含不同启动子的腺病毒载体介导的人β2肾上腺素能受体基因在心肌细胞的表达

目的观察不同的启动子cmv、mlc-2v对腺病毒载体介导的人β     

腺病毒介导的血管内皮生长因子体外靶向性转染心肌细胞

目的:构建以鼠心肌轻链蛋白基因(mlc-2v)为启动子、携带人血管内皮生长因子hVEGF165基因的重组腺病毒载体,检测该重组腺病毒载体对心肌细胞转染的靶向性.方法:酶切腺病毒载体PDC315自身启动子CMV,构建腺病毒载体PDC317,分别接入hVEGF165、mlc-2v基因,构建腺病毒载体PDC-mlc-hVEGF165.鉴定正确后,将PDC-mlc-hVEGF165与Lipofectamine共转染至293细胞,经同源重组获得以mlc-2v为启动子、携带hVEGF165基因的重组腺病毒Ad-mlc-hVEGF165,同步构建无靶向性的重组腺病毒Ad-hVEGF165.扩增并测定滴度后,Ad-hVEGF165、Ad-mlc-hVEGF165分别转染体外培养的心肌细胞、骨骼肌细胞及平滑肌细胞,利用ELISA、Western印迹分析等方法检测Ad-mlc-hVEGF165转染心肌细胞的靶向性.结果:构建的病毒正确,病毒滴度为2.8×109 pfu/ml.转染3 d后,Ad-hVEGF165组在各培养细胞的上清液及细胞内均检测到VEGF的表达,而Ad-mlc-hVEGF165组仅在心肌细胞中有VEGF的表达,且Ad-mlc-hVEGF165组心肌细胞分泌的VEGF要少于Ad-hVEGF165组.结论:成功构建了以mlc-2v为启动子、携带人VEGF基因的重组腺病毒Ad-mlc-hVEGF165;该重组载体能体外靶向性转染心肌细胞并使之表达VEGF.     

重组腺病毒转染心衰大鼠心肌细胞cAMP水平变化

目的测定重组腺病毒Ad5-ADRbeta2-EGFP转染体外培养心衰大鼠心肌细胞48h后环磷酸腺苷（cAMP）的水平变化。方法重组腺病毒Ad5-ADRbeta2-EGFP转染心衰大鼠心肌细胞,培养48h后,检测心肌细胞β2-AR蛋白表达和cAMP的水平。结果①心肌细胞β2-AR蛋白表达测定：心衰组与对照组（假手术组）相比没有明显差异（P〉0．05）,心衰＋β2组与心衰组、对照组相比均明显升高（P〈0．05）。②心肌细胞cAMP水平：心衰组与对照组（假手术组）相比明显减少（P〈0．01）,心衰＋β2组与心衰组相比明显升高（P〈0．01）,心衰＋ISO组与心衰组相比明显升高（P〈0．01）,心衰＋ISO＋β2组与心衰组相比明显升高（P〈0．01）,心衰＋ISO＋β2组与心衰＋β2组、心衰＋ISO组相比没有明显差异（P〉0．05）。结论重组腺病毒Ad5-ADRbeta2-EGFP转染体外培养心衰大鼠心肌细胞后,β2-AR蛋白表达上调,同时伴有基础cAMP逆转恢复正常水平,其可能与β2-AR活性的内在活化有关。     

β2-AR过表达对大鼠心肌细胞存活率的影响及机制

目的观察β2肾上腺素能受体（β2-AR）过表达对成年大鼠心肌细胞在不同浓度的异丙肾上腺素（isoproter-enol,ISO）刺激下的保护作用及机制。方法分离培养成年大鼠心肌细胞,随机分为3组：正常组、转染EGFP基因的腺病毒组和转染人β2-AR．EGFP基因的腺病毒组。心肌细胞培养48h后,用异丙肾上腺素在不同剂量（10～mol／L,10^-5mol／L,10^-4mol／L）下刺激2h,观察细胞在刺激前后的存活率的变化。采用Westernblot检测心肌细胞β2-AR的表达。结果Westernblot结果显示转染β2-AR-EGFP基因的腺病毒组β2-AR蛋白的表达量明显高于其余两组（P〈0．05）,在10^-6mol／L,10^-5mol／L浓度的ISO刺激下3组细胞的存活率校正值无显著性差异,在高浓度（10^-4mol／L）ISO刺激下β2-AR过表达组细胞存活率校正值明显高于其余两组（P〈0．05）。结论β2-AR过表达对高浓度（1014mol／L）ISO刺激的成年大鼠心肌细胞的死亡有保护作用,其可能的机制是通过β2-AR与Gi蛋白的耦联作用。     

