人源性抗核抗体Fab片段的筛选及鉴定

目的:制备人源性抗核抗体Fab片段.方法:通过4轮淘筛,富集已构建的人源性抗核抗体Fab片段噬菌体抗体库,间接ELISA法鉴定4轮淘筛后抗核抗体Fab片段噬菌体抗体;提取阳性克隆的噬菌粒DNA,切除gⅢ基因片段,自连接后转化大肠杆菌XL1-Blue,以IPTG诱导表达可溶性人源性抗核抗体Fab片段;应用间接ELISA法及荧光免疫法对表达上清进行鉴定.结果:第4轮洗脱的噬菌体滴度较第1轮增加200条倍,从抗核抗体Fab片段噬菌体库中筛选出2株阳性克隆,切除gⅢ基因后自连的噬菌粒DNA经XhoⅠ单酶切证实连接成功.间接ELISA法检测结果显示:制备的可溶性人源性抗核抗体Fab片段均呈现抗dsDNA阳性,具有抗原特异性;免疫荧光法结果显示:Hep2细胞和猴肝脏组织细胞核显示均质型荧光,绿蝇短膜虫的动基体显示均质型荧光.结论:成功制备具有抗原特异性的可溶性、人源性抗核抗体Fab片段,为高亲和力抗核抗体Fab片段的制备奠定了基础.     

从人天然抗体库中克隆IgM抗角蛋白抗体

目的：克隆制备人源性抗角蛋白IgM抗体。方法：以人表皮角蛋白为抗原,从人源性天然抗体库中筛选IgM型抗角蛋白人抗体,并用ELISA对所获抗体进行抗原结合活性和特异性等鉴定。结果：通过3轮“亲和吸附－洗脱－扩增”筛选,获得2株IgM型抗角蛋白噬菌体抗体。酶切去掉载体上的噬菌体基因Ⅲ,得到 了可溶性表达的Fab段。经过鉴定,所获抗体具有良好的抗原结合活性和特异性。结论：从人源性天然抗体库中成功筛选出2株抗角蛋白IgM抗体,为进一步研究抗角蛋白抗体的特性提供了条件。     

人源天然Fab噬菌体抗体库的构建及抗c-Met抗体的筛选?鉴定

目的:构建大容量人源天然Fab噬菌体抗体库,筛选抗c-Met特异性抗体并进行初步鉴定.方法:采集20位健康成人的骨髓淋巴细胞.用PCR扩增人Fab片断抗体基因,插入载体pComb3XSS内,构建人源天然Fab抗体库.以固相化的抗原对抗体库进行6轮筛选后,随机挑选60个克隆用Phage ELISA、BstO I酶切片断分析进行检测,阳性克隆作可溶性表达和鉴定.结果:构建的Fab噬菌体抗体库的库容为1.2×109,从中筛选到1株与c-Met特异性结合的人源抗体克隆,命名为AM2-26.DNA序列分析证明为人免疫球蛋白可变区基因,Western blot证实为人源抗c-Met Fab抗体片段,ELISA特异性鉴定阳性.结论:构建了大容量人源天然Fab抗体库,从中获得1株抗c-Met人源Fab抗体片段,有望为抗肿瘤药物的研制提供新的候选分子.     

从噬菌体抗体库中筛选D二聚体特异的Fab抗体

目的:从人源性噬菌体抗体库中筛选抗人D二聚体单克隆抗体并进行可溶性表达。方法:以人D二聚体标准品为抗原对所构建的噬菌体抗体库进行两轮“吸附-洗脱-扩增”筛选,从中筛选出抗人D二聚体的噬菌体Fab抗体,并在大肠杆菌细胞中进行可溶性表达。结果:经4次电转化构建了库容为2. 8×108cfu的抗体库,滴度为4. 1×1014PFU/mL,Fab基因重组率为46%。经过淘筛后携带抗人D二聚体抗体的特异性噬菌体得到了明显的富集。用试剂盒以ELISA法检测对人D二聚体的结合活性,得到抗人D二聚体单克隆抗体。筛选出的阳性克隆成功在大肠杆菌细胞中可溶性表达筛选出的阳性克隆在大肠杆菌细胞中可溶性表达。结论:人D二聚体标准品对抗体库的淘筛,富集了表面呈现抗人D二聚体的单克隆抗体的噬菌体,成功构建了人源性抗D二聚体噬菌体抗体,并成功在大肠杆菌细胞中可溶性表达。     

