水蛭素基因毕赤酵母表达载体的构建及高拷贝稳定整合菌株的筛选

目的:构建水蛭素变异体HV1毕赤酵母分泌型表达载体,获得高拷贝稳定整合菌株,为大量获得重组水蛭素蛋白,进行功能研究及其临床应用奠定基础. 方法:根据水蛭素HV1的氨基酸序列及毕赤酵母偏爱的密码子,设计HV1编码基因,分为8条寡核苷酸片段进行DNA合成,经磷酸化、退火、连接和PCR扩增得到优化的HV1全长基因,测序结果正确的基因序列插入酵母表达载体pPIC9α分泌信号下游,再亚克隆入高拷贝载体pPIC9K,得到pPIC9K-HV1,SalI线性化后转化毕赤酵母GSM1168,G418梯度筛选转化菌,PCR鉴定目的基因的整合. 结果:获得了水蛭素毕赤酵母表达载体pPIC9K-HV1,G418抗性筛选得到高拷贝菌株,PCR证实目的基因已整合到毕赤酵母染色体中. 结论:成功构建了水蛭素HV1毕赤酵母表达载体,获得了高拷贝稳定整合菌株,为大规模表达纯化HV1蛋白及其临床应用奠定了基础.     

重组小鼠凝血因子Ⅶ毕赤酵母表达载体的构建及整合体的筛选

目的构建无凝血活性的重组小鼠凝血因子Ⅶ(rmFⅦ)毕赤酵母分泌型表达载体,获得高拷贝稳定整合菌株,为大量获得rmFⅦ蛋白,进行功能研究及其临床应用奠定基础。方法PCR扩增得到mFⅦ基因片段,插入酵母表达载体pPIC9K的α分泌信号开放阅读框的下游,再经定点突变(K341A)得到pPIC9KrmFⅦ,测序正确的质粒用SacⅠ线性化后电穿孔转化毕赤酵母GS115,G418梯度筛选转化菌,PCR鉴定目的基因的整合。结果获得了rmFⅦ毕赤酵母表达载体pPIC9KrmFⅦ,G418抗性筛选得到高拷贝菌株,PCR证实目的基因已整合到毕赤酵母染色体中。结论成功构建了rmFⅦ毕赤酵母表达载体,获得了高拷贝稳定整合菌株,为大规模表达纯化rmFⅦ蛋白及后续研究奠定了基础。     

sBLyS突变体酵母表达载体的构建

目的构建人BLyS胞外区(soluble BLyS)突变体毕赤酵母分泌型表达载体,为深入探讨BLyS的功能和机制创造条件。方法以构建的人pUC19/sBLyS及其突变体重组质粒为模板,利用PCR扩增出6×his/sBLyS及其突变体的cDNA,克隆入酵母表达载体pPIC9K;行序列测定后电转化入毕赤酵母GS115,并进行表型筛选和G418抗性筛选;对高拷贝整合的重组酵母表达菌株进行PCR鉴定。结果构建得到酵母融合重组表达载体pPIC9K/6×his/sBLyS及其突变体;经G418浓度梯度筛选及PCR鉴定得到高拷贝整合的重组酵母表达菌株。结论成功构建了人sBLyS及其突变体的真核表达载体,为获得人sBLyS及其突变体蛋白,进一步研究BLyS的生物学功能奠定了基础。     

人羧肽酶A1活性中心的毕赤酵母可溶表达

目的:应用毕赤酵母表达系统表达人羧肽酶A1(carboxypeptidase A1,CPA1)活性中心蛋白.方法:从pGEX-CPA1重组质粒中扩增出CPA1活性中心基因,亚克隆入酵母表达载体pPIC9K,酵母重组表达载体pPIC9K-CPA1active center电转化入毕赤酵母SMD1168,并进行营养缺陷筛选和G418抗性筛选,对高拷贝整合的重组酵母表达菌株诱导表达.结果:构建得到酵母融合重组表达载体pPIC9K-CPA1active center,经G418浓度梯度筛选得到串联整合16个重组表达载体的重组酵母表达菌株,诱导表达后得到CPA1活性中心蛋白.结论:在毕赤酵母中成功表达了CPA1活性中心,为进一步研究CPA1活性中心在抗体导向酶-前体药物疗法中的应用打下了基础.     

人胰岛新生相关蛋白基因的克隆及在毕赤酵母中的表达

目的 在毕赤酵母中分泌表达具有生物学活性的重组人胰岛新生相关蛋白(rhINGAP),用于INGAP的生理功能研究和动物试验.方法 通过PCR扩增INGAP基因,插入到重组质粒α/pUC18的α因子信号序列的Xho Ⅰ和EcoR Ⅰ酶切位点处,再将融合基因αINGAP重组到表达质粒pPIC9K的BamH Ⅰ和EcoR Ⅰ之间,Sal Ⅰ酶切线性化重组质粒INGAP/pPIC9K,电穿孔转化毕赤酵母,营养缺陷型培养基MD和G418筛选含高拷贝表达盒的酵母转化子,PCR鉴定是否含有目的 基因INGAP,甲醇诱导rhINGAP的表达,SDS-PAGE和Western blotting鉴定目的 蛋白,用ELISA定量测定生物学活性.结果 成功构建了重组表达质粒INGAP/pPIC9K,筛选得到3个阳性转化子,表达产物具有良好的抗原活性.结论 实现了rhINGAP在毕赤酵母中的高效分泌性表达.     

