反义寡核苷酸对淋巴瘤细胞Livin基因表达及细胞凋亡的影响

目的 分析凋亡抑制蛋白Livin基因的反义寡核苷酸(ASODN)对人淋巴瘤细胞株(Raji)Livin mR-NA表达的抑制作用及其对细胞凋亡的影响.方法 合成Livin特异性全硫代修饰的ASODN及其对照错义寡核苷酸(MSODN),通过脂质体转染Raji细胞,MTT法检测细胞的增殖抑制率;RT-PCR法检测转染前后Livin mRNA表达水平的变化;Hoechst染色观察细胞凋亡的形态学改变;流式细胞仅检测细胞的凋亡率.结果 Livin ASODN能显著抑制Raji细胞的增殖(P<0.01),并呈现一定的剂量效应关系.0.6μmol/L的A-SODN作用Raji细胞48h后,Livin mRNA的表达水平明显下调,细胞出现凋亡的形态学改变,细胞凋亡率为(37.54±2.65)%,与对照组比较,差异有统计学意义(P<0.01).结论 Livin ASODN能有效降低Livin mRNA表达水平,抑制Raji细胞增殖,并增强细胞凋亡的敏感性,有望成为淋巴瘤基因治疗的分子靶向.     

bcl-2 RNAi表达载体的构建、鉴定及其对Raji细胞的干扰效果

目的 构建bcl-2特异性RNAi真核细胞质粒表达载体,并研究其对B细胞淋巴瘤Raji细胞的干扰效果.方法 根据GenBank提供的bcl-2 cDNA序列,设计并合成两对短发夹结构的互补DNA序列,经退火形成互补双链,克隆至载体pGenensil-1构建重组质粒,予以酶切和DNA测序鉴定.脂质体介导重组质粒转染Raji细胞,采用RT-PCR法观察重组质粒对bcl-2 mRNA表达的影响,流式细胞仪测定细胞凋亡.结果 酶切及测序鉴定表明,成功构建bcl-2 shRNA的质粒表达载体pGenesil-1-bcl-2-544和pGenesil-1-bci-2-1009.转染Raji细胞48 h后,RT-PCR结果显示Raji细胞中bcl-2 mRNA表达分别下调了(31.95±3.02)%和(47.57±2.88)%,与空白对照组相比,差异有统计学意义(P<0.05);流式细胞仪检测结果显示Raji细胞凋亡率分别为(14.25±0.84)%和(11.96±0.79)%,明显高于对照组,差异有统计学意义(P<0.05).结论 成功构建bcl-2特异性RNAi真核细胞质粒表达载体,它能有效沉默bcl-2基因在B细胞淋巴瘤中的表达并促进B细胞淋巴瘤Raji细胞凋亡.     

siRNA沉默Pokemon基因抑制人非霍奇金淋巴瘤Raji细胞的增殖

目的采用siRNA干扰技术沉默人非霍奇金淋巴瘤Raji细胞中Pokemon基因,观察Raji细胞增殖活性的变化,并探讨其可能的分子机制。方法构建靶向Pokemon基因的siRNA重组慢病毒载体并转染Raji细胞。采用实时定量PCR法和Western blotting法检测Raji细胞Pokemon基因的沉默效果,在Raji细胞中沉默Pokemon基因的表达后采用实时定量PCR法和Western blotting法检测bcl-6和突变型p53表达水平的变化;采用流式细胞术检测Pokemon基因沉默后Raji细胞凋亡的情况。结果利用Pokemon靶向siRNA重组慢病毒载体感染Raji细胞有效沉默Raji细胞Pokemon基因表达(P<0.05)。沉默Pokemon基因后,bcl-6和突变型p53基因和蛋白表达均显著降低(P<0.05),细胞凋亡率增高(P<0.05)。结论 siRNA沉默Pokemon基因有效降低了人非霍奇金淋巴瘤Raji细胞的bcl-6和突变型p53基因和蛋白表达,促进细胞凋亡,抑制其增殖。Pokemon基因有望成为非霍奇金淋巴瘤治疗的新靶点。     

