Survivin表达抑制影响鼻咽癌细胞的增殖与凋亡

目的研究Survivin表达抑制对鼻咽癌细胞增殖和凋亡的影响,探讨Survivin在鼻咽癌中的生物学功能。方法采用脂质体将Survivin特异性SiRNA(small interfering RNA)转入鼻咽癌细胞株5-8F,培养48h后,收集细胞。逆转录-聚合酶链式反应(RT-PCR)、流式细胞仪(flow cytometry,FCM)分别检测Survivin mRNA和蛋白在细胞中表达。流式细胞仪检测细胞凋亡与周期,显微镜下计数检测细胞增殖抑制。结果SiRNA抑制5-8F细胞Survivin表达后,细胞凋亡指数显著高于对照组(P<0.01);S/G1比值显著高于对照组(P<0.01);细胞增殖抑制率也显著高于对照组(P<0.01);脂质体对照组与阴性SiRNA对照组比较,细胞凋亡、周期和增殖差异无显著性(P>0.05)。结论Survivin表达抑制,诱导鼻咽癌细胞凋亡并抑制其增殖。     

RNAi抑制Survivin基因的表达对乳腺癌SKBr-3细胞的影响

目的 应用RNA干扰技术抑制人Survivin基因表达,观察其对乳腺癌SKBr-3细胞增殖和凋亡的影响.方法 设计、合成靶向Survivin基因的siRNA基因片段,应用脂质体包埋转染乳腺癌SKBr-3细胞,采用MTT比色法检测细胞增殖率、RT-PCR和Western blot法观察乳腺癌细胞Survivin mRNA和蛋白质的表达、流式细胞仪检测细胞凋亡率.结果 MTt检测显示Survivin-siRNA组对细胞增殖有明显抑制作用(P<0.05),其细胞最高抑制率为(43.1±O.3)%;RT-PCR检测显示Survivin-siRNA组mRNA表达明显下调(P<0.01),其相对表达量分别为0.203 ±0.018、0.229±0.019,抑制率分别为55.2%、49.4%;Western blot检测显示Survivin-siRNA组蛋白表达下调(P<0.01),其蛋白相对表达量分别为0.702±0.007、0.684±0.016,抑制率分别为34.8%、36.4%;流式细胞仪检测显示Survivin-siRNA组细胞凋亡率明显增高(P<0.01),分别为(14.2±1.4)%、(16.8±0.6)%.结论 Survivin-siRNA能有效封闭Survivin的表达,抑制SKBr-3细胞增殖并诱导细胞凋亡,推测Sur-vivin基因可能成为乳腺癌基因治疗的一个新靶点.     

SIRT1基因沉默对鼻咽癌CNE-1细胞放射敏感性的影响

目的 探究SIRT1基因沉默对人鼻咽癌细胞CNE-1放射敏感性的影响,为鼻咽癌临床治疗提供新的思路.方法 通过Western blot检测鼻咽癌组织及鼻咽癌细胞中SIRT1蛋白的表达情况.通过脂质体转染SIRT1 siRNA至CNE-1细胞;利用CCK-8法和流式细胞术检测不同剂量(0、2、4、6、8 Gy)的X射线放射处理对CNE-1细胞增殖和凋亡的影响.采用Western blot检测SIRT1沉默后,X射线放射对凋亡相关蛋白Bcl-2、Bax和Cleaved-Caspase3表达的影响.结果 鼻咽癌组织和鼻咽癌细胞中SIRT1蛋白的表达量高于癌旁组织及鼻咽正常上皮细胞NP69中的表达量(P<0.05).靶向SIRT1基因的siRNA转染能特异性的抑制细胞中SIRT1的表达(P<0.05);4、6、8 Gy剂量的X射线放射处理能降低SIRT1 siRNA组CNE-1细胞增殖率,并增加凋亡率(P<0.01).用X射线照射SIRT1基因沉默的CNE-1细胞,能增加促凋亡蛋白Bax和Cleaved-Caspase3表达量,降低抑凋亡蛋白Bcl-2的表达量(P<0.05).结论 SIRT1基因沉默可能通过抑制CNE-1细胞增殖,并诱导细胞发生凋亡,从而增强CNE-1细胞对放疗的敏感性,SIRT1基因有望成为鼻咽癌治疗的新靶点.     