基因工程技术制备和鉴定抗TNF-α蛋白Fab抗体

目的 利用基因工程技术制备人源性抗肿瘤坏死因子-α(TNF-α)蛋白的可溶性Fab抗体,并鉴定其抗原结合活性及特异性.方法 用固相化的TNF-α蛋白从人天然Fab噬菌体抗体库中筛选出表达抗TNF-α蛋白Fab抗体的阳性克隆,经酶切及连接反应构建可溶性表达噬菌粒并转化至大肠杆菌BL21(DE3)pLysS中,IPTG高效可溶性表达,SDS-PAGE及Western blot分析鉴定抗体表达情况、ELISA鉴定其抗原结合活性和特异性.结果 成功筛选并表达了人源性抗TNF-α蛋白的Fab抗体,在非还原SDS-PAGE中形成相对分子质量47 kD的条带;Western blot分析证实表达的蛋白即Fab抗体.ELISA筛选出2株抗体(TA-04,TA-13)具有良好的抗原特异性和抗原结合活性,与小牛血清白蛋白无交叉反应.结论 成功表达并鉴定了人源性抗TNF-α蛋白的可溶性Fab抗体,构建了一种基因工程抗体的有效制备途径.为基因工程抗体在肿瘤免疫治疗方面的临床应用研究奠定了基础.     

大容量天然人源Fab抗体库的构建及日本血吸虫抗独特型抗体的筛选、鉴定

目的:构建大容量天然人源Fab抗体库,筛选全人源日本血吸虫抗独特型抗体并进行初步鉴定。方法:采集15位健康成人的骨髓淋巴细胞,构建天然人源Fab抗体库。并以日本血吸虫可溶性虫卵抗原(SEA)及成虫抗原(SWAP)免疫鼠血清IgG包板进行筛选,经ELISA鉴定后,对阳性克隆进行可溶性表达。结果:构建了2×109大容量天然人源Fab抗体库,经核苷酸序列分析,证实插入片段为Fab基因片断。经过6轮筛选后,从富集的次级抗体库中随机挑取80个克隆,用ELISA法鉴定得到4个可与免疫鼠血清(Ab1)特异性结合,而不与SEA及SWAP结合的单克隆抗体(C12、C19、D1、D2),其中C12经SDS-PAGE电泳显示插入片断正确。结论:成功构建了大容量天然人源Fab抗体库,并从中获得人源日本血吸虫抗独特型抗体C12,为研制用于人体血吸虫病免疫预防的抗独特型抗体疫苗奠定了基础。     

人源日本血吸虫Fab抗体库的构建及抗独特型抗体的筛选、鉴定

目的:构建人源日本血吸虫Fab抗体库,筛选出日本血吸虫抗独特型抗体并进行初步鉴定。方法:以3名晚期血吸虫患者切除的脾脏为源,构建人源日本血吸虫Fab抗体库。并以日本血吸虫可溶性虫卵抗原(SEA)及成虫抗原(SWAP)免疫鼠血清IgG包板进行筛选,经ELISA鉴定后,对阳性克隆进行可溶性表达。结果:构建了人源日本血吸虫Fab抗体库,库容为4.3×106,经核苷酸序列分析,证实插入片段为Fab基因片断。经过5轮筛选后,从富集的次级抗体库中随机挑取60个克隆,用ELISA法鉴定得到4个可与免疫鼠血清(Ab1)特异性结合,而不与SEA及SWAP结合的单克隆抗体(A3、B6、C4、C17),其中B6经SDS-PAGE电泳显示插入片断正确。结论:成功构建了人源日本血吸虫Fab抗体库,并从中获得人源日本血吸虫抗独特型抗体B6,为研制用于人体血吸虫病免疫预防的抗独特型抗体疫苗奠定了基础。     

