phP53-luc质粒的构建及其用于检测化学物质遗传毒性的初步分析

目的:构建2个分别由p53基因启动子和核心启动子驱动的荧光素酶报告基因表达质粒,并探讨其用于检测遗传毒性化学物质的可行性.方法:通过PCR扩增人p53基因启动子片段,然后采用重组DNA技术克隆入pGL3-BASIC的荧光素酶报告基因的上游而获得phP53-luc表达质粒.用脂质体转染法将phP53-luc转染入NIH 3T3细胞并分别用5-氟尿嘧啶(5-FU)处理,最后裂解细胞用Dual-Luciferase Reporter Assay System检测荧光素酶报告基因表达.结果:构建了2个分别由p53基因启动子和核心启动子驱动的荧光素酶报告基因表达质粒,位于这2个质粒上的荧光素酶报告基因在NIH 3T3细胞内的表达显著地受遗传毒性化学物质的诱导,其表达比对照的pGL3-BASIC载体上的荧光素酶报告基因高近240倍.结论:基于人p53基因启动子的荧光素酶报告基因分析系统可以用于快速检测环境毒物的遗传毒性.     

Survivin基因siFNA表达载体的构建及其在食管癌EC109细胞中的表达

目的为研究survivin基因的功能及食管癌的基因治疗,构建针对survivin基因的siRNA的表达载体,转染细胞EC109后观察其对survivm基因表达的抑制作用.方法利用网站选择适当位点设计并合成针对survivin基因nRNA的寡核苷酸序列,插入质粒pBAsi-hU6中形成重组载体pBAsi-survlvin.重组载体经测序鉴定后转染食管癌细胞EC109,western blot检测转染前后survivin蛋白的表达情况.结果测序结果与所合成的寡核苷酸完全一致,证实成功构建了针对sLurvivin基因的siRNA表达载体.Westem blot检测显示转染食管癌细胞EC109后有效抑制了survivin基因的表达.结论针对surivivin基因的siRNA表达载体成功构建,并能有效抑制食管癌细胞EC109中survivin基因的表达.     

人端粒酶逆转录酶基因启动子调控表达载体的构建和鉴定

目的:构建并鉴定人端粒酶逆转录酶(human telomerase reverse transeriptase,hTERT)基因启动子调控的荧光素酶基因表达载体。方法:用巢式PCR方法克隆hTERT序列长约1100bp的启动子片段,经测序无误后将其插入荧光素酶基因报告载体中,构建pGL3-hTERTp重组质粒,转化JM190细菌得到大量含有hTERT启动子的荧光素酶重组质粒。用双酶切和PCR法鉴定重组质粒,并送测序鉴定。结果:经过酶切和PCR法鉴定成功地构建了携带hTERT启动子的重组荧光素酶基因报告载体。结论:成功获得由hTERT启动子调控的荧光素酶基因表达载体,为研究As₂O₃对HL-60细胞端粒酶作用的分子机制打下基础。     

Survivin基因siRNA表达载体的构建及其在食管癌EC109细胞中的表达

目的 为研究survivin基因的功能及食管癌的基因治疗，构建针对survivin基因的siRNA的表达载体，转染细胞EC109后观察其对survivin基因表达的抑制作用。方法 利用网站选择适当位点设计并合成针对survivin基因mRNA的寡核苷酸序列，插入质粒pBAsi-hU6中形成重组载体pBAsi-survivin。重组载体经测序鉴定后转染食管癌细胞EC109，western blot检测转柒前后survivin蛋白的表达情况。结果 测序结果与所合成的寡核苷酸完全一致，证实成功构建了针对survivin基因的siRNA表达载体。Western blot检测显示转染食管癌细胞EC109后有效抑制了survivin基因的表达。结论 针对surivivin基因的siRNA表达载体成功构建，并能有效抑制食管癌细胞EC109中survivin基因的表达。     

NNMT基因启动子双荧光素酶报告系统的构建及其与SND1靶向关系的验证

目的:构建尼克酰胺-N-甲基转移酶（NNMT）启动子区的双荧光素酶报告质粒并检测其活性,从而验证SND1蛋白对NNMT基因的调控作用。方法:根据NNMT基因启动子区上、下游-1 500至+200区间的序列设计引物,并以HepG2细胞提取的RNA逆转录得到cDNA为模板进行PCR扩增,得到的NNMT启动子区片段经酶切后连接到双荧光素报告载体GLuc-ONTM启动子报告克隆中,得到Gluc-NNMT 启动子重组质粒,分别与pLVX-IRES-Puro-Flag-SND1质粒和pLVX-IRES-Puro-vector质粒共同转染HeLa细胞,并检测启动子活性。然后用免疫共沉淀的方法验证NNMT蛋白是否与SND1蛋白之间存在相互作用。结果:重组质粒构建成功。双荧光素酶活性实验显示,共转染Gluc-NNMT启动子重组质粒和pLVX-IRES-Puro-Flag-SND1过表达质粒的实验组与对照组相比,NNMT启动子区活性明显增强。结合免疫共沉淀实验结果,证明NNMT与SND1存在相互作用。结论:成功构建了NNMT启动子双荧光素酶报告质粒,SND1对NNMT启动子活性有增强作用,SND1与NNMT蛋白之间存在相互作用。     

