表达屋尘螨I类变应原Der p1的基因工程菌的发酵条件探讨

目的：探讨发酵条件对大肠杆菌工程菌表达重组屋尘螨I类变应原（Der p1)的影响。方法：应用摇瓶发酵试验观察不同诱导时间和不同诱导时机对Der p1表达量的影响。结果：（1）Der p 1基因在E.coliBL21（pBVDP8)菌密度A600nm0.6左右时，42℃最佳诱导时间为4h.(2)工程菌E.coli BL21(pBVDP8)在30℃培养至A600nm为0．5－0．7时，42℃诱导4hDerp 1表达量最高，结论：确定了最适诱导时间和诱导时机，初步形成了一套高表达Der p 1的发酵技术。     

诱导重组人生长激素工程菌高效表达的研究

目的 筛选诱导重组人生长激素（rhGH)工程菌高效表达的最佳条件，为中试及工业化发酵培养提供依据。方法 质粒PBV－GH转化4种不同宿主菌，比较6种不同培养基在不同pH值、氧含量改变情况下，rhGH工程菌的生长和表达差异，筛选并确定最适宿主菌、最佳培养基及最佳诱导表达条件。结果 ①E.coli DH5α为最适宿主菌；②TB培养基为最佳培养基；③培养基pH7.5-7.8有利于目的蛋白的表达。④在半对数生长期（培养4－5h)迅速升温，且菌密度控制在D600nm3.0之前诱导可获得较高的重组蛋白表达量，rhGH表达量占菌体总蛋白的40％。发酵时间为10h。结论 E.coli DH5α为rhGH高效表达的最适工程菌，E.coli DH5α/PBV-GH在pH7.5-7.8的TB培养基中发酵生长10h，可使菌量最佳扩增，目的产物rhGH表达效率最高。     

溶氧反馈-补料分批技术高密度培养基因重组MAGE1/HSP70/MAGE3工程菌

目的 建立人黑色素瘤抗原MAGE1/HSP70/MAGE3融合蛋白基因重组工程菌E.coli.BL21/pET-MHM的高密度发酵工艺.方法 以摇瓶发酵结果为基础,扩大至NBS Bioflo Ⅳ 20 L发酵罐发酵,利用溶氧反馈-补料分批培养技术,对影响工程菌生长及目的蛋白表达的因素如发酵培养基、活化时间、诱导浓度及时间、pH值及分批补加营养物质等进行优化.结果 保持培养过程中40%的溶解氧,采用半合成培养基、活化至对数生长期时0.2 mmol/L IPTG诱导5 h以及以甘油为碳源连续流加补料的条件发酵,连续3批重复发酵,最终菌密度D(600)均达45～50时,菌体量可提高至70 g/L以上,目的蛋白的表达量占菌体蛋白总量的38%以上.结论 确定了周期短、产率高且稳定可靠的发酵工艺.     

高密度培养大肠杆菌表达重组人胰高血糖素样肽-1

目的:高密度发酵培养表达重组人胰高血糖素样肽-1(hGLP-1).方法:将构建的重组质粒pGEX-hGLP-1转化大肠杆菌BL21(DE3),得到表达hGLP-1的工程菌株E.coli BL21(DE3)/pGEX-hGLP-1.在500 ml三角摇瓶中进行了诱导条件的摸索实验,之后用5 L自控发酵罐对工程菌进行高密度培养,以获取hGLP-1和谷胱甘肽S-转移酶融合蛋白(GST-hGLP-1).结果:采用分批培养和补料分批培养相结合的技术,控制碳源和氮源的补加,控制溶解氧的量,可使重组工程菌发酵光密度D600值达52,融合蛋白的表达量占菌体总蛋白量的28％,其含量达到2.1 g/L.用ESI质谱鉴定hGLP-1相对分子质量与理论值一致.结论:本研究为大规模生产重组hGLP-1奠定了基础.     

重组人骨形态发生蛋白—2在大肠杆菌中的表达及纯化

目的：用基因工程技术在大肠杆菌中表达人骨形态发生蛋白－2（hBMP-2)，方法：hBMP-2原核表达载体pYR(pBV220-hBMP-2)转化E.coli BL21,SDS-PAGE分析工程菌活化状态以及诱导时间与目的蛋白表达率的关系。离子交换层析DEAE和分子筛S－300纯化重组蛋白，自然缓降复性法对其复性。结果：SDS－PAGE显示在相对分子质量约为13000时出现明显外源蛋白表达带，而且当工程菌30℃活化至D600约为0．45时，其表达效率较高，在此状态下，温度诱导表达4h，目的蛋白表达量最高，以后随着时间的延长，表达量销下降，重组蛋白经纯化后植入小鼠肌肉，组织学观察到肌肉内大量间充质细胞增生以及软骨与骨形成，结论：重组hBMP-2具有良好异位成骨活性。     