心肌特异性表达腺病毒载体的构建及鉴定

目的：构建心肌特异性表达的腺病毒载体,使外源基因在心肌细胞中特异性表达。方法：将心肌特异性肌凝蛋白轻链蛋白－２（ｍｌｃ－２ｖ）启动子克隆至腺病毒穿梭质粒,并将绿色荧光蛋白（ＧＦＰ）基因克隆至此质粒中,同源重组含ＧＦＰ基因的缺陷型腺病毒（ＡｄｍｌｃＧＦＰ）,转染心肌细胞及非心肌细胞。结果：经ＲＴ－ＰＣＲ分析,转染的心肌细胞有ＧＦＰｍＲＮＡ的表达,而转染的非心肌细胞无ＧＦＰｍＲＮＡ的表达;荧光显微镜下观察,可见转染的心肌细胞表达ＧＦＰ,而转染的非心肌细胞不表达ＧＦＰ。结论：构建的心肌特异性表达腺病毒载体可使源基因在心肌细胞内特异性表达。     

乙肝病毒X蛋白通过Wnt/β-catenin信号通路调控Gankyrin表达

目的：研究乙肝病毒X蛋白（hepatitis B virus X protein,HBX）对Gankyrin表达水平的影响以及Wnt/β-catenin信号通路在HBX基因调控Gankyrin表达中的作用。方法：HBX腺病毒表达载体（Ad-HBX-GFP）感染转染2种人肝癌细胞系HepG2、SMMC-7721细胞,Si-HBX分子（Si-HBX oligo）用 LipofectamineTM 2000转染入HepG2.2.15细胞,用RT-PCR和Western blot法检测HBX与Gankyrin的表达情况;β-catenin腺病毒载体转染HepG2、SMMC-7721细胞,用Western blot法检测β-catenin与Gankyrin的表达情况;在沉默β-catenin表达的HepG2、SMMC-7721细胞中过表达HBX基因,Western blot法检测β-catenin与Gankyrin的表达情况。结果：①HepG2.2.15细胞中Gankyrin mRNA与蛋白表达水平均高于HepG2细胞（P＜0.05）。②HepG2与SMMC-7721细胞系中Ad-HBX-GFP组Gankyrin mRNA与蛋白表达水平均高于绿色荧光蛋白腺病毒表达载体（Ad-GFP）组（P＜0.01）。③HepG2.2.15细胞中Si-HBX组Gankyrin mRNA与蛋白表达水平均低于Si-阴性对照（Si-NC）组（P＜0.01）。④在HepG2与SMMC-7721细胞系中过表达HBX后,Ad-HBX-GFP组β-catenin的蛋白表达水平均高于Ad-GFP组（P＜0.01）。⑤在HepG2与SMMC-7721细胞系中过表达β-catenin后,β-catenin腺病毒表达载体（Ad-β-catenin-GFP）组Gankyrin的蛋白表达水平均高于Ad-GFP组（P＜0.01）。⑥在HepG2与SMMC-7721细胞系中沉默β-catenin后,过表达HBX基因,Si-β-catenin腺病毒表达载体（Ad-si-β-catenin-RFP）组与红色荧光蛋白腺病毒表达载体（Ad-RFP）组相比,Gankyrin的蛋白表达水平未见升高（P＜0.01）。结论：HBX-Wnt/β-catenin-Gankyrin信号转导通路存在与肝癌细胞中,抑制β-catenin的功能可阻遏HBX对Gankyrin的上调作用,本结果将可能为肝癌的治疗提供新的思路。     

褪黑素对过氧化氢损伤的心肌细胞自身NO生成的影响

目的：探讨氧化损伤时褪黑素（Melatonin,MT)对心肌细胞自身NO生成的影响以及NO水平与其它氧化指标的关系。方法：分高培养原代心肌细胞，分5组：对照组，H2O2处理组，MT组，MT干预组和N－硝基－L－精氨酸（L-nitro-arginine methylester,L-NAME)干预组；用外源性H2O2造成氧化损伤，用生化法检测培养液中乳酸脱氢酶（LDH）浓度，硫代巴比妥酸法检测细胞中丙二醛（MDA）含量，用试剂盒检测细胞培养液中NO含量。结果：H2O2处理组和L－NAME干预组中NO的含量，LDH含量和MDA含量与对照组相比，均差异显（P＜0．01），只是H2O2处理组中三项均比对照组明显增高，而L－NAME干预组中NO含量比对照组明显低；MT组中这三项指标没有明显的变化。MT干预组中虽然与对照组相比也升高，但低于H2O2处理组（P＜0．01）。MT干预组与L－NAME干预组相比其LDH含量和MDA含量也较低（P＜0．01），与H2O2组相比，加入MT干预后，培养液中NO和LDH及MDA含量明显降低（P＜0．01）。，结论：心肌细胞在受H2O2损伤时，心肌细胞自身产生的NO增加，并且在一定程度上参与了对心肌细胞的氧化损伤；MT不仅能抑制LDH和MDA的升高，还能抑制NO的产生。     

