小鼠精子蛋白Sp17的克隆、表达及其免疫原性研究

目的 获得鼠精子蛋白Sp17基因,构建重组表达载体并实现其在大肠杆菌中的高效表达,为制备免疫性避孕疫苗奠定基础.方法 提取BALB/c小鼠睾丸组织总RNA,通过RT-PCR扩增Sp17基因全长cDNA序列,并将其克隆至pMD 18-T质粒载体中,再经HindⅢ/ XhoⅠ双酶切将Sp17基因片段亚克隆至原核表达载体pET-28a(+)中,转化大肠杆菌BL21(DE3),异丙基-β-硫代半乳糖苷(IPTG)进行诱导表达,采用SDS聚丙烯酰胺凝胶电泳(SDS-PAGE)、Western blot对表达产物进行初步鉴定,并检测重组蛋白Sp17免疫小鼠后血清特异抗体滴度.结果 获得了小鼠Sp17的全长cDNA,成功构建了重组原核表达载体pET/Sp17,在IPTG的诱导下实现了目的蛋白在大肠杆菌中的高效表达,免疫后小鼠血清中检测到特异性抗体.结论 小鼠精子蛋白Sp17基因序列已被克隆至原核表达载体pET-28a(+)中,并在大肠杆菌BL21(DE3)中获得高效、正确的表达,重组蛋白Sp17能够诱导产生特异性免疫反应,具有一定程度的免疫原性.     

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多价疫苗SjGST-FABP/pcDNA3的构建及其保护性免疫研究

目的构建日本血吸虫DNA多价疫苗SjGST-FABP/pcDNA3,用以免疫小鼠,观察其在小鼠抗血吸虫感染中的免疫保护作用。方法根据质粒pGEX-4T-1中SjGST-ORF和SjFABP基因序列,利用基因重组、PCR等技术将SjGST和SjFABP编码基因拼接在一起,得到融合基因SjGST-FABP,将融合基因SjGST-FABP定向克隆到pcDNA3多克隆位点上,转化大肠杆菌,经质粒扩增和DNA序列测定后,进行小鼠动物免疫和日本血吸虫尾蚴攻击感染及免疫保护性评价。结果成功构建了日本血吸虫DNA多价疫苗SjGST-FABP/pcDNA3。免疫小鼠获得42.39%的减虫率和56.09%肝减卵率（P〈0.05）。结论日本血吸虫DNA多价疫苗SjGST-FABP/pcDNA3可诱导部分抗血吸虫尾蚴攻击感染的免疫保护效果,具有疫苗研究与开发价值。     

日本血吸虫核酸疫苗pcDNA3．1／SjMfl粘膜免疫小鼠的保护力研究

目的　探讨日本血吸虫核酸疫苗pcDNA3 1/SjMf1粘膜免疫诱导小鼠抗攻击感染的保护力。　方法　按常规方法构建pcDNA3 1/SjMf1。粘膜免疫采用滴鼻方法。供试小鼠随机分为 5组 ,每组 12只 ,包括MPL、pcDNA3 1、pcDNA3 1 MPL、pcDNA3 1/SjMf1和pcDNA3 1/SjMf1 MPL组。第 3次免疫后 2周 ,每只经腹部感染 ( 4 0± 1)条尾蚴。45d宰杀小鼠 ,以观察减虫率和减卵率。在初次免疫前和攻击感染前尾静脉采血的收集粪便 ,ELISA检测血清内特异性IgA和IgG及粪内sIgA水平。　结果　pcDNA3 1/SjMf1滴鼻免疫可诱导小鼠血清内特异性IgA和IgG及粪内sIgA水平。pcDNA3 1/SjMf1和pcDNA3 1/SjMf1加MPL滴鼻免疫诱导小鼠产生的减虫率分别为 2 7 97%和 3 4 75 % ,减卵率分别为 3 8 64 %和 43 3 7%。二者的减虫率和减卵率差异无显著性 (P >0 0 5 )。　结论　pcDNA3 1/SjMf1滴鼻免疫可诱导小鼠产生全身和粘膜免疫应答及部分抗击感染的保护力     

日本血吸虫（大陆株）融合蛋白保护性免疫力的研究

采用日本血吸虫(大陆株)融合蛋白加福氏佐剂免疫小鼠,攻击感染后,不但可减少虫负荷(减虫率为29.2—51.0%),还可降低血吸虫雌虫的产卵量(减卵率为52.1—74.3%),血清抗体在初次免疫后3周即明显升高,并持续维持较高的水平。结果表明,经大肠杆菌表达的日本血吸虫重组融合蛋白能诱导抗攻击感染的保护性免疫力,可望作为混合多价疫苗的候选组份而大规模的生产。     

