日本血吸虫Sj31核酸疫苗联合IFN-γ重组质粒诱导小鼠保护性免疫的研究

目的　探讨干扰素 γ重组质粒对日本血吸虫组织蛋白酶B核酸疫苗在小鼠抗血吸虫作用的影响。　方法　将小鼠干扰素 γ基因PCR扩增片段克隆入真核表达载体pCDNA3 1以构建重组真核表达质粒pCDNA3 1 IFN γ ,并与日本血吸虫组织蛋白酶B真核表达质粒VR10 12 Sj3 1一同免疫小鼠。小鼠分为 4组 ,其中实验组每鼠同时肌注VR10 12 Sj3 1及pCDNA3 1 IFN γ各 10 0 μg ,3个对照组分别为VR10 12 Sj3 1肌注 10 0 μg ,pCDNA3 1 IFN γ肌注 10 0 μg和载体VR10 12及pCHAN3 1肌注各 10 0 μg。共免疫 3次 ,每次间隔 2周。于末次免疫后两周免疫组化检测表达质粒在小鼠肌细胞的表达 ,于末次免疫后 3周经小鼠皮肤攻击感染 40± 1条日本血吸虫尾蚴。 45d后杀小鼠计算减虫率。　结果　VR10 12 Sj3 1及pCDNA3 1 IFN γ均在小鼠肌细胞表达 ,日本血吸虫Sj3 1核酸疫苗联合IFN γ重组质粒免疫可诱导小鼠产生 2 7 3 7%的减虫率 ,与日本血吸虫Sj3 1核酸疫苗单独免疫组比较减虫率显著 (P <0 0 5 )。　结论　IFN γ表达质粒能增强日本血吸虫组织蛋白酸B核酸疫苗的抗日本血吸虫的作用     

日本血吸虫DNA疫苗辅以FQ2诱导小鼠保护性免疫的实验研究

目的探讨日本血吸虫Sj26GSTDNA疫苗辅以佐剂FQ2共同作用对小鼠的免疫保护作用。方法采用FQ212"g/只分别与Sj26GSTDNA疫苗混合经股四头肌接种BALB/c小鼠,共接种3次,同时设Sj26GSTDNA疫苗组与生理盐水对照组,接种后4周以日本血吸虫尾蚴攻击感染,感染后6周剖杀小鼠,计数减虫率和减卵率。结果FQ2 疫苗组的减虫率与减卵率分别为34.24%与41.12%,而疫苗组的减虫率与减卵率各为28.76%与29.03%,结果显示FQ2 疫苗组的减虫率与减卵率均高于疫苗组,减卵率差异显著(P<0.05)。结论FQ2对Sj26GSTDNA疫苗有明显的增效作用,提高了DNA26Kda疫苗对小鼠的保护作用。     

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-Sj23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有极显著性差异(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.     

重组核酸疫苗诱导小鼠抗血吸虫感染的免疫学研究

目的为日本血吸虫病的防治寻求新的高效联合基因免疫策略。方法构建共表达日本血吸虫相对分子质量23000表膜蛋白(Sj23)基因与鼠IL12基因的核酸疫苗质粒pVIVO2IL12Sj23,转染HEK293细胞,通过RTPCR,Westernblot及ELISA法检测Sj23和IL12蛋白的表达。接种并攻击感染BALB/c小鼠,以减虫率和减卵率评价其免疫保护力,同时设攻击感染对照组、空白质粒pVIVO2组、pVIVO2IL12组和pVIVO2Sj23组。用ELISA法和WesternBlot分析免疫小鼠血清中抗体。以脾细胞培养法检测经SEA刺激后,小鼠脾细胞分泌IFNγ和IL4的水平。并以FCM分析脾细胞亚群。结果经瞬时转染HEK293细胞证明质粒pVIVO2IL12Sj23能在体外进行表达。免疫小鼠后获得了4553%的减虫率和5835%的减卵率,较单价DNA疫苗pVIVO2Sj23免疫效果好(P<005)。ELISA法和WesternBlot检测抗体结果表明免疫小鼠产生了抗Sj23特异性IgG抗体。pVIVO2IL12Sj23免疫组IFNγ的水平较对照组显著升高而IL4水平较对照组低。攻击感染后各实验组小鼠脾细胞的CD4+,CD8+亚群比率无显著差别(P>005)。结论pVIVO2IL12Sj23DNA疫苗具有诱导BALB/c小鼠产生较好的抗血吸虫感染免疫保护效果。细胞因子IL12作为基因佐剂,具有诱导Th1型免疫反应从而增强DNA疫苗免疫保护性的作用。     

