虫草素对人骨肉瘤MG-63细胞凋亡的影响及其机制研究

目的 研究虫草素对人骨肉瘤MG-63细胞凋亡的影响及其机制。方法 采用CCK-8实验检测不同浓度虫草素作用于MG-63骨肉瘤细胞24h和48h后对癌细胞增殖的抑制作用;采用流式细胞术分析虫草素对MG-63细胞周期分布及凋亡的影响;采用Western blot法检测虫草素对MG-63细胞凋亡相关蛋白Bax、Bcl-2、cleaved caspase-9、cleaved caspase-3及NICD1、Hes1蛋白表达的影响。结果 虫草素对人骨肉瘤MG-63细胞有明显的增殖抑制作用,导致MG-63细胞周期阻滞于G₀/G₁期并诱导细胞凋亡;虫草素可上调Bax、cleaved caspase-9和cleaved caspase-3蛋白的表达并下调Bcl-2,NICD1和Hes1蛋白的表达。结论 虫草素通过抑制骨肉瘤细胞增殖,诱导细胞周期阻滞于G₀/G₁期并通过线粒体凋亡途径诱导MG-63细胞凋亡从而发挥抗骨肉瘤作用,可能骨肉瘤细胞中Notch信号通路活性下调有关。     

2-甲基-3-羟基蒽醌诱导人乳腺癌MCF-7细胞凋亡的机制

为探讨2-甲基-3-羟基蒽醌抗肿瘤作用及其机制,本研究采用锥虫蓝法检测细胞活力,流式细胞仪检测细胞周期变化、细胞凋亡率、线粒体膜电位及细胞内游离钙的变化,Western blot方法检测凋亡相关蛋白caspase-4、caspase-7、caspase-9、Bcl-2、Bax、JNK、细胞色素C的表达。结果发现：2-甲基-3-羟基蒽醌时间依赖性地抑制乳腺癌细胞的生长,升高细胞内游离钙含量,降低线粒体膜电位并诱导其凋亡;药物上调Bax并下调Bcl-2蛋白的表达;诱导caspase-4、caspase-7、caspase-9、calpain的活化及细胞色素C的释放。结果提示2-甲基-3-羟基蒽醌可能通过Ca²⁺/calpain/caspase-4途径诱导人乳腺癌MCF-7细胞凋亡。     

苦参碱通过线粒体通路诱导HepG2细胞凋亡的机制研究

目的探讨苦参碱通过线粒体通路诱导Hep G2发生凋亡的机制。方法分别采用MTT法、PI染色法、流式细胞术检测苦参碱对肝癌Hep G2细胞的增殖抑制、周期调控、诱导凋亡作用;采用Western Blot检测苦参碱诱导肝癌Hep G2细胞抑凋亡蛋白Bid、Bcl-2的表达量。结果苦参碱可抑制Hep G2细胞的增殖作用和诱导凋亡,呈时间和剂量依赖性,并可将Hep G2细胞周期阻滞在G0/G1期。苦参碱可引起线粒体膜电位崩溃,同时诱导Bid、Bcl-2表达下调,Bax表达上调。结论苦参碱通过调节细胞内Bid、Bcl-2和Bax蛋白的表达,经线粒体信号转导途径诱导人肝癌细胞系Hep G2发生凋亡。     

紫檀芪通过线粒体途径调控结直肠癌细胞增殖和凋亡

目的 探讨紫檀芪对结直肠癌细胞增殖和凋亡的作用及其机制。方法 使用梯度浓度的紫檀芪体外处理结直肠癌细胞,CCK-8法检测结直肠癌细胞增殖和活性,Hoechst 33258荧光染色法检测细胞凋亡,DCFH-DA荧光探针检测细胞内活性氧(reactive oxygen species, ROS)水平的变化,JC-1荧光探针检测细胞内线粒体膜电位的变化,Western blot法检测线粒体途径相关蛋白表达水平。结果 与对照组比较,紫檀芪对结直肠癌细胞增殖有抑制作用,且呈剂量和时间依赖性(P<0.05);Hoechst 33258染色和流式结果显示,紫檀芪促进结直肠癌细胞凋亡(P<0.05);紫檀芪诱导细胞内ROS产生、降低线粒体膜电位水平(P<0.05),增加Apaf-1、AIF、Bax、Cyt C、cleaved caspase-3和cleaved caspase-9的表达,降低Bcl-2、PCNA和survivin的蛋白水平(P<0.05)。结论 紫檀芪增加结直肠癌细胞内ROS水平、降低线粒体膜电位水平,通过线粒体途径抑制结直肠癌细胞增殖,诱导结直肠癌细胞凋亡。     

