TSLC1基因甲基化与宫颈癌HeLa细胞凋亡相关性的研究

目的研究天花粉蛋白诱导宫颈癌HeLa细胞凋亡过程中肿瘤抑制基因TSLC1的甲基化的变化情况,并探讨其在宫颈癌的发生发展中的作用及细胞凋亡和抑癌基因甲基化的相关性,寻找新型的去甲基化药物。方法应用MTT法检测对HeLa细胞增殖抑制的影响;流式细胞仪分析天花粉蛋白对宫颈癌HeLa细胞诱导凋亡作用;甲基化特异性PCR技术(MSP)检测天花粉蛋白处理前、后宫颈癌HeLa细胞中TSLC1基因启动子区CpG岛的甲基化的变化情况。结果宫颈癌HeLa细胞中TSLC1基因启动子区CpG岛是呈高度的甲基化状态,经天花粉蛋白处理后,HeLa细胞生长明显受抑,流式细胞仪分析出有特征性的亚二倍体凋亡峰,TSLC1基因启动子区CpG岛无异常甲基化表现。结论天花粉蛋白诱导宫颈癌HeLa细胞凋亡过程中对TSLC1基因有明显的去甲基化作用,可能是新型的甲基化抑制剂,肿瘤细胞凋亡与抑癌基因的甲基化失活之间可能存在某些相关性。并且TSLC1基因甲基化的检测有可能成为宫颈癌的早期诊断和指导临床治疗的又一指标。     

天花粉蛋白诱导宫颈癌HeLa细胞凋亡及APC基因去甲基化

目的:研究天花粉蛋白(TCS)抗宫颈癌作用机制。方法:MTT还原法检测TCS对宫颈癌HeLa细胞的抑制作用,流式细胞仪检测TCS对HeLa细胞周期及凋亡率的影响,MSP检测TCS作用前后APC基因启动子甲基化状态。结果:TCS对HeLa细胞的生长具有明显的抑制作用,呈剂量依赖性;TCS可以将HeLa细胞阻滞于S期;TCS诱导HeLa细胞发生凋亡,有剂量依赖性;80 mg/LTCS处理HeLa细胞48 h后,APC基因启动子出现去甲基化。结论:TCS对HeLa细胞有明显抑制作用,抑制机理与将HeLa细胞阻滞于S期、诱导细胞凋亡及APC基因启动子出现去甲基化有关,TCS有良好的抗宫颈癌临床应用前景。     

天花粉蛋白对宫颈癌HeLa细胞凋亡及Bax、Bcl-2基因表达的影响

目的:研究天花粉蛋白对人宫颈癌HeLa细胞的诱导凋亡作用并探讨其对凋亡相关基因Bax和Bcl-2表达的影响。方法:不同浓度天花粉蛋白分别处理Hela细胞24~72h后,MTT法检测细胞的生长活性,显微镜观察细胞形态,流式细胞仪检测细胞周期,WesternBlot检测凋亡相关蛋白Bax,Bcl-2的表达。结果:天花粉蛋白对HeLa细胞的生长具有抑制作用,显微镜下观察到细胞圆缩,染色体凝聚等凋亡细胞的形态学改变,流式细胞仪直方图上亦出现特征性的亚二倍体峰,随着天花粉蛋白浓度的增加,Bax基因的表达增加而Bcl-2的表达减少。结论:天花粉蛋白可诱导宫颈癌HeLa细胞发生凋亡,Bcl-2蛋白的减少与Bax蛋白表达增多可能是HeLa细胞凋亡的机制之一。     

重组天花粉蛋白体外诱导宫颈癌 HeLa 细胞 p27 基因去甲基化的研究

目的 研究重组天花粉蛋白 (recombinant trichosanthin, rTCS) 对宫颈癌 HeLa 细胞中 p27 基因甲基化状态和基因表达的影响。方法 应用不同质量浓度的 rTCS (20、40、80 μg/mL) 处理体外培养的宫颈癌 HeLa 细胞后,MSP 法检测用药前后细胞中 p27 基因的甲基化状态,RealTime PCR 法检测用药前后细胞中 p27 和 DNA 甲基转移酶1 (DNMT1) mRNA 水平的变化,Westernblotting 法检测用药前后细胞中 p27 蛋白表达的变化。结果 HeLa 细胞中 p27 基因为低表达,基因启动子区 CpG 岛呈部分甲基 化状态。40 μg/mL rTCS 处理导致 p27 基因启动子区 CpG 岛去甲基化;在 20、40、80 μg/mL 的 rTCS 作用 48 h 后,p27 基因 mRNA 相对表达水平分别升高 2.22、4.00、6.03 倍,p27 蛋白水平表达也逐渐增加;宫颈癌 HeLa 细胞中 DNMT1 mRNA 高表达,40 μg/mL rTCS 处理 48 h 可至 DNMT1 mRNA 表达水平降低 78%。结论 rTCS 通过抑制 DNMT1,逆转 p27 基因启动子区 CpG 岛的甲基化状态,使宫颈癌 HeLa 细胞中 p27 基因表达活化。rTCS 能逆转宫颈癌 HeLa 细胞 p27 基因甲基化状态,调控 p27 基因和 DNMT1 的表达。     

