携带人Oct4和EGFP基因慢病毒表达载体构建

目的 构建携带人Oct4和EGFP基因的慢病毒表达载体pFUOGW.方法 Sac Ⅱ线性化pLentiEF1a-Oct4-IRES-EGFP,回收片段并补平Sac Ⅱ切口,接着BamH Ⅰ酶切该片段,回收2.565 kb片段而获连接用Oct4-IRES-EGFP;EcoR Ⅰ线性化pFUGW,回收并补平EcoR Ⅰ切口,然后BamH Ⅰ酶切该片段,回收9.174 kb片段获连接用载体片段,最后使用DNA连接试剂盒(TaKaRa)中的Solution Ⅰ将其与连接用Oct4-IRES-EGFP连接,连接产物转化,次日挑选单菌落,提取质粒并行酶切鉴定.所构建载体命名为pFUOGW.获pFUOGW后,按Invitrogen公司推荐的标准程序进行慢病毒包装和确认慢病毒是否成功生产;携带Oct4和EGFP基因的慢病毒感染293FF细胞和鼻咽癌细胞株C666-1、CNEI和6-10B以建立相应病毒感染体系.结果 酶切鉴定证实成功构建了pFUOGW,按标准程序生产的携带Oct4和EGFP基因的慢病毒上清高效率感染鼻咽癌细胞株C666-1、CNE1和6-10B.结论 成功构建携带人Oct4和EGFP基因的慢病毒表达载体pFUOGW,为相关后续的研究打下良好的基础.     

携带人Sox2和EGFP基因慢病毒表达载体构建

目的 构建携带人Sox2和EGFP基因的慢病毒表达载体pFUSGW.方法 Xho Ⅰ线性化pLentiEF1a-SOx2-IRES-EGFP,回收片段并补平Xho Ⅰ切口,接着BamH Ⅰ酶切该片段,回收2.356 kb片段而获连接用Sox2-IRES-EGFP;EcoR Ⅰ线性化pFUGW,回收并补平EcoR Ⅰ切口,然后BamH Ⅰ酶切该片段,回收9.174 kb片段获连接用载体片段,最后使用DNA连接试剂盒(TaKaRa)中的Solution Ⅰ将其与连接用Sox2-IRES-EGFP连接,连接产物转化,次日挑选单菌落,提取质粒并行酶切鉴定.所构建载体命名为pFUSGW.获pFUSGW后,按Invitrogen公司推荐的标准程序进行慢病毒包装和确认慢病毒是否成功生产;携带Sox2和EGFP基因的慢病毒感染小鼠胚胎成纤维细胞(MEFs)、人胚肺成纤维细胞(HLFs)和人神经胶质瘤细胞(U87)以建立相应病毒感染体系.结果 酶切证实成功构建了pFUSGW,按标准程序生产的携带SOx2和EGFP基因的慢病毒上清高效率感染MEFs、HLFs及U87.结论 成功构建携带人Sox2和EGFP基因的慢病毒表达载体pFUSGW,为相关后续的研究打下了良好的基础.     

携带人c-Myc和EGFP基因慢病毒表达载体的构建

目的 构建携带人c-Myc和EGFP基因的慢病毒表达载体pFUMGW.方法 Xho I线性化plenti-EFla-c-Myc-IRES-EGFP,回收片段并补平Xho I,接着BamH I酶切该片段,回收2722 bp片段而获连接用c-Myc-IRES-EGFP;EcoR I线性化pFUGW,回收并补平EcoR I...     

携人PTHLH基因慢病毒表达载体的构建

目的构建携带人甲状旁腺相关激素(PTHLH)基因的慢病毒表达载体pGC-FU/PTHLH。方法酶切载体pGC-FU,根据人PTHLH基因合成特定引物,扩增目的基因片段,将其克隆到pGC-FU质粒(含EGFP基因)上,菌落PCR鉴定及测序分析重组载体,使用Lipofectamine 2000诱导转染pGC-FU、pHelper1.0和pHelper2.0载体三质粒进入293T细胞包装慢病毒,并用带PTHLH的慢病毒感染293T细胞和鼻咽癌CEN1细胞确认慢病毒包装是否成功。结果菌液PCR产物琼脂糖凝胶电泳鉴定显示,与理论预计值阳性转化子735bp,阴性转化子198bp基本相吻合;PCR鉴定阳性的克隆进行测序和比对分析,结果完全匹配,进一步鉴定载体构建成功。分别将质粒包装系统共转染293T细胞,包装产生空白对照慢病毒(pGC-FU)和过表达PTHLH的慢病毒(pGC-FU/PTHLH);或用携带PTHLH和EGFP基因的病毒上清感染CNE1细胞,48h后,倒置荧光显微镜下观察293T和CNE1细胞,均可见绿色荧光,转染成功。结论成功构建携人PTHLH基因慢病毒表达载体,为进一步研究PTHLH基因在鼻咽癌转移中的作用及鼻咽癌发病分子机制奠定了基础。     