小鼠STING基因启动子的克隆鉴定及功能初步分析

目的:克隆小鼠干扰素基因刺激因子(STING)基因启动子,并对其启动子活性和转录调控机制进行初步探讨?方法:利用PCR方法扩增小鼠STING基因5′上游1 005 bp(-927~+77)的片段,亚克隆至pGL3-basic质粒,然后通过步移缺失构建不同缺失片段的重组质粒?双荧光素酶报告活性分析检测重组质粒在NIH3T3中的活性,并利用生物信息学方法预测转录因子结合位点?结果:经酶切?测序鉴定,成功构建了小鼠STING启动子荧光素酶报告基因重组质粒?与pGL3-basic质粒相比,STING启动子重组质粒的相对荧光素酶活性增加(P < 0.05)?通过生物信息学软件预测小鼠STING启动子区域(-177~-48)可能含有GATA?IK2?MZF1?SP1/SP3?STAT等转录因子结合位点?结论:成功构建小鼠STING不同缺失片段的启动子荧光素酶报告基因重组质粒?通过活性比较,推测小鼠STING的核心启动子区位于-177~+77区域,其中可能含有多个潜在的转录因子结合序列?     

Survivin启动子的克隆及其在膀胱癌细胞BIU87中的活性鉴定

目的克隆Survivin启动子的有效片段,并检测其在人膀胱癌细胞株BIU87和人正常膀胱上皮细胞SV-HUC-1中的转录活性。方法用PCR扩增Survivin基因的启动子片段,克隆入荧光素酶报告质粒pGL3-Basic,构建pGL3BSurvivin重组质粒,用脂质体法瞬时转染BIU87及SV-HUC-1中,检测Survivin启动子在细胞中的转录活性。同时构建含CMV启动子的pGL3BCMV重组质粒作为阳性对照。48h后收集转染细胞与荧光素酶底物反应,检测荧光素酶活性。结果成功克隆440 bp的Survivin基因启动子,并构建了携带有Survivin基因启动子的pGL3Basic真核表达载体,转染BIU87细胞后的荧光素酶活性为2 286.98±440.21,而SV-HUC-1荧光素酶活性为12.32±1.16,两者差异有统计学意义（〈0.01）。结论本实验成功克隆的Survivin启动子在BIU87细胞中表现出较高的肿瘤特异性活性,其有可能作为调控元件用于膀胱癌的靶向性基因治疗。     

人FAT1基因启动子的克隆鉴定及其功能浅析

目的：克隆人脂肪非典型钙黏蛋白1（FAT atypical cadherin 1,FAT1）基因启动子,找到核心启动子区域并对其转录调控机制进行初步分析。方法：通过PCR方法获得人FAT1基因5′上游1 163 bp（-1 029~+134 bp）的片段,亚克隆至pGL3?basic载体;通过步移缺失构建不同缺失片段的重组质粒。双荧光素酶报告活性分析检测各重组质粒在A549细胞和HEK293T细胞中的活性,找到核心启动子区域。利用生物信息学方法预测核心区域的转录因子结合位点。结果：经测序、酶切鉴定,成功构建了人FAT1启动子荧光素酶活性报告基因重组质粒。与pGL3?basic质粒相比,人FAT1启动子重组质粒的相对荧光素酶活性增加（P < 0.05）。通过生物信息学软件预测人FAT1启动子区域（-233~-110 bp）可能含有TFAP2C、KLF5等转录因子结合位点。结论：成功构建人FAT1启动子不同缺失片段的荧光素酶报告基因重组质粒。通过荧光素酶活性比较,推测人FAT1的核心启动子区域位于-233~+134 bp区域,其中可能含有若干转录因子结合位点。     

CEA siRNA载体的构建和转染人食管癌EC9706细胞的沉默作用

目的:应用RNA干扰技术,构建针对CEA的siRNA载体,抑制人食管癌EC9706细胞CEA基因的表达.方法:依据设计siRNA的原则,针对人CEA的mRNA序列,设计并合成编码siRNA的两对寡核苷酸序列,经退火成互补双链,克隆入载体pRNAT-U6.2中构建重组体pRNAT-U6.2-CEA,筛选、鉴定.然后转染重组体至人食管癌EC9706细胞中,经G418筛选,获得阳性克隆,并以空质粒转染和未进行转染的EC9706作对照,运用PCR和Western Blot检测CEA的表达.结果:测序鉴定证实目的寡核苷酸片断已被克隆到pRNAT-U6.2载体中,pRNAT-U6.2-CEA转染人食管癌EC9706细胞后,CEA基因表达在mRNA和蛋白质水平都受到显著抑制.结论:成功构建了针对人食管癌EC9706细胞的siRNA载体,可有效抑制CEA的表达,为进一步从分子水平探讨CEA的功能奠定了基础.     

人SMYD3基因启动子荧光素酶报告载体的构建与分析

目的:克隆5'长度不同的SMYD3基因启动子序列并进行活性鉴定.方法:采用PCR方法克隆出SMYD3基因转录起始位点上游不同长度的基因片段,构建T载体并利用酶切与测序进行鉴定;将该基因亚克隆至pGL3-Basic荧光报告基因载体上;瞬时转染Hep3B细胞后用双荧光报告检测系统检测不同片段的荧光活性.结果:T载体酶切与测序结果正确,成功构建了pGL3-Basic+370～1 400 bp荧光报告载体并利用双荧光素酶报告基因检测系统分析了重组质粒的荧光活性.结论:pGL3-Basic+370～1 400 bp重组质粒转染组与阳性对照组相比并未表现出显著的荧光活性,本研究认为SMYD3基因的启动子核心区域位于-1 334 bp的上游.     

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.