一种精子蛋白嵌合肽的实验研究

目的：探讨可供两性使用的免疫避孕疫苗的可能性。方法：设计含有小鼠精子／睾丸特异性抗原SP17,Cyritestin中特异性氨基酸序列的新抗原35肽,经合成,鉴定及纯化后,免疫雌性Balb/C小鼠,并了解其免疫原性,同时观察其抗生育效应。结果：获得的新抗原35肽经质谱鉴定证实其合成正确,经高压液相色谱（KPLC）纯化后纯度达95％以上,嵌合肽与钥孔血蓝蛋白（KLH）偶联后免疫小鼠获得的特异性抗血清可识别小鼠,大鼠及人睾丸组织蛋白提取物中相对分子质量的蛋白质,与等量弗氏佐剂混悬后免疫小鼠,其血清中特异性IgG抗体滴度最高达1：6000,阴道粘膜冲洗液中特异性IgA抗体滴度最高达1：300,免疫后小鼠受孕率降至61．11％,平均每胎产仔量也减至4．3只,结论：新抗原35肽可在雌性Balb/C小鼠体内激发出较强的特异怀体液和粘膜免疫,使小鼠受孕率和每胎产仔数明显下降。     

以小鼠精子/睾丸特异性抗原为基础的合成肽免疫避孕效应研究

目的：研制以精子/睾丸特异性抗原为基础的多价嵌合肽避孕疫苗。方法：选择小鼠精子/睾丸特异性抗原Sp17、Gy-ritestin作为对象，采用分子设计的方法与外源性牛核糖核酸酶非选择性T细胞表位组合合成新型嵌合肽-35肽，并对其免疫效应进行研究。结果：该嵌合肽可刺激小鼠产生较高效价的血清和阴道粘膜特异性抗体，诱导脾脏淋巴细胞增殖，促进细胞因子IL-4的分泌；将其与佛氏佐剂混悬，免疫雌性BALB/c小鼠可明显降低受孕率和每胎产仔率，其抗血清可显著抑制体外精-卵结合和融合。结论：新型嵌合肽具有较明确的抗生育作用，提示以分子设计、蛋白质优化方案进行多价避孕疫苗的研究是可行的。     

结核杆菌多价核酸疫苗的构建及其免疫原性的初步研究

目的构建包含结核杆菌3种抗原蛋白Ag85A、ESAT-6和CFP-10真核表达质粒的多价DNA疫苗,并对其在小鼠体内的免疫原性进行初步分析。方法PCR扩增获得结核杆菌ag85 a、esat-6和cfp-10基因片段,分别克隆入真核表达质粒VR1020的BamHⅠ位点构建DNA疫苗,以此多价DNA疫苗免疫BALB/c小鼠,检测小鼠体内的特异性抗体应答和细胞免疫反应,并与BCG免疫组、质粒VR1020免疫组和生理盐水对照组相比较。结果多价DNA疫苗免疫小鼠后能产生特异性的细胞和体液免疫应答,DNA疫苗免疫组小鼠脾细胞IFN-γ的表达量显著高于BCG免疫组,而DNA疫苗免疫组小鼠所产生的特异性抗体水平低于BCG免疫组。结论成功构建了结核杆菌多价DNA疫苗,免疫小鼠后检测到特异性细胞和体液免疫应答。     

Fmoc固相直接法合成Aβ1-15肽疫苗及其免疫活性

 【目的】探讨实验室合成Aβ1-15肽疫苗的方法，并对其进行鉴定。【方法】选取Aβ42主要包含B细胞抗原决定簇的片段Aβ1-15，采用8分支多重抗原肽系统，以Fmoc固相直接法合成MAP Aβ1-15；用MAP Aβ1-15疫苗免疫接种C57BL／6小鼠，ELISA检测血清特异性抗Aβ抗体。【结果】用Fmoc法直接合成方法成功制备MAP Aβ1-15疫苗，质谱检测其主要离子峰的相对分子质量为15458，与理论相对分子质量15451十分接近。但还有其它不同分子质量的离子峰存在。以合成的MAP Aβ1-15免疫C57BL／6小鼠后。可产生高滴度的特异性抗Aβ越抗体，第3次接种后抗体平均滴度为1：467±196，第5次免疫接种后。平均滴度为1：4367±1120。对照组的血清抗体检测基本无明显变化。【结论】Fmoc固相直接法可成功合成出MAP Aβ1-15，合成出的MAP Aβ1-15具有很好免疫活性。     