建立慢病毒介导的miR-145稳定过表达的鼻咽癌细胞株

目的 构建过表达miR-145的慢病毒载体,建立稳定过表达miR-145的鼻咽癌细胞株.方法 构建慢病毒表达质粒pLVTHM/miR-145;293FT细胞进行慢病毒包装,生产的慢病毒感染鼻咽癌细胞株CNE1和CNE2,经流式细胞仪筛选后,建立稳定表达miR-145的鼻咽癌细胞株;最后应用荧光定量PCR检测病毒感染后鼻咽癌细胞株CNE1和CNE2中miR-145的表达水平.结果 荧光定量PCR结果和测序验证pLVTHM/miR-145重组质粒构建成功;包装生产的慢病毒感染鼻咽癌细胞CNE1和CNE2后,与阴性对照组比较,其表达水平分别升高4000倍和4500倍.结论 成功构建了pLVTHM/miR-145慢病毒重组质粒,建立稳定表达miR-145的鼻咽癌细胞株CNE1和CNE2,为研究miR-145在鼻咽癌发生发展过程中的基因调控和相应的作用机制提供了有用的细胞模型.     

p38丝裂原激活蛋白激酶基因重组慢病毒载体的构建及其在建立人前列腺癌稳定细胞株中的应用

目的构建p38丝裂原激活蛋白激酶基因(MAPK)重组慢病毒载体并建立表达外源性p38基因的人前列腺癌稳定细胞株。方法采用限制性内切酶酶切、T4 DNA连接酶连接等方法,将EGFP/p38融合基因插入慢病毒载体pTYF-EF1α-IRES-EGFP中,构建启动子EF1α调控的EGFP/p38共表达慢病毒载体pTYF-EF1α-EGFP/p38,经酶切鉴定后,利用慢病毒三质粒系统,通过脂质体法转染包装细胞HEK 293T细胞,收集病毒上清后转导人前列腺癌DU145细胞株,经有限稀释法筛选重组EGFP/p38稳定细胞株,用Western blotting检测细胞总p38的表达,用计数法绘制细胞的生长曲线。结果经酶切鉴定,成功构建EGFP/p38重组慢病毒载体,并包装慢病毒,检测病毒悬液的滴度为4.7×106 TU/ml,利用此病毒悬液感染DU145细胞,成功筛选到EGFP/p38稳定表达细胞株,Western blotting显示该细胞株能稳定表达外源性EGFP/p38融合蛋白,这种过表达p38的细胞株的生长速度明显低于对照组。结论成功构建p38 MAPK重组慢病毒载体并建立了表达外源性p38 MAPK基因的稳定细胞株EGFP/p38-DU145,细胞内p38过表达能够抑制DU145细胞的增殖。     

稳定过表达miR-26a肝癌细胞株的建立

目的建立稳定过表达miR-26a的肝癌细胞株。方法以CTE049质粒为模板,PCR扩增miR．26a序列418bp,片段两侧添加XbaI和BamHI酶切位点,In．Fusion克隆至pHAGE．fullEFla．MCS．IZsGreen中,次日挑选单菌落,提取质粒并进行测序和酶切鉴定;所构建的载体命名为pLVT．miR26a。获得pLVT．miR26a后,按Invitrogen公司推荐的标准程序进行慢病毒包装和确认慢病毒是否成功生产;携带miR．26a和ZsGreen基因的慢病毒感染小鼠肝癌细胞（Hepl．6）、人肝癌细胞（BEL．7402、HepG2、Sk—Hep．1、HepG2．1uc）,以建立稳定过表达miR．26a的肝癌细胞系：qRT．PCR检测所建立肝癌细胞系中miR26a的表达水平。结果测序和酶切证实成功构建了pLVT．miR26a,按标准程序生产的携带miR．26a和ZsGreen基因的慢病毒上清成功感染小鼠及人肝癌细胞。结论成功建立稳定过表达miR．26a的肝癌细胞株,为相关后续研究打下了良好基础。     