日本血吸虫重组BCG—Sj26GST疫苗免疫小鼠血清IgG动态观察及免疫保护力的研究

为检测血吸虫重组BCG－Sj26GST疫苗免疫小鼠血清IgG动态变化和免疫保护力，采用10^6CFU疫苗皮下1次和3次接种小鼠，接种后8周用日本血吸虫尾蚴感染。感染后6周剖杀小鼠，计算减虫率和减卵率，同时设有BCG对照组。结果发现：实验组免疫后血清IgG抗体迅速升高并持续高于水平；减虫率分别为39.74%和39.74%，减卵率分别为62.86%和55.62%，与对照组相比均有非常显著的差异（P＜0.01）。而免疫3次和免疫1次小鼠血清IgG抗体水平及减虫率、减卵率均无显著差异。血吸虫重组BCG－Sj26GST疫苗皮下注射1次即能诱导小鼠较高水平的IgG抗体和免疫保护力，是一种比较理想的新型疫苗，值得进一步研究。     

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。     

日本血吸虫复合DNA疫苗的组织表达及免疫保护效果研究

目的 :观察本课题组所构建日本血吸虫大陆株复合DNA疫苗VR10 12 SjGST和VR10 12 SjGST Sj32在小鼠肌肉组织中的表达 ,并进行免疫保护效果测定。方法 :用纯化质粒免疫昆明鼠 :4 8只小鼠分为 4组 ,两个对照组的小鼠分别于股四头肌注射生理盐水 10 0 μl或空质粒VR10 12 10 0 μg,两个实验组的小鼠则同法分别注射VR10 12 SjGST和VR10 12 SjGST Sj32各 10 0 μg。末次免疫后 ,每组解剖两只小鼠 ,以间接免疫荧光法观察SjGST、Sj32在肌肉组织中的表达。其余每只鼠经腹部感染 10条尾蚴 ,4 5d后剖杀计数各小鼠成虫数和肝卵数。结果 :小鼠肌肉组织表达出抗原特异性蛋白质抗原分子。与生理盐水组比较 ,两个实验组的减虫率分别为 33.9%和及 2 7.14 %(均为P <0 .0 5 ) ,减卵率分别为 6 1.86 %和 6 8.87% (均为P <0 .0 0 1)。与VR10 12 SjGST组相比 ,VR10 12 SjGST Sj32组的减卵率为 18.5 1% (P <0 .0 5 )。结论 :DNA疫苗VR10 12 SjGST和VR10 12 SjGST Sj32能在组织中正常表达 ,能诱导小鼠产生一定水平的抗日本血吸虫感染保护作用 ,复合疫苗的保护效果要大于单价疫苗     

日本血吸虫Mf2基因的亚克隆表达与免疫保护效果研究

目的 在大肠杆菌中高效表达日本血吸虫(Schistosoma japonicum，Sj）Mf2蛋白，并对表达产物进行免疫保护效果测定。方法 将SjMf2基因亚克隆至pCEX—5X—3原核表达载体，以GST融合蛋白的形式在ER2688中表达，表达产物免疫小鼠，免疫用抗原剂量为50μg/次.鼠，对照组分别注射FCA或PBS，在0、2、6周共免疫3次。第3次免疫后2周进行攻击感染，42d后杀鼠，观察减虫和减卵效果。结果 在IPTG诱导下，表达载体中的SjGST基因与重组的SjMf2基因在大肠杆菌中得以高效表达，形成了SjGST—Mf2融合蛋白，用这种融合蛋白免疫小鼠，诱导产生了27．75％的减虫率和45．7％的每雌肝组织虫卵减少率(LEPF)。结论 SjMf2基因亚克隆至pGEX—5X—3载体后可在大肠杆菌中高效表达，表达产物能诱导小鼠产生一定程度的抗日本血吸虫保护性免疫力。     

Sj31与鼠IFN-γ基因融合DNA疫苗免疫保护效果的研究

目的构建表达mIFN-γ与Sj31抗原融合蛋白的DNA疫苗,并探讨其免疫保护作用。方法在Sj31基因上游引物与mIFN-γ下游引物的5’分别设计与克隆载体相匹配的酶切位点,在mIFN-γ上游引物与Sj31基因下游引物的5’设计相同的酶切位点。分别进行PCR扩增,再通过引物的5’端相同的酶切位点进行2个PCR产物的酶切与连接,以连接产物为模板,应用Sj31的上游引物和mIFN-γ下游引物进行PCR扩增,将此次PCR产物经常规方法克隆入载体pcDNA3.0(简称为pc),构建成多价DNA疫苗pc-Sj31-mIFN-γ。对重组质粒鉴定后,大量制备纯化质粒免疫昆明小鼠,48只小鼠分为A、B、C、D4组,对照组的小鼠于股四头肌注射质粒pcDNA3.0100μg,3个免疫组的小鼠分别注射pc-Sj31、pc-mIFN-γ和pc-Sj31-mIFN-γ质粒DNA各100μg。在0、2、6周免疫共3次,第3次免疫后2周,每只小鼠经腹部贴片感染尾蚴40±1条,感染后第42天剖杀,计数各小鼠检获成虫数及肝卵数。结果pc-Sj31-mIFN-γ(D组)免疫组的减虫率和肝组织减卵率分别为28.56%和60.20%,但是表达鼠mIFN-γ与Sj31抗原融合蛋白的DNA疫苗并未明显优于单纯表达日本血吸虫组织蛋白酶B(Sj31)DNA疫苗的免疫保护作用。结论pc-Sj31-mIFN-γ多价DNA疫苗构建成功,但其诱导小鼠抗血吸虫病免疫保护效果不明显优于pc-Sj31。     

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.     