小干扰RNA抑制高迁移率族蛋白B1基因表达对肺癌细胞增殖和侵袭的影响

目的 研究小干扰RNA(siRNA)抑制高迁移率族蛋白B1(HMGB1)基因表达对肺癌细胞L9981增殖和侵袭能力的影响.方法 L9981细胞分3组,其中2组分别转染靶向HMGB1基因的siRNA(HMGB1-siRNA组)和无关序列siRNA(无关siRNA组),1组为空白对照组.转染后72 h,采用实时荧光定量PCR和蛋白质印迹法检测各组细胞HMGB1 mRNA和蛋白表达水平;细胞活力计数仪测定各组细胞活力.分别在转染后24、48、72和96 h应用噻唑蓝(MTT)法检测各组细胞生存状态.应用Boyden小室实验检测转染后72 h各组细胞的体外侵袭力.结果 HMGB1-siRNA组HMGB1mRNA相对表达量(1.0±0.0)明显低于无关siRNA组(12.8±1.3,P<0.05)和空白对照组(12.1± 1.0,P<0.05),HMGB1蛋白表达水平也明显受到抑制;细胞活力(44.7%±1.7%)明显低于无关siRNA组(73.7%±2.1%,P<0.01)和空白对照组(77.3%±1.9%,P<0.01).MTT测定显示转染后不同时间HMGB1-siRNA组L9981细胞生长均受到明显抑制.Boyden小室实验显示HMGB1-siRNA组穿膜细胞数(个/高倍视野,18.3±2.2)明显少于无关siRNA组(127.7±9.3,P<0.01)和空白对照组(132.3±10.7,P<0.01).结论 利用siRNA技术抑制HMGB1基因表达可有效抑制肺癌细胞的体外增殖和侵袭.     

化学合成siRNA沉默宫颈癌SiHa细胞E6基因表达的进一步研究

目的 探讨靶向HPV16 E6基因特异性siRNA对SiHa细胞E6和E7基因表达的沉默效果,以及沉默作用对pRb、P53、Survivin和VEGF-C蛋白表达的影响.方法 化学合成靶向HPV16 E6基因的siRNA,脂质体法转染SiHa细胞.半定量RT-PCR检测siRNA对SiHa细胞E6和E7基因转录表达的影响;Western Blotting法检测pRb、P53、Survivin和VEGF-C蛋白的表达.结果 RT-PCR检测结果显示,转染siRNA后24、48和72 h组SiHa细胞E6和E7 mRNA表达水平均明显低于对照组(F=80.71、182.18,P<0.05).Western Blotting结果显示,与对照组相比,转染组pRb和P53蛋白表达明显升高(F=59.78、29.00,P<0.05),VEGF-C蛋白表达明显降低(F=7.58,P<0.05),Survivin蛋白的表达则没有发生明显变化(F=2.28,P>0.05).结论 HPV16 E6特异性siRNA能同时抑制SiHa细胞内E6和E7基因的表达,进而引起P53、pRb以及VEGF-C蛋白表达水平的改变.     