大鼠线粒体膜蛋白Nix基因过表达与shRNA慢病毒载体的构建与鉴定

目的 构建线粒体膜蛋白Nix基因过表达与shRNA慢病毒载体.方法 PCR扩增获得Nix基因序列,与经BamH Ⅰ、EcoR Ⅰ双酶切的pHBLV-CMVIE-IRES-Puro载体连接获得Nix过表达重组慢病毒载体;以Nix为靶基因,合成寡核苷酸,退火形成双链DNA,与经BamH Ⅰ、EcoR Ⅰ双酶切的pHBLV-U6-Scramble-Puro载体连接获得Nix shRNA重组慢病毒载体;将病毒包装辅助质粒与上述重组慢病毒载体共转染293T细胞,收集上清液并浓缩获得慢病毒浓缩液,3倍梯度稀释法测定病毒滴度;用病毒颗粒感染PC12细胞,经嘌呤酶素筛后得到稳定感染细胞株;荧光显微镜检测Nix荧光水平,Western blot检测Nix蛋白的表达.结果 成功构建Nix基因过表达和shRNA慢病毒载体,并在293T细胞中包装获得病毒.重组病毒感染PC12细胞后,荧光显微镜检测Nix基因过表达细胞荧光水平增加,shRNA感染细胞荧光水平降低;Western blot检测显示Nix基因过表达PC12细胞中Nix蛋白表达显著升高,shRNA慢病毒感染细胞中Nix蛋白表达明显降低,而Nix shRNA1干扰效果最好.结论 成功构建Nix基因过表达与shRNA慢病毒载体,并获得Nix过表达与干扰细胞模型.     

NOC2L基因重组慢病毒表达载体的构建及其在293T细胞中的表达

目的构建NOC2L基因重组慢病毒并使其在293T细胞中表达。方法通过设计引物及PCR扩增获得NOC2L全基因片段,将pCDH-GFP载体分别用XbaⅠ和BamHⅠ双酶切制备线性化载体;NOC2L全基因片段和线性化载体经切胶回收后,通过同源重组反应将NOC2L全基因片段连接到线性化的pCDH-GFP载体上,构建pCDH-NOC2L-GFP慢病毒表达载体;通过菌落PCR、质粒双酶切、测序等方法对构建的NOC2L基因重组慢病毒表达载体进行鉴定,利用构建的pCDH-NOC2L-GFP包装NOC2L慢病毒并使用该慢病毒感染293T细胞。结果菌落PCR、质粒双酶切和测序结果显示成功将NOC2L基因连接到pCDH-GFP载体上,使用构建成功的pCDH-NOC2L-GFP转染293T细胞,能够成功转染及包装NOC2L基因重组慢病毒,同时包装好的NOC2L基因重组慢病毒能够成功感染293T细胞并过表达293T细胞中的NOC2L基因。结论采用本研究方法能成功构建NOC2L基因重组慢病毒表达载体。     

改建型STAT1-CC基因慢病毒载体的构建及其在肺癌A549细胞中的表达

目的:构建携带STAT1基因和突变型STAT1-CC基因的慢病毒表达载体,转染肺癌A549细胞并检测STAT1和STAT1-CC基因在肺癌A549细胞内的表达.方法:采用PCR技术从质粒模板中扩增得到目的基因STAT1和STAT1-CC,将其接入含有CMV启动子和绿色荧光蛋白(EGFP)的慢病毒载体中,构建慢病毒载体表达载体Lenti-STAT1和Lenti-STAT1-CC,双酶切和测序鉴定.分别将重组子Lenti-STAT1、LentiSTAT1-CC和包装质粒共转染293T细胞,包装产生慢病毒颗粒,感染非小细胞肺癌细胞株A549.荧光显微镜观察A549细胞EGFP表达,Western Blotting验证STAT1在A549细胞株中的表达.结果:酶切及测序结果显示成功构建重组慢病毒表达载体Lenti-STAT1和Lenti-STAT1-CC,将慢病毒颗粒感染A549细胞48 h后,观察到宿主细胞内EGFP表达,Western Blotting证实STAT1蛋白在A549细胞中过表达.结论:本实验成功构建了携带STAT1基因和STAT1-CC基因的慢病毒表达载体,并在A549细胞中过表达STAT1基因.     

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.