SARS冠状病毒E蛋白的表达与纯化

目的研究SARS冠状病毒E蛋白对SARS诊断和预防的价值，利用原核表达系统克隆和表达E蛋白并纯化。方法采用RT—PCR从SARS冠状病毒RNA中扩增出编码N蛋白的基因；经克隆和测序分析后，亚克隆至表达载体DGEX-4T-2，转化大肠杆菌BL21（DE3），PCR和双酶切鉴定；阳性菌株经IPTG诱导，SDS—PAGE分析，进一步以SARS病人血清进行免疫印迹分析；大量诱导表达N蛋白，亲和层析予以纯化。结果RT—PCR扩增出E蛋白基因的特异片段，获得的阳性克隆序列与GenBank中登录的SARS冠状病毒的E蛋白基因序列同源性为100％；E蛋白基因被亚克隆至表达载体pGEX-4T-2，在BL21中获得表达，表达产物能被SARS病人血清识别。表达的E蛋白经亲和层析获得纯化。结论成功构建了SARS冠状病毒E蛋白的重组表达质粒，在大肠杆菌中表达的E蛋白的融合蛋白具免疫活性。     

恶性疟原虫谷氨酸脱氢酶的表达及免疫活性鉴定

目的：在大肠杆菌中表达恶性疟原虫谷氨酸脱氢酶（GDH）与谷胱甘肽S-转移酶融合蛋白，测定重组蛋白的免疫活性。方法：采用PCR方法特异性扩增恶性疟原虫（海南株）GDH基因，双酶切后克隆入pGEX-4T-1载体中进行诱导表达，重组蛋白纯化后免疫小鼠制备特异性血清，并用琼脂双向扩散法检测效价，ELISA、Westernblotting检测重组抗原的免疫活性。结果：获到了重组表达的抗原蛋白，表达产物能与鼠抗恶性疟原虫血清发生特异反应，并能诱导小鼠产生特异性体液免疫应答，免疫琼脂扩散法检测抗体滴度为1：16。结论：恶性疟原虫GDH在大肠杆菌中获得高效表达且表达产物具有良好的抗原性。     

胞嘧啶脱氨酶的原核表达和多克隆抗体的制备

目的：在原核系统中表达并纯化大肠杆菌胞嘧啶脱氨酶（cytosine deaminase,CD)，制备鼠抗大肠杆菌CD多克隆抗体，方法：亚克隆CD基因到原核表达载体pMAL-c2和pBV222中，并转化入大肠杆菌DH5α内，诱导表达并纯化MBP-CD和6his-CD融合蛋白，用纯化的MBP-CD融合蛋白免疫小鼠制备多克隆抗体。结果：通过重组质粒的酶切筛选出重组阳性克隆，成功地表达和纯化出MBP-CD和6his-CD融合蛋白，用纯化的MBP-CD成功制备了鼠抗CD多克隆抗体，并用6his-CD和GST-CD重组蛋白进行Western印迹分析，证实了抗体的正确性，结论：应用多克隆抗体可以检测体内外CD基因的表达，为临床前和临床上深入开展CD基因的生物治疗研究提供重要的实验材料。     

小鼠TSR2-3/TSP-1基因片段的克隆及其GST融合蛋白的表达和纯化

目的：获取小鼠TSR2—3／TSP-1基因片段，并高效表达和纯化GST—TSR2—3融合蛋白。方法：应用RT—PCR技术，从小鼠肺总RNA中，获得编码TSR2—3的基因片功能片段，测序后，通过酶切克隆至表达载体pGEX-5X-2，构建重组表达载体，并导入E.coli BL21宿主菌中，IPTG诱导表达重组的GST融合蛋白，亲和层析纯化表达产物，SDS—PAGE和Westem blot进行分析鉴定。结果：获得小鼠TSR2-3基因片段，测序结果与GenBank的基因序列一致，重组GST融合蛋白经SDS-PAGE和Westem blot分析，在相对分子量39kDa处，出现特异性蛋白条带，重组蛋白经GST亲和层析柱纯化后，得到了高纯度的融合蛋白。结论：成功克隆小鼠TSR2—3基因片段，并在Ecoli BL21中高效表达，亲和层析后获得高纯度GST—TSR2—3融合蛋白。     

恶性疟原虫复制蛋白PfRPA2的原核表达及多抗制备

目的克隆表达恶性疟原虫复制蛋白PfRPA2亚基,制备多抗,为该蛋白的功能研究奠定基础。方法 PCR扩增目的基因片段,克隆到表达载体pGEX-KG中,构建PfRPA2/pGEX-KG原核表达载体,IPTG诱导表达,SDS-PAGE电泳分析表达产物,谷胱甘肽柱纯化蛋白,Westernblot检测其抗原性,以纯化的GST-PfRPA2免疫小鼠,制备抗PfRPA2的多抗,间接ELISA法检测鼠血清效价,Westernblot鉴定多抗特异性。结果成功构建了重组PfRPA2/pGEX-KG原核表达质粒,并在大肠杆菌中以可溶性形式高效表达,纯化表达产物,制备抗PfRPA2的鼠多抗,效价为10-7,Westernblot证实此抗体可识别恶性疟原虫内与PfRPA2蛋白位置对应的特异性条带。结论恶性疟原虫复制蛋白PfRPA2亚基在大肠杆菌中获得可溶性高效表达,纯化表达产物能诱导小鼠生产能识别天然蛋白的特异性抗体。     

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.