DNA甲基转移酶1基因沉默对HT-29细胞凋亡的影响

目的:观察DNA甲基转移酶1(DNMT1)基因沉默对结肠癌HT-29细胞凋亡的影响.方法:HT-29细胞分为3组:未处理组、对照siRNA组及DNMT1 siRNA组,后2细胞转染对照siRNA和DNMT1 siRNA,24 h后,通过RT-PCR和Western blot观察3组细胞DNMT1、Caspase-3/9、bcl-2及Bax mRNA和蛋白表达的变化,通过流式细胞术分析HT-29细胞凋亡,并测定Caspase-3/9的活性.结果:DNMT1 siRNA组中DNMT1 mRNA和蛋白的相对表达量为(0.273±0.016)和(1.127±0.019),低于未处理组[分别为(1.729±0.023)和(2.741±0.031)]和对照siRNA组[分别为(1.751±0.018)和(2.673±0.027)](F分别为5822.683和3656.560,P均<0.001).DNMT1 siRNA组细胞凋亡率为(22.65%±1.67%),高于未处理组(7.92%±1.29%)和对照siRNA组(8.13%±1.21%)(F=108.460,P<0.001).DNMT1 siRNA组细胞的Caspase-3/9活性[分别为(2671.83±57.63)和(2187.71±68.35)]高于未处理组[(196.47±16.73)和(511.35±34.90)]和对照siRNA组[(215.29±14.38)和(534.78±41.70)](F分别为4790.975和1089.891,P均<0.001).与未处理组和对照组相比DNMT1 siRNA组Bcl-2 mR-NA和蛋白的表达降低,但Caspase-3/9和Bax mRNA和蛋白的表达升高(P<0.05).结论:DNMT1的下调能诱导HT-29细胞凋亡,其可能的作用机制与caspase-3/9、bcl-2和bax表达的变化密切相关.     

生存素下调多药耐药蛋白增强人鼻咽癌细胞株对紫杉醇敏感性的研究

目的 研究生存素（Survivin）下调多药耐药蛋白（MRP）的表达与鼻咽癌紫杉醇（PTX）耐药性关系,探讨鼻咽癌PTX耐药的分子机制.方法 采用免疫组化检测Survivin和MRP在42例鼻咽癌PTX耐药患者与24例PTX非耐药患者中的表达;采用浓度递增法持续诱导建立鼻咽癌化疗耐药细胞株5-8F-PTX（＋）,绘制细胞生长曲线,测定细胞生长的倍增时间,检测肿瘤细胞的周期分布;采用siRNA技术干扰5-8F-PTX（＋）中Survivin的表达后,Western blot法检测Survivin和MRP表达变化,MTT检测不同抗肿瘤药物PTX、顺铂（cDDP）、5-氟尿嘧啶（5-FU）、长春新碱（VCR）耐药敏感性的变化.结果 Survivin在鼻咽癌化疗耐药患者中阳性表达率为83.3％,明显高于非耐药患者（41.7％）,差异有高度统计学意义（P＜0.01）;MRP在鼻咽癌PTX耐药患者中表达阳性率为88.1％,明显高于非耐药患者（37.5％）,差异有高度统计学意义（P＜0.01）.5-8F-PTX（＋）较5-8F的细胞生长速度明显减慢,5-8F-PTX（＋）细胞生长的倍增时间为21h,亲本5-8F细胞生长的倍增时间为15 h;5-8F-PTX（＋）的G2/M期细胞百分比[（23.1±1.3）％]显著高于亲本5-8F细胞[（13.5±0.9）％];Survivin和MRP在PTX耐药细胞株5-8F-PTX（＋）细胞株中的表达水平明显高于非耐药的5-8F,siRNA干扰5-8F-PTX（＋）中Survivin的表达后,Survivin和MRP表达明显下调,PTX、cDDP、5-FU、VCR耐在siRNA-5-8F-PTX（＋）中的IC50值不同程度地下降.结论 Survivin可通过下调MRP的表达增强鼻咽癌细胞对PTX的敏感性.     

Survivin siRNA对人脐静脉内皮细胞的survivin基因及蛋白表达与凋亡的作用

[摘要] 目的 探讨Survivin siRNA对人脐静脉内皮细胞(HUVEC)的Survivin基因与蛋白表达与凋亡的影响。方法 合成Survivin siRNA转染混合物,以不同浓度转染HUVEC细胞, MTT法测定细胞生长抑制率, RT-PCR、Western blot检测HUVEC细胞Survivin mRNA及蛋白的表达变化,流式细胞术检测细胞的凋亡率。结果 HUVEC细胞转染荧光标记的FAM-siRNA后,细胞内有绿色荧光表达。分别以10,30,50nmol/L Survivin siRNA转染细胞后,细胞生长抑制率、Survivin mRNA表达、Survivin蛋白表达与对空白照组、阴性对照组、脂质体对照组比较差异有统计学意义(P<0.001)。细胞凋亡率与对照组比较明显下降(P<0.01)。随着Survivin siRNA浓度的增加,细胞抑制率与细胞凋亡率呈上升趋势,Survivin mRNA及Survivin 蛋白的表达呈逐渐降低趋势。结论 Survivin siRNA能抑制HUVEC的Survivin基因表达与并促进其凋亡。     

