可溶性AZURIN分子重组子构建对人骨肉瘤U2OS细胞的杀伤和诱导凋亡

目的:研究可溶性细菌氧化还原蛋白AZURIN对人骨肉瘤U2OS细胞的生长抑制效应及凋亡诱导作用.方法:用PCR方法由假单胞菌染色体DNA扩增AZURIN基因片段,将其融合插入原核表达载体pQE30中,并在M15大肠杆菌表达可溶性的AZURIN蛋白.该蛋白经纯化和复性后,诱导人骨肉瘤U2OS细胞,通过MTT法、Hoechest 33258荧光染色法及倒置显微镜观察凋亡细胞;流式细胞仪分析细胞周期、凋亡率、线粒体膜电位变化和DNA断裂实验检测细胞增殖和细胞凋亡.结果:AZURIN的蛋白纯度达99.1%以上.用50～200 mg/L的蛋白诱导人骨肉瘤U2OS细胞12 h以上细胞的生长和增殖被显著抑制,并且观察到典型的凋亡细胞、凋亡小体、凋亡峰及DNA的片段化等凋亡的特征性变化,线粒体跨膜电位随着AZURIN作用的时间和浓度的增加而逐渐降低,对照组和50 mg/L AZURIN、100 mg/L AZURIN、200 mg/L AZURIN组细胞线粒体跨膜电位分别降低(7.76±0.47)%、(9.01±0.87)%、(24.13±2.12)%、(37.48±3.59)%(P<0.01).细胞的凋亡率也呈剂量依赖和时间依赖关系,作用48 h时凋亡率达峰值(35.8±3.78)%.结论:利用大肠杆菌制备的小分子蛋白AZURIN对人骨肉瘤U2OS细胞有明显的增殖抑制效应和凋亡诱导作用,线粒体途径可能是AZURIN诱导细胞凋亡的机制之一.     

胶质瘤细胞系对γ射线辐射敏感性实验研究

目的:探讨γ射线杀伤胶质瘤细胞的剂量-效应关系、时间-效应关系和杀伤作用的机制,比较两种不同种属来源的胶质瘤细胞系对γ射线照射的敏感性.方法:以胶质瘤细胞系C6和SHG-44为实验对象,应用60Co放射源产生的γ射线行单次照射,设对照组、4 Gy,8Gy,16 Gy,32 Gy和64Gy组.照射后光镜下观察细胞形态改变并进行细胞凋亡检测,流式细胞术测定照射后不同时间和不同剂量照射时瘤细胞坏死、凋亡和存活的比例.结果:照射后24 h,32 Gy和64Gy组两种细胞均可见多数细胞出现明显的类似凋亡的细胞形态学改变,Hoechst33342染色可见细胞核呈致密浓染.随着照射后观察时间的延长,两种细胞的凋亡率逐渐提高,6 h后各时间点细胞凋亡率均高于对照组,差异有非常显著性(P＜0.01).两种细胞在照射后48 h凋亡率与48 h前各时间点相比明显增高,差异有非常显著性(P＜0.01).48 h后各个时间点的凋亡率与48 h相比,无显著差异(P＞0.05).随着照射剂量的增加,照后48 h两种细胞凋亡率均逐渐增高,与对照组相比差异有非常显著性(P＜0.01),但32Gy以上剂量照射后细胞凋亡率趋于稳定.在各种照射剂量下两种胶质瘤细胞坏死率与对照组相比均无显著差异(P＞0.05).SHG-44细胞与C6细胞相比,在吸收剂量为4 Gy、32 Gy和64Gy时凋亡率较高,有非常显著差异(P＜0.01).结论:γ射线照射可以诱导体外培养的C6和SHG-44胶质瘤细胞发生凋亡.细胞凋亡高峰出现在照射后48 h左右.随着剂量的增大,凋亡的比例增加,但凋亡率提高至一定的水平(50%左右),将照射剂量继续增加则不能引起凋亡率的明显提高.与C6细胞相比,SHG44细胞对γ射线更加敏感.     

