转录病毒载体可以被限制性内切酶EcoRⅠ和BamHⅠ切开 ,电泳分离显示两条阳性条带 ,分别出现于 0 .8kb和 5 .9kb处 ;重组载体转染包装细胞 ,经G4 18筛选 ,可形成抗性克隆 ,并测得病毒滴度为 4× 10 5cfu/ml,提示构建携带人NT - 3基因的逆转录病毒载体以及稳定的包装细胞系成功。结论 :构建携带人NT - 3基因的逆转录病毒载体可行 ,并且可望成为一种很好的对神经系统疾病进行基因治疗的方法。     

人CD59 RNAi逆转录病毒载体系统的构建与鉴定

目的 利用siRNA表达载体法构建并筛选携带针对CD59基因pSUPER retro RNAi逆转录病毒载体及稳定产毒的细胞克隆.方法 用DNA重组技术,将3条60 bp能转录产生靶向CD59小发夹RNA(shRNA)的寡核苷酸序列定向克隆入逆转录病毒载体pSUPER retro,脂质体法转染包装细胞系Phoenix A,建立产生逆转录病毒的细胞克隆.结果 重组载体经PCR及限制性内切酶酶切鉴定初步成功后测序序列正确;重组载体转染包装细胞,可表达绿色荧光蛋白,表明包装成功.结论 特异性沉默CD59基因的pSUPER retro RNAi逆转录病毒载体以及稳定产毒的细胞系构建成功,为后续进行肿瘤细胞的研究奠定了基础,可望为肿瘤治疗开辟新途径.     

骨形成蛋白9腺病毒表达载体的构建及鉴定

目的 构建骨形态发生蛋白9 (BMP9)基因腺病毒表达载体及稳定产毒细胞系,为观察其对T2DM糖、脂代谢异常的干预效应奠定基础.方法 质粒pCMV/BMP9双酶切克隆至腺病毒载体Ad5.F,构建重组腺病毒表达载体Ad5.BMP9.F,酶切测序鉴定;转包装细胞,经抗性筛选检测病毒滴度,挑选滴度较高的稳定产毒细胞克隆进行逆转录PCR(RT-PCR)鉴定.结果 建立BMP9基因腺病毒表达载体Ad5.BMP9.F经酶切及测序证实目的基因插入位点及读码框架正确;建立稳定产毒细胞克隆最高病毒滴度达7.4×105 CFU/mL,RT-PCR证实BMP9基因整合其中并稳定表达.结论 成功构建含BMP9基因的腺病毒表达载体及其稳定产毒细胞系.     

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.     

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…     

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.     

Possible Spread of adenovirus type 3 from poultry to humans ：indirect evidence from an outbreak in China

Objective:To explore the epidemiology and etiology for an outbreak of acute respiratory tract infection that occurred in one county of Jiangsu Province, China 2004. Methods: Only cases meeting the case definition were included in the study. We reviewed the medical records of the cases who were admitted to the local hospitals, interviewed cases by a standard questionnaire, and then described the epidemiotogic features and analyzed risk factors by means of a case-control study. We collected pharyngeal swab specimens and sent them to different laboratories for isolation and culture. The laboratory used different detection methods such as DIP, PCR, electron microscope examination and microneutralization assay, to identify and then type the positive specimens. Results:A total of 871 cases were reported during the period from April 18 to July 4,2004. The distribution of onset times presented two peaks, one in late May and another in middle June. The epidemic occurred mainly in the elementary and junior high schools in ten townships of one county, and the mean age of the cases was 12 years (range 7 months to 18 years). The course of the disease was acute, and was characterized by fever accompanied with sore throat and tonsillitis. The WBC count of cases was normal or elevated. The mean duration of illness was 5 days (range 2 to 12 days). No fatalities from illness were reported. A case-control study indicated that the possible risk factors were close contact with a case and/or poultry before onset and sharing of towels among members of the family. The typical CPE was observed through inoculating pharyngeal swab specimens into the HEP-2 cell cultures in different laboratories. An infection of adenovirus type 3 was verified by detecting positive specimens in different methods. Conclusion:This investigation demonstrated that the acute respiratory infection in cases was caused by adenovirus type 3. Cases occurred in over 70 schools in ten townships in 2004, and the route of transmission was possibly close contact with cases or droplet transmission.