自噬基因Beclin 1 shRNA表达质粒的构建及其对宫颈癌细胞HeLa生长的作用

目的 构建自噬基因Beclin 1小发夹RNA(short hairpin RNA,shRNA)表达质粒,探讨Beclin 1抑制后对宫颈癌HeLa细胞生长的作用.方法 根据人Beclin 1 mRNA编码序列,设计RNA干扰靶点,构建Beclin 1 shRNA表达质粒,脂质体法转染人宫颈癌HeLa细胞,通过荧光定量PCR(RT-PCR)和Western blot检测其对HeLa细胞自噬基因Beclin 1 mRNA及蛋白表达的影响.MTT法分析其对细胞增殖、流式细胞仪检测其对细胞周期和细胞凋亡的影响.结果 构建的shRNA表达载体可以使HeLa细胞中自噬基因Beclin 1的mRNA及其蛋白含量降低,转染质粒的细胞生长增殖速度加快,凋亡率降低.结论 成功构建了针对自噬基因Beclin 1的shRNA表达载体,通过转染HeLa细胞,可有效抑制细胞中Beclin 1的表达,并促进细胞生长,抑制凋亡.     

苦参碱诱导Raji细胞凋亡及其对Bcl-2表达的影响

目的:观察苦参碱(Matrine,Mat)对体外人淋巴瘤Raij细胞增殖及细胞凋亡的影响,探讨 Mat 诱导 Raji 细胞凋亡的作用机制.方法:四甲基偶氮唑蓝(MTT)比色法观察Mat对Raji细胞增殖的影响;流式细胞术检测凋亡率;免疫细胞化学技术检测 Mat对Raji 细胞株 Bcl-2 蛋白表达的影响;RT-PCR法检测Bcl-2 mRNA表达.结果:不同浓度的 Mat 对 Raji 细胞均有一定的增殖抑制作用,且呈量效和时效关系;Mat能明显诱导Raji细胞凋亡;免疫细胞化学结果显示 Bcl-2 在实验组中的表达显著低于对照组.RT-PCR 显示 Bcl-2 mRNA 表达明显减弱,且随剂量增加越明显.结论:Mat 能抑制人淋巴瘤 Raji 细胞的增殖,促进细胞凋亡,其作用机制可能与下调 Bcl-2 表达有关.     

人pre-miR-15a真核表达载体的构建及其对Raji细胞生长的抑制作用

目的 构建人pre-miR-15a真核表达载体,并研究其对Raii细胞的生长抑制作用.方法 将pre-miR-15a与目标载体(PGCSIL-GFP)定向连接,转化细菌感受念细胞,PCR鉴定阳性克隆,并测序.利用脂质体法将该载体转染Raji细胞,实验分为空白对照组、阴性对照质粒组和pre-miR-15a组(n=5).RT-PCR检测Bel-2 mRNA表达,间接免疫荧光法检测Bcl-2蛋白表达,台盼蓝细胞计数法检测细胞增殖活性.结果 PCR 阳性克隆鉴定及测序结果 均与日的序列一致.倒置荧光显微镜下见绿色荧光表达;RT-PCR法示各组间Bcl-2 mRNA表达差异无显著性(P>0.05);间接免疫荧光法示pre-miR-15a组的Bcl-2蛋白表达量较空白对照组和阴性对照质粒组显著降低(P<0.05);台盼蓝拒染法示pre-miR-15a组Raji细胞生长受抑.结论 本实验成功构建了per-miR-15a真核表达载体,且pre-miR-15a可以抑制Raji细胞生长.     

靶向bcl-2基因的shRNA对肝星状细胞凋亡的影响

目的 研究短发夹RNA(shRNA)重组载体介导抑制大鼠肝星状细胞(HSCs)bcl-2基因的表达,观察其对HSCs凋亡的影响.方法 重组质粒载体pGPU6-GFP-shRNA由脂质体介导转染HSC-T6细胞株.荧光定量PCR和Western blotting鉴定bcl-2表达;CCK-8法观察质粒转染对HSCa生长和增殖的影响;Hoechst 33258染色观察细胞形态学改变;流式细胞术及caspase-3和caspase-9酶活性检测分析转染后细胞凋亡情况.结果 pGPU6-GFP-shRNA能抑制bcl-2 mRNA和蛋白表达;转染后HSC-T6体外生长明显受到抑制;转染后72 h时细胞凋亡率为33.34%,caspase-3和caspase-9酶活性显著增高.结论 shRNA重组载体pGPU6-GFP-shRNA能有效地抑制HSC-T6中bcl-2的表达,抑制细胞生长并促进凋亡.实验为进一步探索肝纤维化基因治疗提供了实验依据.     