B细胞淋巴瘤Fab噬菌体抗体库的构建

目的构建B细胞淋巴瘤Fab噬菌体抗体库。方法从人B细胞淋巴瘤细胞系Raji细胞免疫的BALB/c小鼠脾细胞中提取总RNA,RT-PCR扩增免疫球蛋白轻链κ基因及重链Fd片段,然后经酶切、纯化、连接等步骤将轻链κ基因和重链Fd片段依次克隆入噬菌体载体pComb3H-SS中,并电转化大肠杆菌XL1-Blue。结果构建了抗人B细胞淋巴瘤Fab噬菌体抗体库,轻链κ基因、重链Fd片段与载体的重组率分别为100%和78%,库容量为2.18×107。结论成功构建了抗人B细胞淋巴瘤Fab噬菌体抗体库,方便进一步筛选抗人B细胞淋巴瘤抗体。     

人源抗EB病毒潜伏膜蛋白1特异性基因工程抗体Fab的筛选与特性

目的:应用噬菌体展示技术制备抗EB病毒编码的潜伏膜蛋白1(latert membrare protern 1,LMP1)特异性?高亲和力的全人抗体Fab片段?方法:利用稳定转染表达LMP1的SUNE-1细胞及原核表达的LMP1胞外区蛋白作为抗原对大容量人源Fab抗体库进行差减筛选及固相筛选,经过5轮细胞筛选和3轮固相筛选,随机挑选30个克隆经酶联免疫吸附法鉴定,阳性克隆进行可溶性表达,用LMP1阳性表达的人鼻咽癌细胞株HNE2/LMP1鉴定抗LMP1抗体Fab的结合活性?结果:Western blot?免疫荧光结果显示,从大容量人源Fab抗体库中筛选出1株抗LMP1抗体Fab,细胞ELISA?荧光激活细胞分类术?免疫荧光分析?免疫组化检测结果显示Fab与人鼻咽癌细胞表面LMP1分子有较好的结合活性?结论:从人源Fab抗体库中筛选的Fab抗体能够与鼻咽癌细胞表面LMP1分子特异性结合,为研制用于EBV相关肿瘤如鼻咽癌生物治疗的靶向药物,提供了候选分子?     

基因工程技术制备的人源性抗Mr 48×103角蛋白抗体Fab片段的纯化与活性鉴定

目的用基因工程技术制备人源性抗角蛋白抗体Fab片段,并对其纯度、抗原结合活性及特异性等进行鉴定. 方法将从半合成噬菌体抗体库中成功地筛选到的特异表达抗角蛋白Fab片段的阳性克隆转化大肠杆菌,IPTG诱导表达,产物经金属鏊合层析纯化,并用ELISA鉴定抗原结合活性和特异性、SDS-PAGE鉴定纯度和蛋白印迹检测抗角蛋白抗体Fab片段识别的抗原组分. 结果可溶性表达的抗角蛋白抗体Fab片段经金属鏊合层析可有效地纯化,蛋白印迹明确纯化产物为人Fab片段.所纯化的Fab片段在非还原SDS-PAGE中形成Mr 50×103单一条带,在还原SDS-PAGE中可见Mr 23×103,Mr 25×103两条带. ELISA和Western blot证实该片段具备良好的抗原结合活性和抗原特异性. 结论成功表达并鉴定了人源性抗Mr 48×103角蛋白的Fab可溶性片段,为人源性抗角蛋白抗体工程化、进一步研究该抗体的生物活性并提高其临床应用价值奠定了良好的基础.     

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.