N-乙酰半胱氨酸萝卜硫素通过上调Bak和下调Bcl-2蛋白表达诱导前列腺癌细胞凋亡

目的探究N-乙酰半胱氨酸萝卜硫素(D,L-sulforaphane N-Acetyl-L-cysteine,SFN-NAC)诱导前列腺癌细胞凋亡的分子机制。方法将前列腺癌细胞DU145和PC-3分别培养在6孔板中,当细胞密度达到70%-80%时,分别随机分为对照组(不加SFN-NAC)和加药组(加入终浓度为30μmol/L的SFN-NAC),继续在37℃培养24 h。利用AnnexinⅤ异硫氰酸荧光素/碘化丙啶(FITC/PI)双染流式细胞术检测细胞凋亡程度;利用Western blot检测Bak、Bax、Bcl-2和cleaved Caspase-3蛋白表达水平。结果与对照组相比,加药组的细胞凋亡率增高(P<0.01)。加药组细胞Bak蛋白表达较对照组升高(P<0.05);Bax蛋白表达也有少量增加,但与对照组相比,无统计学差异(P>0.05);Bcl-2蛋白表达下降(P<0.05);Bax/Bcl-2比值增加(P<0.01);细胞cleaved Caspase-3表达水平升高(P<0.01)。结论 N-乙酰半胱氨酸萝卜硫素通过上调Bak、下调Bcl-2蛋白表达,活化Caspase-3,诱导前列腺癌细胞凋亡,说明N-乙酰半胱氨酸萝卜硫素通过线粒体途径发挥抗癌作用。     

姜黄油诱导人肝癌细胞凋亡及其机制

探讨姜黄油的抗肝癌作用及其作用机制,采用二甲基噻唑蓝比色法(MTT)检测肝癌细胞的活力,流式细胞仪检测细胞凋亡率以及线粒体膜电位的变化,Western blot方法检测凋亡相关蛋白Bax、Bcl-2、caspase-3、caspase-9以及细胞色素C的表达。结果发现,姜黄油能时间依赖性地抑制肝癌细胞的生长,降低线粒体膜电位并诱导其凋亡;升高细胞内Bax与Bcl-2的比值;诱导细胞色素C从线粒体中的释放并激活caspase-9和caspase-3。结果提示姜黄油可能通过线粒体途径诱导肝癌细胞的凋亡。     

α-茄碱通过ROS/线粒体途径促进鼻咽癌细胞凋亡的分子机制

目的：通过体外实验探讨α-茄碱对人鼻咽癌细胞CNE-2的作用及其潜在机制。方法：CCK-8法检测α-茄碱处理后CNE-2 细胞的活性;流式细胞术检测细胞凋亡;Real-time PCR和Western blot实验检测CNE-2细胞内Bax、Bcl-2等凋亡相关基因和蛋白的表达水平。Mito-SOX流式细胞术检测线粒体ROS水平的变化;透射电子显微镜观察细胞线粒体超微结构。结果：α-茄碱显著抑制细胞活性,诱导细胞凋亡,呈时间与剂量依赖性。与对照组相比,10 μmol/L α-茄碱作用于CNE-2细胞48 h后,细胞内Aif和Caspase-8的表达量显著增加（P ＜0.01）;Bcl-2表达和Bcl-2/Bax比值显著降低（P ＜0.05）。α-茄碱引起线粒体内ROS水平显著升高,透射电镜结果显示了CNE-2细胞中线粒体的损伤。结论：α-茄碱能抑制细胞增殖活性,引起ROS水平升高,进而导致线粒体损伤,最终通过线粒体途径诱导CNE-2细胞凋亡。     

