HIF-2琢对自噬介导的肝癌细胞顺铂耐药的影响及机制

目的探究低氧诱导因子-2琢（HIF-2琢）对自噬介导的人肝癌阿霉素耐药细胞株（HepG2/ADM）顺铂（cisplatin）耐药的影响及机制,以期为疾病的临床治疗提供新思路。方法将人肝癌细胞HepG2和HepG2/ADM分为7组并加入相应试剂：（1）HIF-2琢-siRNA＋cisplatin组;（2）HIF-2琢-siRNA组;（3）NC-siRNA组;（4）3-甲基腺嘌呤（3-MA）＋cisplatin组;（5）cisplatin组;（6）3-MA组;（7）对照组。采用Westernblot法检测微管相关蛋白1轻链3-Ⅱ（LC3-Ⅱ）、Beclin1、HIF-2琢蛋白表达水平,流式细胞仪检测细胞凋亡率,CCK-8试剂盒检测细胞增殖抑制率。结果cisplatin组HepG2、HepG2/ADM细胞中LC3-Ⅱ、Beclin1、HIF-2α蛋白表达水平及细胞凋亡率较对照组均显著升高（均P＜0.05）。3-MA组HepG2/ADM细胞中LC3-Ⅱ、Beclin1蛋白表达水平较对照组均显著下降（均P＜0.05）。3-MA＋cisplatin组HepG2/ADM细胞凋亡率较cisplatin组、3-MA组和对照组均显著升高（均P＜0.05）;cisplatin组HepG2/ADM细胞凋亡率较3-MA组和对照组均显著升高（均P＜0.05）。HIF-2琢-siRNA组HepG2/ADM细胞中HIF-2琢、LC3-Ⅱ、Beclin1蛋白表达水平较NC-siRNA组均显著下降（均P＜0.05）。HIF-2琢-siRNA＋cisplatin组HepG2/ADM细胞凋亡率较HIF-2琢-siRNA组、NC-siRNA组和cisplatin组均显著升高（均P＜0.05）;HIF-2琢-siRNA组和NC-siRNA组HepG2/ADM细胞凋亡率较cisplatin组均显著降低（均P＜0.05）。沉默HIF-2琢表达后,随cisplatin处理浓度增加,HepG2/ADM细胞增殖抑制率升高。结论HIF-2琢可能通过调节自噬影响肝癌细胞HepG2/ADM的cisplatin耐药性,因此临床上可通过降低HIF-2α表达水平,提高cisplatin对肝癌细胞HepG2/ADM的杀伤作用,为晚期肝癌治疗提供新思路。     

阿霉素对人肝癌细胞株HepG2中Survivin表达的影响

目的探讨阿霉素(ADM)对人肝癌细胞株HepG2中Survivin表达的影响。方法用不同浓度的ADM对人肝癌细胞株HepG2作用不同时间,然后以免疫细胞化学和RT-PCR方法检测HepG2细胞中Survivin蛋白和SurvivinmRNA表达的改变。结果未加药物干预之前,免疫化学中,HepG2细胞被Survivin抗体染成棕黄色,核内染色深于胞浆;琼脂糖凝胶电泳中,可见SurvivinmRNA明亮条带;各种浓度的ADM均可降低HepG2细胞Survivin免疫染色和Sur-vivinmRNA在凝胶电泳中的灰度;并且随干预浓度的增加或作用时相的延长,降低更明显。结论人肝癌HepG2细胞表达Survivin,且核内Survivin蛋白表达比胞浆表达强;ADM可降低HepG2细胞中Survivin表达,并呈浓度和时间依赖性;ADM可能通过下调Survivin表达而参与诱导癌细胞凋亡而发挥其抗癌作用。     

NF-κB decoy寡核苷酸联合阿霉素诱导裸鼠肝癌细胞HepG_2凋亡

目的:探讨NF-κB decoy寡核苷酸协同阿霉素对裸鼠肝癌细胞HepG2的作用及机制。方法:接种传代培养的人肝癌细胞HepG2于裸鼠皮下,局部注射NF-κB decoy寡核苷酸和/或阿霉素进行治疗。观测裸鼠瘤体大小变化,计算抑瘤率;HE染色观察肝癌组织结构和细胞形态改变;TUNEL法检测细胞凋亡。结果:NF-κB decoy寡核苷酸联合阿霉素治疗组裸鼠肝癌细胞的生长受到明显抑制,抑瘤率为80.52%;肿瘤重量、体积明显小于单用NF-κBdecoy寡核苷酸、阿霉素治疗组及对照组;联合组癌组织凋亡增加,凋亡指数为(39.8±1.6)%,与其余3组比较差异均有统计学意义(P<0.05)。结论:NF-κB decoy寡核苷酸协同阿霉素能抑制NF-κB活性,促进肝癌细胞凋亡,增加其对化疗药物的敏感性。     