灵芝多糖对γ射线照射后NIH3T3成纤维细胞细胞周期及细胞增殖的影响

目的探讨γ射线对成纤维细胞细胞周期及增殖的影响,以及从细胞水平探讨灵芝多糖的抗辐射作用。方法加不同剂量灵芝多糖的NIH3T3细胞在60Coγ射线15Gy照射12h后收集,以流式细胞仪检测细胞各周期百分率及凋亡率。结果照射剂量为15Gy阳性对照细胞组G0～G1期细胞数占细胞总数的52.76%,与阴性对照组(90.09%)对比,P＜0.01;加药剂量50mg/L组、100mg/L组、150mg/L组的G0～G1期细胞数分别占细胞总数66.17%、71.73%、76.1%,与阳性对照组对比,50mg/L组为P＜0.05,100mg/L组和150mg/L组P＜0.01有很显著性差异。阴性对照组细胞凋亡率0.4%,照射后各组细胞凋亡率均为0。结论15Gy60Coγ射线照射促进NIH3T3细胞增殖,0～15Gy照射后12h内不能促进细胞凋亡;加不同剂量灵芝多糖后均对细胞增殖有抑制作用,从而使受损细胞不能增殖,实现抗辐射作用。     

沉默MagT1影响大鼠心肌细胞内Mg2+水平和细胞凋亡的研究

目的 使用siRNA沉默大鼠心肌细胞的镁离子转运蛋白1(MagT1),检测细胞内Mg2+浓度变化和细胞凋亡情况.方法 设计并合成MagT1 siRNA序列,转入原代培养大鼠心肌细胞沉默MagT1,使用反转录PCR和Western blot检测MagT1mRNA和蛋白表达情况,荧光显微镜检测细胞内Mg2+浓度变化情况,流式细胞法检测细胞凋亡情况.结果 相比阴性siRNA组,MagT1 siRNA转染大鼠心肌细胞48 h,MagT1 mRNA的沉默效率为51.83％ (P＜0.05),MagT1蛋白的沉默效率为56.75％(P＜0.05),胞内Mg2+浓度降低29.13％ (P＜0.05),细胞凋亡率为31.18％ (P＜0.01);转染60 h,MagT1 mRNA的沉默效率为86.91％ (P＜0.01),MagT1蛋白质的沉默效率为83.85％ (P＜0.01),细胞内Mg2+浓度降低41.32％ (P＜0.01),细胞凋亡率为40.61％ (P＜0.01).结论 MagT1被显著沉默后,细胞内Mg2+浓度显著降低,细胞凋亡显著提高,细胞生命活动受到较大影响.     

X射线对BGC-803胃癌细胞凋亡及Survivin蛋白表达的影响

目的分析X射线对BGC-803胃癌细胞凋亡及Survivin蛋白表达的影响。方法将BGC-803胃癌细胞分为对照组、实验组;实验组分别给予1、2、5 Gy X射线照射。流式细胞仪检测各组细胞凋亡及Survivin蛋白表达情况。结果随着X射线剂量增大,BGC-803细胞凋亡率增大;在2 Gy、5 Gy组,随X射线照射时间延长,细胞凋亡率增大,而1 Gy组无明显改变。大剂量(5 Gy)X射线引起BGC-803胃癌细胞Survivin蛋白表达增加,而小剂量X射线(1 Gy、2 Gy组)对BGC-803胃癌细胞Survivin蛋白表达无显著影响。结论 X线辐射引起BGC-803胃癌细胞凋亡具有辐射剂量、时间依赖性;大剂量X射线照射使Survivin蛋白表达增加,可能与细胞周期、DNA同源重组修复有关。     

片仔癀对人骨肉瘤细胞U2OS细胞周期的影响

目的:探讨片仔癀对体外培养的人骨肉瘤U2OS细胞周期的作用及机制.方法:0.8、1.2、1.6mg/ml片仔癀干预U2OS细胞24、48、72h后,MTT检测片仔癀对U2OS细胞的生长抑制作用;片仔癀干预U2OS细胞48h后流式细胞术分析细胞周期的变化;Western blot检测Survivin蛋白的表达.结果:与对照组比较,不同浓度的片仔癀作用24、48、72h后,不同程度的抑制了U2OS细胞增殖,且呈时间和剂量依赖性(P＜0.05);片仔癀干预U2OS细胞48h后细胞发生G2/M阻滞,Survivin蛋白表达较对照组明显降低(P＜0.05).结论:片仔癀可抑制骨肉瘤U2OS细胞的生长,诱导U2OS细胞发生G2/M周期阻滞,周期阻滞的发生与Survivin蛋白表达减少有关.     

PDTC影响骨肉瘤U-2 OS细胞凋亡的研究

目的:观察在NF-KB抑制剂PDTC诱导骨肉瘤细胞发生凋亡的过程中,NF-KB活性抑制后,Livin和aspase-3表达水平变化.方法:以不同浓度(0、50、100、200、400 μmol/L)PDTC作用于骨肉瘤U-2 OS细胞不同时间(24小时,48小时)后,采用MTT法和流式细胞仪测定U-2 OS细胞凋亡,采用Western Blot检测U-2 OS细胞Livin和caspase-3蛋白表达.结果:①不同浓度PDTC作用24小时、48小时后对U-2 OS细胞生长有明显抑制作用,并呈时间、剂量依赖性.②50、100、200和400μmol/L PDTC作用24小时后的凋亡率与对照组相比差异有显著性,且各浓度组之间差异均有显著性(P＜0.01).③PDTC作用于U-2 OS细胞后可降低Livin蛋白的表达,增加caspase-3蛋白表达,呈现剂量依赖性.结论:PDTC可明显抑制人骨肉瘤U-2 OS细胞,并使细胞周期受到阻滞,进一步诱导肿瘤细胞凋亡,抑制细胞与诱导凋亡呈剂量依赖关系.     