双氢青蒿素诱导前列腺癌 PC-3 细胞凋亡及其机制研究

目的 研究双氢青蒿素对前列腺癌 PC-3 细胞的凋亡诱导作用,并探讨其可能的机制。方法 MTT 法测定不同浓度双氢青蒿素对 PC-3 细胞增殖的影响,流式细胞仪 (FCM)、透射电镜 (TEM) 观察双氢青蒿素对 PC-3 细胞的凋亡诱导作用,免疫组化 (SP) 检测双氢青蒿素对 PC-3 细胞内 Bax、Bcl-2 蛋白阳性表达的影响。结果 双氢青蒿素对 PC-3 细胞有明显增殖抑制效应,能诱导 PC-3 细胞凋亡且具有浓度-时间依赖性,导致 PC-3 细胞线粒体肿胀、核碎裂、凋亡小体形成;随着双氢青蒿素浓度的增加,Bcl-2 蛋白阳性表达降低,而 Bax 蛋白阳性表达增加。结论 双氢青蒿素可能通过上调 Bax,下调 Bcl-2 蛋白表达诱导前列腺癌 PC-3 细胞凋亡。     

鱼藤素对乳腺癌细胞MDA-MB-231线粒体通透性转换孔的作用

目的研究鱼藤素对乳腺癌细胞MDA-MB-231线粒体通透性转换孔的作用。方法MTT法检测鱼藤素对MDA-MB-231细胞的增殖抑制作用;AnnexinⅤ-FITC/PI双染流式细胞术检测细胞凋亡率;罗丹明123单染法观察线粒体膜电位变化;蛋白免疫印迹法检测细胞质中Cytc的表达;分光光度法检测caspase-3蛋白活性;Fluo-3/AM荧光指示剂检测细胞内钙离子浓度变化;RT-PCR和蛋白免疫印迹法检测Bcl-2和Bax mRNA及其蛋白表达。结果鱼藤素对MDA-MB-231细胞增殖具有明显的抑制作用,呈时效和量效依赖关系(P0.05);鱼藤素处理细胞后,细胞线粒体膜电位降低,细胞质内Cytc蛋白表达水平升高,caspase-3活性明显提高,与未处理组相比差异具有统计学意义(P0.01)。流式细胞术检测显示细胞凋亡率和细胞内钙离子浓度随鱼藤素浓度的增加而增多;RT-PCR和蛋白印迹检测结果发现鱼藤素能使Bcl-2 mRNA和蛋白表达减少,而Bax表达增加。结论鱼藤素对乳腺癌MDA-MB-231细胞的增殖抑制、诱导凋亡作用可能与细胞内钙超载,Bcl-2和Bax表达改变影响线粒体通透性转换孔开放,诱导线粒体膜电位降低及Cytc释放有关。     

小檗碱通过ROS及caspase通路对肝癌HepG2细胞凋亡的作用

目的 探究小檗碱(BBR)对肝癌HepG2细胞的增殖抑制及作用机制.方法 采用噻唑蓝(MTT)法检测不同浓度(0、10、20、40、80 μmol/L) BBR对肝癌HepG2细胞的抑制作用;采用Hoechst 33342染色观察处理后HepG2细胞形态学变化;用流式细胞仪检测HepG2细胞凋亡率;间接荧光标记法检测BBR对细胞内活性氧簇(ROS)水平的影响;JC-1染色法检测细胞内线粒体膜电位的变化;应用Western blot法检测凋亡相关蛋白Bax、caspase-3、Cleaved-caspase-3的表达变化.结果 MTT法显示BBR可呈剂量、时间依赖性抑制肝癌HepG2细胞活力(P＜0.05);Hoechst 33342染色发现细胞形态发生明显变化,0μmol/L BBR处理的细胞呈淡蓝色,处理组细胞核固缩,形成凋亡小体,细胞凋亡率与药物浓度呈正相关(P＜0.05).流式细胞术发现BBR能使细胞内ROS升高,线粒体膜电位水平下降.Western blot法显示随着药物浓度升高,Bax、Cleaved-caspase-3、cleaved-聚腺苷二磷酸核糖聚合酶表达增强,Bcl-2蛋白表达降低,凋亡相关蛋白caspase-3表达下降.结论 BBR在体外可能通过增加ROS、提高促凋亡蛋白Bax并激活caspase-3表达抑制肝癌HepG2细胞增殖并诱导细胞凋亡.     

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.