姜黄素对肝癌耐药细胞HepG2/ADM的增殖和阿霉素耐药性的影响

目的分析姜黄素对肝癌耐药细胞HepG2/ADM的增殖和阿霉素耐药性的影响。方法 (1)将HepG2/ADM细胞分为不同浓度药物组(加入5μg/mL、10μg/mL、20μg/mL、40μg/mL、60μg/mL的姜黄素)、阴性对照组(接种细胞但不加药物)及空白对照组(不接种细胞,只加培养基),培养24、48及72 h后检测细胞增殖情况。(2)将HepG2细胞、HepG2/ADM细胞分为药物组、阴性对照组(接种细胞但不加药物)及空白对照组(不接种细胞,只加培养基)。HepG2细胞药物组加入不同浓度阿霉素(0.1μg/mL、0.2μg/mL、0.4μg/mL、0.8μg/mL、1.6μg/mL);HepG2/ADM细胞药物组分两个亚组,一组加入不同浓度阿霉素(1.5μg/mL、3μg/mL、6μg/mL、12μg/mL、24μg/mL),另一组加入5μg/mL姜黄素和不同浓度阿霉素(1.5μg/mL、3μg/mL、6μg/mL、12μg/mL、24μg/mL)。培养48 h后检测细胞增殖情况,计算姜黄素的逆转倍数以及HepG2/ADM细胞耐药指数。(3)将HepG2/ADM细胞分为对照组(...     

凋亡素联合阿霉素与顺铂对肝细胞癌的凋亡作用研究

目的 探讨凋亡素联合阿霉素(ADM)、顺铂(DDP)对肝癌的凋亡作用,为肝癌治疗提供实验依据.方法 通过腺病毒载体介导凋亡素基因导入Hepa1-6肝癌细胞中,联合应用ADM、DDP在细胞水平上观察凋亡形态,检测凋亡率,评估联合治疗凋亡效果.结果 无凋亡素介导下10 μmol/ L 的DDP 联合及1.26 μmol/ L 的ADM作用36 h 效果最好,该浓度下有凋亡素介导的实验组Hepa1-6的凋亡率要高于对照组.结论 带有特异性甲胎蛋白启动子的重组腺病毒载体,介导凋亡素基因导入肝癌细胞后, 联合阿霉素、顺铂对能合成甲胎蛋白的肝癌细胞具有特异性靶向性致凋亡作用.     

DADS对人肝癌细胞株HepG2增殖和凋亡的影响

目的：探讨二烯丙基二硫（DADS）对人肝癌细胞株HepG2增殖和凋亡的影响.方法：采用MTT、细胞计数检测DADS对人肝癌细胞株HepG2增殖的影响;应用流式细胞仪检测DADS对HepG2细胞周期和细胞凋亡率的影响.结果：DADS使细胞增殖明显受抑制且抑制率呈药物浓度时间依赖性（P＜0.05）,DADS组细胞的倍增时间延长（P＜0.05）.与对照组比较DADS处理组G2/M期细胞比例显著增加,而G0/G1期细胞比例则明显下降,细胞的凋亡率显著增加（P＜0.05）.结论;DADS对人肝癌细胞株具有抗增殖作用,且与药物浓度及作用时间相关.DADS能阻滞细胞周期在G2/M期,并诱导细胞凋亡.     

硒化麒麟菜多糖对肝癌细胞株生长和凋亡的影响

 【目的】 探讨硒化麒麟菜多糖在体外对肝癌HepG2.2.15细胞株生长和凋亡的作用及机制?【方法】 处于生长期的人肝癌细胞HepG2.2.15分为3组,对照组?硒化麒麟菜多糖组和顺氨氯铂组?用MTT法检测各组肿瘤细胞增殖的情况,流式细胞仪检测细胞周期?细胞调亡以及凋亡基因Fas的表达情况?【结果】 硒化麒麟菜多糖在体外可抑制肿瘤细胞的增殖,最高抑制率为97.2%,并且有时间依赖性?硒化麒麟菜多糖可阻滞肝癌HepG2.2.15细胞的生长使之停留在S和G2/M期,从而抑制了肿瘤细胞的增殖,同时通过促进Fas表达14.4% ± 0.11%,明显高于对照组1.5% ± 0.01% (P < 0.05),从而诱导HepG2.2.15细胞凋亡?【结论】 硒化麒麟菜多糖可能是通过阻滞肿瘤细胞的生长及诱导细胞凋亡等机制,从而对肝癌细胞的增殖有一定的抑制作用?     