天花粉蛋白对HeLa细胞抑制作用的实验研究

目的:研究天花粉蛋白(TCS)对人宫颈癌HeLa细胞生长的抑制作用,探讨TCS抗肿瘤的作用机制。方法:采用MTT法检测TCS对Hela细胞的抑制作用,形态学观察、DNA电泳检测TCS对HeLa细胞的诱导凋亡作用。结果:TCS对HeLa细胞的增殖具有明显的抑制作用,DNA凝胶电泳呈梯级格局,DNA细胞出现断裂,细胞凋亡现象,凋亡比例具有剂量和时间依赖性。结论:天花粉蛋白通过诱导细胞发生凋亡而抑制宫颈癌He-La细胞的增殖。     

组蛋白去乙酰化酶1 siRNA对HeLa细胞生长和凋亡的影响

目的 研究组蛋白去乙酰化酶1(histone deacetylases-1,HDAC-1)siRNA对宫颈癌HeLa细胞HDAC-1蛋白表达、细胞生长和凋亡的影响.方法 HDAC-1 siRNA沉默HDAC-1蛋白;Western blot技术检测HDAC-1蛋白;MTT法和克隆形成实验检测细胞生长抑制情况;流式细胞仪检测细胞凋亡.结果 HDAC-1 siRNA转染48 h可明显下调HeLa细胞HDAC-1蛋白表达;下调HDAC-1 表达明显降低HeLa细胞克隆形成率,抑制细胞增殖;下调HDAC-1诱导HeLa细胞凋亡.结论 HDAC-1 siRNA可能通过诱导凋亡抑制HeLa细胞生长.     

冬凌草甲素作用PI3K/AKT通路诱导HeLa细胞凋亡

 【目的】研究冬凌草甲素诱导HeLa细胞凋亡的作用及其机制。【方法】MTT法测定冬凌草甲素对HeLa细胞的生长抑制实验,Hoechst 33342荧光染色等观察细胞核形态学变化;LDH法研究细胞死亡的途径。流式细胞仪检测细胞的凋亡,免疫印迹实验检测相关蛋白的表达。【结果】25μmol/L以上的冬凌草甲素对宫颈癌HeLa细胞具有明显的生长抑制作用,并能诱导细胞发生凋亡,随着药物浓度的增加和作用时间的延长,细胞的生长抑制率及细胞凋亡率均明显升高,冬凌草甲素抑制细胞生长及诱导细胞发生凋亡的过程中,细胞端粒酶Akt、FKHRL、GSK3表达水平及活性显著降低。【结论】冬凌草甲素能够通过诱导人子宫颈癌HeLa细胞发生凋亡而发挥抑制HeLa细胞生长作用,抑制Akt和GSK3的活性是冬凌草甲素体外诱导人子宫颈癌HeLa细胞发生凋亡的重要作用机制之一。     

宫颈癌细胞FHIT基因启动子CpG岛甲基化状态分析

FHIT基因是一种与凋亡有关的肿瘤抑制基因,广泛存在于各种细胞凋亡系统中,在多种肿瘤及细胞系中失活,其启动子部位CpG岛甲基化是FHIT基因失活的主要机制之一。本研究应用甲基化特异性PCR(Methylation specific PCR,MSP)对不同宫颈鳞癌细胞系FHIT基因启动子CpG岛甲基化状态进行检测,在分子水平上了解FHIT异常甲基化与宫颈癌的相关性,为宫颈癌去甲基化治疗提供依据。     

冬凌草甲素作用PI3K/AKT通路诱导HeLa细胞凋亡

【目的】研究冬凌草甲素诱导HeLa细胞凋亡的作用及其机制。【方法】MTT法测定冬凌草甲素对HeLa细胞的生长抑制实验,Hoechst 33342荧光染色等观察细胞核形态学变化;LDH法研究细胞死亡的途径。流式细胞仪检测细胞的凋亡,免疫印迹实验检测相关蛋白的表达。【结果】25μmol/L以上的冬凌草甲素对宫颈癌HeLa细胞具有明显的生长抑制作用,并能诱导细胞发生凋亡,随着药物浓度的增加和作用时间的延长,细胞的生长抑制率及细胞凋亡率均明显升高,冬凌草甲素抑制细胞生长及诱导细胞发生凋亡的过程中,细胞端粒酶Akt、FKHRL、GSK3表达水平及活性显著降低。【结论】冬凌草甲素能够通过诱导人子宫颈癌HeLa细胞发生凋亡而发挥抑制HeLa细胞生长作用,抑制Akt和GSK3的活性是冬凌草甲素体外诱导人子宫颈癌HeLa细胞发生凋亡的重要作用机制之一。     

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.     

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.