特异性SiRNA沉默小鼠NK细胞Ly49 C基因的实验研究揪

目的探讨特异性的SiRNA对小鼠NK细胞Ly49C基因表达的影响。方法将特异性的SiRNA作用小鼠NK细胞后,SYBRReal-time PCR及流式检测NK细胞Ly49C基因mRNA及其蛋白质表达。结果特异性siRNA片段作用NK细胞后能有效降低Ly49C mRNA和蛋白质水平的表达(P<0.05)。结论特异性的SiRNA可有效降低小鼠NK细胞中Ly49C mRNA水平,明显下调Ly49C蛋白表达。     

MTA1靶向RNA干扰对人肝癌HepG2细胞生物学行为影响的研究

目的 观察转移相关基因1(MTA1)特异性siRNA对肝癌细胞株HepG2细胞生物学行为的影响.方法 体外构建2个MTA1特异性siRNA表达载体(pRNAi-1、pRNAi-2),实验分为脂质体对照、空质粒转染对照及MTA1 siRNA重组质粒转染组,转染肝癌细胞株HepG2细胞,定量RT-PCR检测MTA1 mRNA表达情况,检验其对细胞的RNA干扰效果;采用MTT法分析其对细胞增殖的影响,流式细胞仪检测其对细胞周期的影响.结果 测序表明MTA1干扰序列完全正确;pRNAi-1与pRNAi-2表达质粒有效抑制肝癌细胞株HepG2细胞中MTA1的表达,MTT 法检测结果显示转染重组质粒组细胞体外生长的抑制率高于脂质体对照组和空质粒对照组(P<0.01);流式细胞术检测的转染重组质粒组细胞凋亡率高于脂质体对照组和空质粒对照组(P<0.01).结论 成功构建了MTA1干扰真核表达载体,重组MTA1特异性siRNA质粒可抑制肝癌细胞中MTA1的表达,并抑制肝癌细胞生长,促进其凋亡.     

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.     

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…     

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.     

Acupuncture Combined with Spinal Tui Na for Treatment of Primary Dysmenorrhea in 30 Cases

Objective: To observe the therapeutic effects in acupunture treatment of primary dysmenorrhea combined with spinal Tui Na, and study its mechanism. Methods: Thirty cases of the treatment group were treated by acupuncture combined with spinal Tui Na, and thirty cases in the control group were treated by routine acupuncture. Results: The total effective rate was 93.3% in the treatment group, and 73.3% in the control group, with a significant difference between the two groups (P<0.05). Conclusions: Acupuncture combined with spinal Tui Na has good prospects for treatment of primary dysmenorrhea.     

Luo Lingjie's Experience in Treating Chronic Nephropathy

In treating chronic nephropathy,Luo Lingjie,a chief physician,pays attention to regulating the balance between yin and yang,treating infection if present,and removing pathogenic factors.He prescribes gentle drugs and uses carefully strongly warming-tonifying ones,emphasizes the importance of persuading the patient to persist in treatment with medication and nurse one's health for recuperation,and is good at combined use of TCM and western medicine therapy and brings the merits of various therapies into full play,with obvious theraoeutic effects.     

Dr.Zhang Ren's Experience in the Acupuncture Treatment of Different Diseases with the Same Therapeutic Principle

Dr.Zhang Ren,the chief physician,is the chairman of Shanghai Acupuncture and Moxibustion Association.Having been engaged in medicine for about 40 years,he is experienced in treating various intractable diseases.In his long years of clinical practice,he advocates taking the TCM differentiation as the basis to seek for the acupuncture method for treatment of modern intractable diseases.The author of this essay had the fortune to follow Dr.Zhang in study.The following is a summary of Dr.Zhang's experience in the acupuncture treatment for different intractable diseases with the same therapeutic principle.     

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.