重组褐藻胶裂解酶表达体系的构建及诱导条件优化

目的对褐藻胶裂解酶基因aly-cob进行外源表达,并优化诱导条件以实现褐藻胶裂解酶的高效表达。方法以产褐藻胶裂解酶菌株Cobetia sp.WG-007为出发菌株,对克隆得到的褐藻胶裂解酶基因进行稀有密码子改造,构建重组质粒pET-28a(+)-aly-cob,在宿主Escherichia coli BL21pLysS中进行诱导表达,对发酵培养基、异丙基 -D-硫代半乳糖苷(IPTG)、诱导温度、诱导时机、诱导浓度及诱导时间进行系统优化以提高酶活。结果构建了重组菌E.coli BL21pLysS/pET-28a(+)?aly-cob,最适培养基为SB培养基,最佳诱导条件为OD600为1.0时加入终浓度为0.1mmol/L的IPTG,在22℃下诱导24h后酶活可达2 403.93U/mL。结论成功对优化后的褐藻胶裂解酶基因aly-cob进行外源表达,经诱导表达条件优化后酶活为野生菌酶活的15倍,更具有工业化应用的潜力。     

谷胱甘肽合成酶系基因协调表达体系的构建

通过 PCR获得 E.coli B谷胱甘肽合成酶系基因 ( gsh I,gsh II)片段 ,结合定点突变稀有起始密码子 ,设定 gsh I与 gsh II位置与距离 ,构建双顺反子重组表达载体 p Trc99A/gsh I- gsh II,建立GSHI、GSH- II蛋白表达体系。结果表明 :以 0 .0 8mmol/L IPTG于 2 8℃诱导工程菌 E.coli BL2 1( DE3) ( p Trc99A/gsh I- gsh II) ,GSH- I、GSH- II蛋白比为 4.5∶ 1 ( m∶ m)时谷胱甘肽合成能力最高 ,达到每克湿菌体 8.5 mg。通过构建单顺反子重组表达载体 p Trc99A/gsh I、p Trc99A/gsh II测定GSH- I与 GSH- II蛋白的最适配比 ,结果表明 :在 GSH- I、GSH- II蛋白总量恒定的情况下 ,要提高谷胱甘肽产率 ,两者比例以 3∶ 1～ 6∶ 1为宜     

重组人血管内皮细胞生长因子工程菌高密度发酵工艺研究

目的建立人血管内皮细胞生长因子(BL21/pET-24a/hVEGF121)工程菌的高密度发酵工艺.方法采用发酵罐发酵,对影响工程菌生长及目的蛋白表达的因素如培养基、诱导时间及补料进行优化.结果采用M9复合培养基、活化至对数生长期时诱导4 h以及以甘油为碳源连续流加补料的条件发酵,可使菌体量提高至68 g/L,rhVEGF121的表达量达菌体蛋白总量的23%.结论该发酵工艺提高了工程菌的产量和rhVEGF121的表达水平.     

重组人骨形态发生蛋白-2在大肠杆菌中的表达及纯化

目的用基因工程技术在大肠杆菌中表达人骨形态发生蛋白-2(hBMP-2)。方法hBMP－2原核表达载体pYR(pBV220－hBMP-2)转化 E coli BL21,SDS－PAGE分析工程菌活化状态以及诱导时间与目的蛋白表达率的关系。离子交换层析 DEAE和分子筛 S－300纯化重组蛋白,自然缓降复性法对其复性。结果SDS－PAGE显示在相对分子质量约为13000时出现明显外源蛋白表达带,而且当工程菌30℃活化至D600约为0．45时,其表达效率较高;在此状态下,温度诱导衣达4h,目的蛋白表达量最高,以后随着时间的延长,表达量稍下降。重组蛋白经纯化后植人小鼠肌肉,组织学观察到肌肉内大量间允质细胞增生以及软骨与骨形成。结论重组hBMP－2具有良好异位成骨活性。     

重组SRH融合蛋白工程菌发酵的工艺研究

目的通过对影响表达SRH融合蛋白工程菌发酵的主要因素的研究,建立适宜SRH融合蛋白工程菌的发酵工艺。方法通过对发酵过程中的pH、溶氧调控、诱导温度、诱导时机及诱导时间进行优化建立SRH蛋白的发酵工艺,利用SDS-PAGE电泳分析表达情况。进一步利用离子交换和凝胶过滤层析对目的蛋白进行纯化,并对纯化后目的蛋白的溶栓活性和抗凝活性进行检测。结果优化后发酵条件为:发酵pH为7.0、溶氧为45%、菌体浓度达到OD600=8时开始诱导,诱导温度为41℃,诱导时间为4.5h。该发酵条件下得菌量为25g/L。SRH蛋白表达量为45%,并且90%以上为可溶性表达,纯化后蛋白纯度达95%以上,纯品SRH蛋白溶栓活性为1.80×104AU/mg,抗凝活性为100AU/mg。结论建立了SRH融合蛋白工程菌稳定的发酵工艺,该条件下SRH工程菌发酵蛋白表达量较高,多为可溶性表达,且杂蛋白较少,发酵产物具有生物学活性。     

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.     

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.