EGCG逆转人耐药肝癌细胞株多药耐药的体内实验研究

目的:研究表没食子儿茶素没食子酸酯(EGCG)体内逆转人肝癌细胞多药耐药性的作用及可能的机制.方法:MTT法检测肝癌耐药细胞株BEL-7404/ADR与其亲本细胞BEL-7404耐药倍数,采用BEL-7404/ADR种植于裸鼠皮下,建立肿瘤耐药模型,两周后EGCG灌胃,腹腔注射阿霉素(ADM),联合治疗两周;荧光分光光度法检测瘤体内ADM含量,HE染色观察形态学改变;RT-PCR检测瘤组织mdr1 mRNA的表达,免疫组化法检测瘤组织P糖蛋白(P-gp)的表达.结果:低、中、高EGCG联合ADM组肿瘤生长速度明显低于ADM组,瘤重明显低于阿霉素组;EGCG联合ADM可增加瘤体内ADM的含量 (P<0.01);三个EGCG联合 ADM组与阿霉素组相比HE病理图片显示肿瘤组织纤维包裹,炎细胞浸润; mdr1 mRNA和P-gp的表达明显低于对照组和阿霉素组 (P<0.01).结论:EGCG在体内具有逆转BEL-7404/ADR的耐药作用,机制可能是EGCG下调了肝癌细胞mdr1基因的表达,使P-gp的表达降低,瘤组织内药量增高.     

survivin反义寡核苷酸对肝癌耐药细胞的作用及与化疗的关系

目的研究survivin反义寡核苷酸对人肝癌耐药细胞株的增殖和凋亡情况,以及对阿霉素化疗敏感性的影响。方法将人肝癌耐药细胞系SMMC-7721/ADM分为脂质体转染组、阿霉素组、正义寡核苷酸转染组、正义寡核苷酸转染 阿霉素组、反义寡核苷酸转染组、反义寡核苷酸转染 阿霉素组共6组。MTT法检测细胞相对存活率,流式细胞仪分析细胞凋亡率变化,RT-PCR和Westernblot印迹法检测细胞survivinmRNA和蛋白表达。结果survivin-ASODN作用后的人肝癌耐药细胞SMMC-7721/ADM的细胞凋亡率明显增高(P<0.05)。survivinmRNA和survivin蛋白表达:脂质体转染对照组、阿霉素组、正义寡核苷酸转染对照组、正义寡核苷酸转染对照 阿霉素组之间survivin蛋白表达无明显差异(P>0.05)。400ng/mlsurvivingASODN组和400ng/mlASODN 阿霉素组survivinmRNA较其他4组显著降低(P<0.05),但其两组之间表达无明显差异(P>0.05)。结论survivin反义寡核苷酸能降低肝癌耐药细胞survivin表达,增强人肝癌耐药细胞对阿霉素的化疗敏感性。     

半乳糖化白蛋白磁性阿霉素纳米粒对肝癌细胞株侵袭力的影响的实验研究

目的研究半乳糖化白蛋白磁性阿霉素纳米粒对肝癌细胞株HepG2侵袭力的影响.方法应用半乳糖化白蛋白磁性阿霉素纳米粒于HepG2细胞,并设A组:RPMI-1640对照组、B组:普通盐酸阿霉素组(ADM)、C组:磁性白蛋白阿霉素纳米粒联合磁场(MADM M)组、D组:半乳糖化白蛋白阿霉素纳米粒(Gal-ADM)组、E组:半乳糖化白蛋白磁性阿霉素纳米粒联合磁场(Gal-MADM M)组.药物作用后以β-actin基因作为参照,半定量的逆转录-聚合酶链反应(RT-PCR)检测Cathepsin B-mRNA的表达差异,应用Transwell 小室检测体外侵袭力的变化,并用裸鼠肝癌皮下转移模型检测对体内侵袭力的影响.结果半乳糖化白蛋白磁性阿霉素纳米粒作用HepG2肝癌细胞后Cathepsin B-mRNA表达降低较其他各组更为明显且有显著性,侵过小室滤膜细胞数目减少于其他各组且差异有显著性(P＜0.01),裸鼠肝癌生长明显受到抑制(P＜0.01).结论半乳糖化白蛋白磁性阿霉素纳米粒抑制肝癌细胞株HepG2的侵袭力的作用较阿霉素、磁性白蛋白阿霉素纳米粒和半乳糖化白蛋白阿霉素纳米粒为强.     

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.