滋养层细胞促融合表位肽抗生育作用的初步研究

目的 合成多价抗原肽,观察其体液免疫效果,并初步评估其抗生育潜力.方法 选择人滋养层细胞促融合表位模拟肽与一种通用辅助T细胞表位(PADRE)作为骨架,合成多价抗原肽(MAP)结构的免疫原,与等量弗氏佐剂混悬后免疫雌性C57BL/6小鼠,观察体液免疫反应的特点及抗血清干扰滋养细胞融合的效果.结果 在431A 固相自动肽合成仪上成功地合成了MAP多肽, 并经HPLC纯化、质谱鉴定证实其纯度达95%以上;其在小鼠血清中诱导出的特异性抗体的滴度最高达1∶1 024,且可以特异识别滋养层细胞相关抗原表位;在1∶10 稀释的抗血清存在时,forskolin诱导的人绒癌细胞(BeWo)间融合显著减少(P＜0.01).结论 由含有人滋养层细胞促融合表位模拟肽、具有特异性氨基酸序列的新抗原所制备的抗血清可识别滋养层细胞相关天然抗原表位, 并在小鼠体内激发出较理想的特异性体液免疫, 同时,抗血清对BeWo细胞间融合具有一定抑制作用.     

恶性疟MSP-2、CSP多价DNA疫苗免疫小鼠应答类型研究

目的：探讨恶性疟原虫FCC-1/HN株裂殖子表面蛋白-2(MPS-2)和环子孢子蛋白(CSP)组成的DNA多价疫苗在小鼠体内诱导的免疫应答的特性及抗感染的保护性免疫机制。方法：将重组真核表达质粒pBK/CSP和pBK/MSP-2经骨骼肌注射BALB/c小鼠，小鼠经多价疫苗免疫8wk后，用流式细胞仪分析脾脏T淋巴细胞的分化，并体外培养脾脏细胞，用夹心ELISA法测定IFN-γ和IL-2的产生；用血清学方法测定免疫鼠IgG抗体的动态变化。结果：与对照组相比，疫苗组CD^4 cd^8 淋巴细胞有显的增高，体外培养的脾脏细胞IFN-γ有一个高浓度的分泌。同时，免疫鼠血清对疟原虫抗原都表现了一个较高水平的IgG类抗体反应。结论：恶性疟原虫FCC-1/HN，pBK/MSP-2和pBK/CSP多价疫苗诱导了一个以细胞免疫为主的免疫应答类型。     

多价精子抗原表位多肽-免疫刺激复合物的抗生育作用研究

目的 提高多价小鼠精子抗原表位多肽的免疫性避孕效应。方法 应用固相化学合成法合成具有两种精子抗原蛋白关键表位的35肽，纯化后的多肽与皂甙Quil A及磷脂、胆固醇混合，透析制备多肽－免疫刺激复合物（ISCOMs)，并用于免疫雌性C57小鼠。结果 合成后的35肽经纯化后纯度大于95％，经质谱仪鉴定质荷比与理论值一致；透析法制备的多肽－ISCOMs在电镜下呈规则的蜂巢样、由多个亚单位构成的开放型结构；免疫后小鼠的血清及阴道冲洗液中特异性IgG和IgA最高分别为1：18000和1：1600；同时受孕率降为20.0%，显著低于对照组（P≤0.01)。结论 多肽－ISCOMs复合物具有独特的蜂巢样结构，其应用可显著提升多肽的免疫效应，尤其是粘膜免疫，获得较好的免疫性避孕效果，具有广阔的应用前景。     