5－氮杂－2′－脱氧胞苷对肺癌 SPC －A －1细胞RAR －β基因去甲基化及其表达的作用

目的：观察5－氮杂－2′－脱氧胞苷（5－Aza－CdR）对肺腺癌SPC－A－1细胞视黄酸受体β（ RAR－β）基因甲基化状态的影响,探讨不同浓度5－Aza－CdR对RAR－β基因表达缺陷的调控机制。方法将体外培养的人肺腺癌SPC－A－1细胞株分为4组,分别是A组（未加药）及B、C、D组（分别加入3、6、12μmol／L的5－Aza－CdR）。采用甲基化特异性PCR（MSP）方法检测4组细胞RAR－β基因甲基化状态的差异,RT－PCR法检测4组细胞RAR－βmRNA表达水平的差异,Western blot检测4组细胞RAR－β蛋白表达水平的差异,流式细胞仪技术检测各组细胞周期及细胞凋亡率的差异。结果 A组标本检测出RAR－β基因为甲基化状态,而B、C、D组均检测出RAR－β为去甲基化状态。肺腺癌SPC－A－1细胞RAR－βmRNA及RAR－β蛋白表达水平低下,经5－Aza－CdR处理后,其表达水平明显增强（P＜0．01）。5－Aza－CdR处理后（B、C、D组）的细胞凋亡率均较A组明显提高,差异有统计学意义（ P＜0．01）。结论肺癌RAR－β基因因甲基化出现表达缺陷,5－Aza－CdR具有明显去甲基化作用,可诱导RAR－β基因重新激活表达,促进肺癌细胞凋亡及抑制癌细胞增殖,从而发挥抗癌作用。     

美喘清、地塞米松对小儿支气管哮喘β受体和cAMP的影响

目的　研究美喘清、地塞米松对支气管哮喘小儿β受体和cAMP水平的影响。方法　采用放射配基受体结合分析方法，对支气管哮喘急性发作期患儿应用单一口服美喘清以及美瑞清和静注地塞米松联合治疗前后外周血淋巴细胞β－AR和血浆cAMP水平及临床治疗效果进行了观察。结果美喘清组：β－AR治疗前为3911．9±306．9site／cell，治疗后为2672．8±343．7site／cell（P＜0．01）；cAMP治疗前为18．9±1．gpm／mL，治疗后为19.6±1．5pm／mL（P＞0．05）；美喘清＋地塞米松组：β－AR治疗前为4018．8±344．5site／cell，治疗后为2227．9±208．2site／cell（P＜0．01）；cAMP治疗前为17．4±1．5pm／mL，治疗后为21．3±2．1pm／mL（P＞0．05）。结论美喘清在体内激活了β-AR，对哮喘急性发作期治疗有效，而联合用药时，静注地塞米松未见明显加强美喘清激活β-AR的作用。     

维拉帕米阻断TGF-β1诱导的α1(Ⅰ)胶原基因启动子的激活

目的：探讨维拉帕米对α1(Ⅰ）胶原基因启动子活性的影响。方法：将人α1(Ⅰ）胶原基因启动子重组体pCOLH1.5、pCOLH2.5转染至大鼠肾小球纱膜细胞，分别经不同浓度的转化生长因子β1(TGF-β1）和纺拉帕米处理后，ELISA法测定细胞CAT表达水平。结果：TGF－β1作用后，pCOLH1.5和pCOLH2.5的CAT活性明显增加，分别为对照组的1.498和1.551倍；维拉帕米对两上重组体的CAT表达均有抑制作用，与单纯TGF－β1组比较，维拉帕米加TGF－β1组pCOLH2.5的CAT活性明显降低（维拉帕米低浓度组P＜0.05，高浓度组P＜0.01）。结论：维拉帕米不仅直接抑制启动子的活性，而且还部分阻断TGF－β1诱导的α1(Ⅰ）胶原基因启动子激活。     

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.     

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.     

Scientific principles and rigorous processes should be followed in developing clinical guidelines for therapeutic interventions of integrative medicine

A clinical guideline for the therapeutic interventions of integrative medicine may be defined as a written document which states a series of recommendations on therapeutic interventions of integrative medicine for a special disease or condition. The guideline may provide assistance to medical professionals in making clinical decisions aimed at improving the clinical outcome of patients and reducing the costs of medical care（~＇4~. Recommendations issued by a guideline should be based on the best available evidence in both Western and Chinese medicine. For fulfilling this purpose, the development of clinical guidelines for therapeutic interventions in the field of integrative medicine should follow scientific principles and undergo a rigorous processes.