中华眼镜蛇蛇毒因子的简易提纯及鉴定

目的探索从中华眼镜蛇（Ｃｈｉｎｅｓｅ ｃｏｂｒａ,Ｎａｊａ ｎａｊａ ａｔｒａ）蛇毒中一次性快速分离提纯具有抗补体活性及溶血活性的中华眼镜蛇蛇毒因子子质量测定,并鉴定其活性与毒性。结果本法ＣＣＶＦ得率高于２％,电泳图谱呈单一区带,由３个亚基组成,相对分子质量为１４５ ８００,抗补体及溶血活性强,毒性低。结论本法操作简单,时间短,纯度高,为一实用高效快速提纯方法。     

中华眼镜蛇蛇毒因子的简易提纯及鉴定

目的 勃勃从中华眼镜蛇（Ｃｈｉｎｅｓｅ ｃｏｂｒａ,Ｎａｊａ ｎａｊａ ａｔｒａ）蛇毒中一镒性快速分离提纯具有抗补体活性及溶血生的中华眼镜晕 毒因子（Ｃｈｉｎｅｓｅ ｃｏｂｒａ ｖｅｎｏｍ ｒａｃｔｏｒ,ＣＣＶＦ）的方法。方法 用倒流法将粗毒９经ＤＥＡＥ－ｓｅｐｈａｄｅｘ Ａ－５０柱层析分离提纯。用ＳＤＳ－ＰＡＧＥ进行纯度鉴定及相对分子质量测定,并鉴定其活性与毒性。结果 本法ＣＣＶＦ得率高于２％,     

蛇毒因子溶血试验简介

蛇毒因子(Cobra Venom FactorCOF)是从眼镜蛇蛇毒中提纯的一种低毒性,高分子量蛋白质,其分子量为144,000。它能直接激活血清中补体的第3组分(C_3),进而激活补体末端组分(C_5-C_9),破坏靶细胞。在国外,蛇毒因子溶血试验是研究补体溶血机制常用的方法之一,但仅局限于实验室应用。1978年我们从中华眼镜蛇(Naja Naja)蛇毒中提纯了蛇毒因子,改进并建立了蛇毒因子溶血试验(COFT)。我们建立的COFT有如下几个特点:     

舟山眼镜蛇毒蕈碱样多肽的分离及其对M胆碱受体的激动效应

目的 从舟山眼镜蛇Naja atra蛇毒中分离毒蕈碱样多肽,探讨其与M胆碱受体的关系.方法 应用分子筛凝胶Sephadex G-50、Sephadex G-100、离子交换凝胶CM-Sepharose F.F.分离纯化目的组分;SDS-PAGE鉴定目的蛋白的纯度并测定其相对分子质量;放射受体分析法测定该物质与大鼠脑组织M胆碱受体的亲和力;选用离体豚鼠回肠纵行肌观察其对M胆碱受体的效应.结果 从舟山眼镜蛇蛇毒中获得一种与大鼠脑组织M胆碱受体具有高亲和力的多肽(Ki为10.12 nmol/L),其相对分子质量约14 kD.该多肽可引起离体豚鼠回肠纵行肌收缩,此效应可被阿托品阻断.结论 舟山眼镜蛇蛇毒中获得与M胆碱受体具有高亲和力的多肽,该多肽对M胆碱受体具有激动效应.     

中华眼镜蛇毒腺cDNA表达文库的建立和分析

目的: 为研究开发眼镜蛇毒药用基因工程产品,筛选克隆及表达相关药用功能基因,构建中华眼镜蛇毒腺cDNA表达文库. 方法:一步法提取总RNA,Oligo(dT)纤维素层析柱纯化mRNA,逆转录PCR合成双链cDNA,分级分离除去小片段后,收集大于500 bp的cDNA片段,取10 ng cDNA与质粒载体pSPORT1连接、转化. 结果:获得克隆总数为2×105的眼镜蛇毒腺cDNA表达文库,重组率97%. 利用PCR技术从该文库扩增了心脏毒素-3(CTX-3)和磷脂酶A2(PLA2)基因的cDNA. 通过对文库克隆的序列测定和初步生物信息学分析,获得24个中华眼镜蛇毒腺EST序列. 结论:所建立的毒腺cDNA表达文库质量较高,可用于进一步筛选、克隆蛇毒药用功能新基因.     