抗人肝癌单克隆抗体嵌合轻链在巴氏毕赤酵母的高效分泌表达

目的: 通过整合,用巴氏毕赤酵母表达抗人肝癌mAb HAb18的cFab的嵌合轻链.方法: 将抗人肝癌mAb cFab/HAb18原核表达载体pET32a/cFab中的cL亚克隆到酵母表达载体pPIC9K,构建成重组质粒pPIC9K/cL测序鉴定.重组质粒pPIC9K/cL整合到酵母菌GS115的染色体上,经G418筛选得到高拷贝转化子及Mut表型鉴定后,用含5 mL/L甲醇的培养基诱导表达.结果: 该系统成功表达了抗人肝癌mAb HAb18cFab的嵌合轻链,表达水平为22 mg/L;Western Blotting证实,表达产物具有良好的HAb18GE结合活性和特异性.结论: 嵌合轻链/HAb18在巴氏毕赤酵母获得成功表达,为高效分泌表达cFab抗体奠定了基础.     

人溶菌酶基因的克隆及其在毕赤酵母中的表达

目的：利用巴斯德华赤（Pichia pastoris）酵母真核表达系统,构建真核表达载体pPIC9K与人溶菌酶基因（humanlysozyme,hLY）的重组质粒pPIC9K-hLY,表达出具有活性的人溶菌酶。方法：此项研究在本实验室构建的人溶菌酶（hLY）基因与pUC19的重组质粒pUC19-hLY的基础上,通过设计一对引物,用多聚酶链式反应（PCR）扩增出人溶菌酶全基因片段后,将其克隆到巴斯德毕赤酶母分泌型表达载体pPIC9K中,构建重建质粒pPIC9K-hLY,经Bg1Ⅱ酶切线性化后,用电穿孔法将其转入毕赤酵母细胞内。通过G418筛选,选到的高拷贝转化子经PCR检测人溶菌基因与毕赤酵母染色体是否稳定整合,阳性克隆经甲醇诱导进行表达。结果：序列测定,经PCR扩增后的人溶菌酶基因与献发表的序列相比,缺失4个碱基。我们决定采用重组PCR法予以纠正,经序列测定结果正确。用PCR检测,人溶菌酶基因与毕赤酵母染色体稳定整合。用甲醇诱导表达并以SDS－PAGE分析,在Mr为15000左右出现一条蛋白带,表达量约占上清总蛋白的0．47。Western Blot证实表达产物具有天然人溶菌酶的抗原性。用黄色小球菌平板溶圈法鉴定表态产物具有人溶菌酶的活性。结论：成功的构建了重组质粒pPIC9K-hLY并在毕赤醇母中表达了人溶酶基因。     

SARS病毒M蛋白编码基因真核载体的构建及酵母表达菌株的建立

目的:构建SARS冠状病毒M蛋白N端编码基因1~129bp的真核表达载体,并分析其在毕赤酵母GS115中的表达情况。方法:用PCR方法从质粒pGEX-6P-1+SARS-M上扩增出M编码基因片段,克隆至真核表达载体pPIC9上,构建重组质粒pPIC9+SARS-M。重组质粒经酶切鉴定和核苷酸测序鉴定后,转化毕赤酵母GS115,用甲醇诱导重组M蛋白片段表达。结果:酶切鉴定及核苷酸序列测定表明重组质粒的构建完全正确。对甲醇诱导后重组毕赤酵母培养上清行SDS-PAGE电泳分析,在相对分子量约为10000处有重组蛋白的表达。结论:SARS冠状病毒M蛋白N端1~43aa在毕赤酵母中获得了高效、分泌性表达。     

SARS病毒S蛋白N端片段真核载体的构建及酵母表达菌株的建立

目的：构建SARS冠状病毒S蛋白N端1～501bp的真核表达载体,并分析其在毕赤酵母GS115中的表达情况.方法： 用PCR方法从质粒pGEX-6P-1＋SARS-S上扩增出S编码基因片段,克隆至真核表达载体pPIC9上,构建重组质粒pPIC9＋SARS-S.重组质粒经酶切鉴定和核苷酸测序鉴定后,转化毕赤酵母GS115,用甲醇诱导重组S蛋白片段表达.结果： 酶切鉴定及核苷酸序列测定表明重组真核表达质粒的构建完全正确.对甲醇诱导后重组毕赤酵母培养上清行SDS-PAGE电泳分析,在相对分子量约为41 000处有重组蛋白的表达.结论： SARS冠状病毒S蛋白N端1～167aa在毕赤酵母中获得了高效、分泌性表达.     

大鼠DAF蛋白真核表达载体的构建及生物活性鉴定

目的 构建大鼠促衰变因子(DAF)的酵母真核表达载体,诱导其表达并对生物学特性进行鉴定.方法 采用RT-PCR技术从大鼠肝脏组织中扩增出DAF基因并克隆到毕赤酵母表达载体pPIC9K质粒中.经酶切鉴定后,将pPIC9K-DAF转化至毕赤酵母菌GS115,通过G418-YPD平板筛选高拷贝转化子.转化子经甲醇诱导表达后,对表达的DAF蛋白进行SDS-PAGE及Western blot鉴定并检测重组蛋白的生物活性.结果 成功克隆大鼠DAF基因,并筛选出pPIC9K载体中的DAF阳性克隆,证实所获得相对分子质量约为70×103大小的重组蛋白具有大鼠DAF蛋白的抗原性和生物学活性.结论 采用分子克隆的方法成功构建大鼠DAF蛋白的毕赤酵母真核表达载体,能为补体及免疫研究提供高效的大鼠DAF蛋白来源.     

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.