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.     

Expression of OPGmRNA and ODFmRNA in the patients of hip fracture due to osteoporosis

Objective：To investigate the gene expression of osteoprotegerin（OPG） and osteoclast differentiation factor（ODF） in the bone tissue of patients with hip fracture due to osteoporosis. Methods：OPGmRNA and ODFmRNA in the bone tissue in 50 cases of osteoporosis sufferers（over 50 years old） with hip fracture（Observer Group） and 30 cases of hip facture sufferers with no osteoporosis（Control group） were analyzed with the Semi-Quantitative RT-PCR method. Results：The mRNA expressed of ODF, OPG were both high in the patients with hip fracture. In the control group, the expression of OPG mRNA was observed, while the expression of ODF mRNA was very slight. Conclusion：Aged patients contained all signals including OPG, ODF that are essential for inducing osteoclastogenesis and promoting bone resorption.     

The clinicopathological and immuohistochemical analysis of solid-pseudopapillary tumor of the pancreas： report of 9 cases

Objective：To investigate the clinical features, pathological characteristics and immunophenotype of solid-pseudopapillary tumor of the pancreas（SPTP）. Methods：Nine surgically treated cases of SPTP were retrospectively reviewed. Hematoxylin and Eosin（HE） staining and immunohistochemical staining were used to analyze all cases, and the general clinical data was collected. Results：Six patients were asymptomatic except for a palpable mass. Two patients complained of vague-epigastric pain. One patient appeared jaundice. The tumor was encapsulated and solid tissues alternately with cystic tissues. Histologically, the histological structure of solid portion was pseudopapillary with a fibrovascular core. Tumor cells were uniform and medium-sized which were arranged in sheets ets or nests or pseudopapillary patterns. Immunohistochemical studies demonstrated that SPTP proved positive in vimentin（9/9 cases）, AAT（9/9 cases）, NSE（9/9 cases）, ACT（7/9 cases）, CK20（2/9 cases）, CgA（1/9 cases）, S-100（3/gcases）, PR（4/gcases）, Syn（3/9 cases） and CD56（5/9cases）, negative in CEA and ER. Conclusion：SPTP is a tumor predominantly occurring in young women frequently without special symptoms. This tumor has various characteristical histological patterns with different immunophenotype.     

Dynamic Changes in Serum Estradiol and Progesterone levels in Patients of Premenstrual Syndrome with Adverse Flow of Liver-qi

Objective:To probe into the influence of changes of ovarian hormones on the pathogenesis of the specific sub-type premenstrual syndrome(PMS)and reveal partial microcosmic mechanisms of adverse flow of liver-qi.Methods:Estradiol(E2)and progesterone(P)levels in serum were determined at different phases of menstrual cycle by radioimmunoassay.Results:In the group of PMS with adverse flow of liver-qi.the secretive peak value Of E2 and P at the follicular phase significantly decreased,and the secretive peak value at the luteal phase did not come into being.Conclusions:Low E2 and P secretive peak at the follicular phase and absence of secretive peak at the luteal phase is one of the microcosmic mechanisms of PMS with adverse flow of liver-qi.One of the pathophysiologic mechanisms of specific sub-type PMS is probably the continuous low level of E2and P.     

Real-time Three-dimensional Echocardiography in Assessment of Left Ventricular and Right Ventricular Volumes

Real-time three-dimensional echocardiography (RT3DE)is a new ultrasound technique that enables dynamic threedimensional visualization and quantification of the heart in real time. Investigation of feasibility and methodology of RT3DE in determining left ventricular (LV) and right ventricular (RV) volumes, RT3DE was performed in 35 normal adults using Philips SONOS 7500 system with a 2-4 MHz matrix array transducer. The 60°×60° "pyramid" volume database was obtained and analyzed on a TomTec echo workstation. Both LV and RV volumes were calculated with four 3DE methods (i.e. apical 2, 4, 8, and 16-plane) through manually tracing ventricular endocardial borders in end diastole and end systole. Stroke volumes were then calculated. LV volume was also measured by 2DE Simpson's rule using GE VIVID 7 ultrasound machine.     

SCREENING FOR A 21-CHROMOSOME ABNORMALITY IN PREIMPLANTED EMBRYOS OF ELDERLY WOMEN

Increasing maternal age is the only etiological factor unequivocally linked to Down's syndrome in humans. The occurrence rate of newborns with Down's syndrome is about 1/220 in women over 35 years old. However, the occurrence rate in embryos fertilized in vitro, of the elder woman is unclear. Using FISH we screened the number of chromosome 21 in preimplanted embryos of 5 elderly women (average age, 38.4 years) to study the feasibility and necessity of screening trisomy 21 in embryos in patients over 35 years old at the in vitro fertilization (IVF) center.