一种新的蝮蛇蛇毒磷脂酶A2同源物的分离纯化及其对Hep3B细胞基因谱的影响

目的从中国蝮蛇蛇毒中分离纯化出一种新的磷脂酶A2（PLA2）同源物,并研究其对Hep3B细胞基因谱的影响。方法用C18反相高效液相层析从蛇毒粗毒中分离纯化出PLA2同源物,并检测其纯度。用电喷雾质谱仪测定PLA2同源物分子量。体外培养Hep3B细胞,以139μg／mlPLA2同源物处理12h,应用基因芯片检测基因表达的改变。结果从蛇毒粗毒中分离纯化出一种新的PLA2同源物,纯度为97．2％,相对分子质量为13900。139μg／mlP乙嘘同源物处理Hep3B细胞12h后．19个基因表达下调,20个基因表达上调。表达改变的基因按功能分主要为以下6类：细胞周期控制和DAN损伤修复类、细胞调亡和衰老类信号转导分子和转录因子、细胞黏附类、血管生成类以及肿瘤细胞入侵和转移类。结论PLA2同源物作用于Hep3B细胞后,可影响细胞多种基因的表达,表达改变的基因主要参与肿瘤细胞的生长和转移。本研究为进一步深入研究PLA2对肿瘤细胞的作用机制提供线索和依据。     

广西眼镜王蛇毒一种碱性蛋白的分离纯化及性质研究

目的:广西眼镜王蛇毒一种碱性蛋白的分离纯化和性质研究.方法:分离纯化采用DEAE-Sepharose CL-6B离子交换柱层析,Sephadex G-75凝胶过滤和CM-Sepharose CL-6B离子交换柱层析;通过电泳,磷脂酶A2(PLA2)酶活力测定,氨基酸组成分析,N端序列测定及药理实验进行性质研究.结果:从广西眼镜王蛇毒中分离到一种电泳纯的碱性蛋白(命名为CM-IV),相对分子质量约为14 000,等电点约为8.65;PLA2的比活力为23 μmol·mg-1/min.氨基酸组成分析表明,其分子中Cys的含量占6.85%;利用蛋白质直接测序N端序列为TKCYVTPDVK.药理实验表明它对实验用小白鼠有致死性(LD50约为0.2 mg/kg),并具有心脏毒性,但无血小板抑制活性.根据Genebank Blast 分析软件,它的N端序列与神经毒素具有高度的同源性.结论:这种碱性蛋白可能为神经毒素,但却拥有PLA2的活力.     

舟山眼镜蛇毒L-氨基酸氧化酶的分离纯化、理化及酶学性质

目的 从舟山眼镜蛇蛇毒中分离L-氨基酸氧化酶(LAO),测定其理化和酶学性质.方法 应用Sephadex G-100凝胶色谱和POROS CM20离子交换色谱分离纯化LAO;Wellner和Lichtenberg法测定LAO活力,SDS-PAGE法测定相对分子质量.结果 舟山眼镜蛇蛇毒经Sephadex G-100凝胶色谱和两次POROS 20离子交换色谱后,获得LAO纯品(暂定名NA-LAO).NA-LAO在非还原和还原条件下相对分子质量均为58 kD左右;其反应最适pH为8.0,最适温度为60℃,对L-苯丙氨酸的米氏常数(Km)为3.08 mmoL/L.结论 应用凝胶过滤色谱和离子交换色谱可从舟山眼镜蛇毒中分离纯化LAO.     

广西眼镜蛇毒中Natrin蛋白的分离及纯化

目的:从广西眼镜蛇毒中分离出Natrin蛋白.方法:广西眼镜蛇毒经SephadexG75凝胶、CM Sepharose CL-6B离子交换层析及FPLC mono-S柱层析分离纯化出Natrin蛋白.结果:从广西眼镜蛇蛇毒中分离纯化得到的Natrin蛋白,经SDS-PAGE电泳测定其分子量为25 ku,质谱测定其分子量...     

广西眼镜王蛇毒酸性磷脂酶A2的分离纯化和性质研究

目的广西眼镜王蛇毒酸性磷脂酶A2的分离纯化和性质研究.方法分离纯化采用SephadexG-75,CM-SepharoseCL-6B和DEAE-SephadexA-50柱层析法;通过电泳,磷脂酶A2活力测定,氨基酸组成测定,N端序列分析及药理实验进行性质研究.结果从广西眼镜王蛇毒中分离到一种酸性的磷脂酶A2(命名为APLA2-1),相对分子质量为14600u,等电点5.4,酶的比活力为108μmol/mg*min-1,N端序列为NLIQFGNMIQCTVPGF.它对实验用小白鼠有致死性(LD506.6mg/kg),具有肌毒性,心脏毒性,抗血小板聚集活性,能诱导水肿形成.结论APLA2-1生化和药理性质的研究,为阐明蛇毒PLA2结构和功能关系,及蛇伤中毒机制,打下了良好基础.     

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.