粗江蓠多糖对辐射损伤小鼠免疫细胞周期的影响

目的 研究粗江蓠多糖(GHPS)对辐射损伤小鼠胸腺细胞周期的影响.方法 采用60Coγ射线2Gy全身照射小鼠制造辐射损伤模型,通过流式细胞术(FCM)检测不同剂量(10mg/kg,20mg/kg,40mg/kg)的GHPS对辐射损伤小鼠胸腺细胞周期的影响.结果辐射对照组小鼠接受60Co γ射线2Gy照射后,胸腺细胞G0/G1期细胞百分数升高(P<0.01),S期和G2/M期细胞百分数下降(P<0.01).经腹腔注射(10,20,40)mg/kg GHPS,再接受γ射线2Gy照射后,小鼠胸腺细胞G0/G1期百分数明显低于辐射对照组(P<0.01),S期和G2/M期细胞百分数明显高于辐射对照组(P<0.01).并呈剂量依赖性.结论 GHPS可以缓解辐射所致的小鼠胸腺细胞G0/G1期阻滞,使S期和G2/M期细胞百分数增加.     

Src激酶在骨肉瘤U2OS细胞失巢凋亡中的作用

【摘要】 目的 探讨Src激酶在骨肉瘤U2OS细胞失巢凋亡中的作用。方法 采用超低黏附板构建骨肉瘤U2OS细胞失巢凋亡抵抗模型,Western blot检测骨肉瘤U2OS细胞和失巢凋亡抵抗细胞中Src激酶蛋白的水平。加入Src激酶抑制剂,Western blot检测U2OS细胞中Src激酶蛋白的水平变化,并利用TUNEL染色和流式细胞双染技术检测细胞凋亡率。结果 成功构建骨肉瘤失巢凋亡抵抗模型细胞（U2OSar）,在骨肉瘤失巢凋亡过程中,p Src的蛋白水平逐渐升高;与骨肉瘤U2OS细胞相比,U2OSar细胞中,p Src/Src蛋白灰度比值023上升至091（P＜005）;加入Src激酶抑制剂PP2后, 骨肉瘤U2OS细胞的p Src的蛋白水平明显降低,凋亡率从4666%下降至2832%（P＜005）。结论 Src激酶参与调控骨肉瘤失巢凋亡,抑制肿瘤细胞凋亡,促进其失巢凋亡抵抗。     

电离辐射致小鼠骨髓基质细胞凋亡的研究

目的探讨电离辐射致骨髓基质细胞(BMSC)凋亡的规律。方法采用流式细胞仪、电镜技术观察不同剂量6OCoγ射线照射后BMSC的凋亡变化。结果经50Gy剂量照射后,BMSC木见有明显的形态学改变,而经100和200Gy照射后 BMSC出现了明显的凋亡改变。流式细胞检测显示,经50Gy剂量照射后,BMSC的凋亡率与正常对照相比变化不明显(P＞0.05);100Gy照射后BMSC的凋亡率出砚了明显改变(P＞0.01),而且这种变化始于照射后4h,至照后24h到达高峰,随后凋亡率慢慢下降;200Gy照射组的凋亡率略高于100Gy组,但差异不明显(P＞0.05)。结论体外培养的BMSC受一定剂量的6OCoγ射线照射后可发生凋亡,并且有较强的辐射抗性。     

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.     

APPLICATION OF LORNOXICAM TO PATIENT-CONTROLLED ANALGESIA IN PATIENTS UNDERGOING ABDOMINAL SURGERIES

FOR anesthesiologis s ,treatingpostoperativepainhas alwaysbeen a problem.Althoughopioidshave been provedtobe effective,theirsideeffectscouldnotbeignored.With thedevelopmentofscienceand pharmacology,many drugs with aspectsof satisfactoryanalgesicefficacyand couldbe welltoleratedby patientshave been developed.And lornoxicamisone of them, which isa non-steroidalanti-inflammatorydrug (NSAID ), with analgesic, anti-infl-ammatory,andantipyreticproperties.Itseliminationhalf-time(3 to 5 hours) isle…     

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.     

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.