地西他滨对淋巴瘤细胞株Raji中P15INK4B基因的去甲基化作用

目的 探讨Burkitt淋巴瘤细胞系Raji中P15INK4B基因甲基化状态,地西他滨对Raji细胞P15INK4B基因去甲基化作用及生长增殖的生物学影响。方法 用不同浓度地西他滨作用淋巴瘤细胞株Raji,用台酚蓝拒染法研究药物对Raji细胞生长曲线的影响,采用流式细胞术检测细胞凋亡率,采用聚合酶链反应(RT PCR)检测P15INK4B表达,用甲基化特异PCR(MSP) 方法检测P15INK4B 基因的甲基化程度。结果 地西他滨对Raji细胞有生长抑制作用,作用后细胞凋亡增加,P15INK4B基因表达增加,地西他滨使其甲基化程度下降。结论 在Raji细胞中P15INK4B高度基因甲基化,并且表达下降,地西他滨通过去甲基化抑制淋巴瘤增殖。     

醋酸棉酚对Raji细胞增殖和凋亡的影响

目的 观察醋酸棉酚对人Burkitt淋巴瘤细胞株Raji细胞增殖和凋亡的影响.方法 采用台盼蓝染色和噻唑蓝(MTT)法观察醋酸棉酚对Raji细胞生长存活的影响,瑞氏染色法观察药物处理后细胞形态学变化,琼脂糖凝胶电泳和Annexin V-FITC标记流式细胞仪检测细胞凋亡,流式细胞仪分析细胞周期、检测细胞凋亡率及Bcl-2蛋白表达水平变化,比色法测定Raji细胞Caspase-3样蛋白酶活性.结果 5 μmol/L以上浓度醋酸棉酚能抑制Raji细胞生长增殖并诱导其凋亡,效应与药物浓度及给药时间呈正相关.醋酸棉酚作用后,Raji细胞被阻滞于G_0/G_1期.比色法显示Caspase-3样蛋白酶活化.流式细胞仪检测到Bcl-2蛋白表达下调,且与Caspase-3活化在时间上存在同步趋势.结论 醋酸棉酚能抑制aaji细胞增殖并诱导凋亡,其原因可能与调节细胞周期及下调Bcl-2蛋白表达有关.     

Cyclin D3正、反义表达质粒在淋巴瘤细胞系Raji中的转染与鉴定

目的 :构建人类cyclinD3正义和反义真核表达质粒 ,分别转染到淋巴瘤细胞系Raji细胞中 ,为探讨cyclinD3基因对淋巴瘤细胞生物学行为的影响提供研究基础。方法 :以Raji细胞总RNA为模板 ,通过RT -PCR获取cyclinD3全长cDNA ,克隆入 pGEM -T载体 ,再亚克隆到真核表达载体pcDNA3 .1中 ,构成含cyclinD3的表达质粒 pcD NA3 -cyclinD3。再运用DNA重组技术将cyclinD3基因反向克隆到pcDNA3 .1中 ,构建成cyclinD3反义表达质粒pcDNA3 -ascyclinD3。用脂质体介导的基因转染方法 ,将cyclinD3正、反义表达质粒和空质粒分别导入Raji细胞 ,经G418筛选建立稳定转染细胞株 ,用WesternBlot方法鉴定cyclinD3基因的表达。结果 :酶切鉴定和测序分析证实 ,实验成功构建了cyclinD3正、反义表达质粒。与转染cyclinD3正义表达质粒和空质粒的细胞相比 ,转染cyclinD3反义表达质粒的Raji细胞中cyclinD3表达水平下降。 结论 :正、反义cyclinD3在淋巴瘤细胞中的转染和表达 ,为进一步研究cyclinD3在淋巴瘤细胞增殖、细胞周期和细胞凋亡中的作用奠定了基础     

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.     

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.