利用Sm抗原模拟表位肽构建狼疮样鼠模型

目的利用Sm抗原模拟表位肽免疫BALB/C小鼠,构建狼疮样鼠模型。方法用鼠Sm单克隆抗体对噬菌体随机12肽库进行亲和筛选,将筛选的噬菌体阳性克隆行双抗体夹心ELISA检测、DNA序列测定并推导其氨基酸序列,初步确定Sm抗原模拟表位;进而用混合噬菌体阳性克隆皮下免疫BALB/C小鼠,ELISA法检测小鼠血清IgG型抗Sm抗体、抗双链DNA（dsDNA）抗体和抗核抗体（ANA）水平;直接免疫荧光检测小鼠肾脏IgG型免疫复合物的沉积以及HE染色观察小鼠肾脏组织病理损伤情况。结果免疫筛选获得的5个ELISA强阳性的噬菌体克隆的氨基酸序列IR、SQ、PP出现频率增加;实验组小鼠血清中第28天出现Sm抗体、dsDNA抗体、ANA抗体,且滴度不断升高;第33天出现蛋白尿;肾脏可以检测到IgG型免疫复合物沉积及明显的肾脏病理改变。对照组小鼠无明显自身抗体产生和肾脏病变。结论利用Sm抗原噬菌体模拟表位肽可成功构建狼疮样小鼠模型。     

日本血吸虫多价DNA疫苗pBK—Sj26（Sj32）—Sj23免疫效果的观察

为了观察血吸虫病多价DNA疫苗的保护力,将小鼠分成5组：空白对照组、空质粒对照组、单价抗原DNA疫苗pBK-CMV-Sj23组、多价抗原DNA疫苗pBK-CMV-Sj26-Sj23和pBK-CMV-Sj32-Sj32组。大量提取各组质粒DNA后,各组于0、3、5周在BALB／c小鼠股四头肌注射相应质粒DNA,9周用血吸虫尾蚴攻击感染,15周剖杀小鼠计算减虫率及减卵率。结果显示：与对照组比较,实验组小鼠减虫率及减卵率及极显著性差异（P＜0.01）;与单价pBK-CMV-Sj23组比较,多价DNA疫苗组的减虫率及减卵率有显著性差异,提示血吸虫多价DNA疫苗诱导小鼠对血吸虫的保护力优于单价DNA疫苗。     

Aβ1-5多价DNA疫苗的构建及其对体液免疫的效果

目的】构建Aβ1-15多价DNA疫苗，研究其诱导体液免疫的效果。【方法】基因合成和PCR技术扩增以多个甘氨酸连接的Aβ1-15二价基因片段；在N端引物延伸加上IgGκ轻链信号肽序列；GSGGSG linker再串连两段Aβ1-15二价基因片段，克隆入pcDNA3．1表达载体，构建peDNA3．1-s4xAβ15真核表达质粒；质粒转染COS-7细胞，Western blot检测4xAβ15的表达；质粒肌肉注射接种BALB／c鼠，共6次，18周加强免疫，ELISA法检测抗体效价以及抗体分型；免疫组织化学和Aβ42肽抗原的中和实验检测抗血清与转基因鼠Tg2576脑切片中老年斑的特异结合。【结果】测序证实所构建的peDNA3．1-s4xAβ15载体序列正确，在COS-7细胞中表达分子质量约8ku的分泌型4xAβ15蛋白。peDNA3．1-s4xAβ15疫苗于第2次加强免疫后产生Aβ抗体，随加强免疫次数增多，抗体滴度增加，在19周，抗体平均滴度为1：6400，抗体以IgG1型为主，且可以与转基因鼠Tg2576脑切片中的老年斑免疫结合并被Aβ42肽抗原的中和。【结论】所构建的peDNA3．1-s4xAβ15疫苗可在小鼠体内产生高效价的Aβ抗体，为下一步免疫老年性痴呆转基因动物的研究提供条件。     

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.