苯丁酸钠通过下调CLDN4基因DNA甲基化对喉鳞状细胞癌增殖与凋亡的作用研究

目的 观察苯丁酸钠对喉鳞状细胞癌(laryngeal squamous cell carcinoma,LSCC)细胞株 Hep-2增殖及凋亡的作用,探讨其作用机制与紧密连接蛋白4(claudin4,CLDN4)基因甲基化的相关性。方法 使用0.5、1.0、2.0、4.0、8.0mmol/L的苯丁酸钠分别处理Hep-2细胞24h,利用四甲基偶氮唑蓝(MTT)比色法、流式细胞术检测Hep-2细胞增殖、细胞周期与细胞凋亡,细胞免疫荧光法检测Hep-2细胞中caspase-3表达,实时荧光定量PCR(qRT-PCR)检测Hep-2细胞中DNA甲基转移酶(DNMT1、DNMT3a)mRNA表达水平,免疫印迹试验(Western blot)法检测Hep-2细胞CLDN4蛋白表达水平,利用甲基化特异性 PCR 法(MSP)分析苯丁酸钠对Hep-2细胞CLDN4基因甲基化的影响。结果 苯丁酸钠呈剂量依赖地抑制Hep-2 细胞增殖,使细胞周期阻滞在 G₀/G₁期,并诱导细胞凋亡;4.0mmol/L苯丁酸钠处理Hep-2 细胞后,caspase-3 蛋白表达明显增加;苯丁酸钠处理Hep-2 细胞后,CLDN4蛋白表达水平随苯丁酸钠浓度增加而升高,而DNMT1、DNMT3a mRNA表达水平随苯丁酸钠浓度增加而下降;苯丁酸钠作用后CLDN4基因的高甲基化被逆转,并呈剂量依赖性。结论 苯丁酸钠可通过抑制甲基转移酶DNMT1、DNMT3a的表达使CLDN4基因去甲基化,从而抑制Hep-2细胞的增殖并诱导其凋亡。     

siRNAi沉默DNMT1基因对肺癌细胞WIF-1启动子甲基化的影响

目的 探讨DNA甲基转移酶(DNMT1)siRNAi对人肺癌A549细胞内抑癌基因wnt抑制因子-1(WIF-1)基因启动子高甲基化的影响.方法 利用pSilencer 4.1-CMV neo载体,通过siRNAi表达载体法制备DNMT1 siRNAi.将DNMT1 siRNAi转染肺癌A549细胞,采用RT-PCR和Western blot检测观察转染前后肺癌A549细胞DNMT1内DNMT1基因的表达改变,并利用甲基化PCR,直接测序法检测WIF-1基因启动子甲基化水平.结果 构建的DNMT1 siRNAi转染肺癌A549细胞后,明显抑制DNMT1基因的表达,mRNA表达下降75%,蛋白表达下调82%;同时,WIF-1基因启动子序列较转染前甲基化水平明显降低.结论 靶向DNMT1的siRNAi可明显下调靶基因DNMT1的表达,并逆转WIF-1基因启动子高甲基化水平.     

藤黄酸抑制人结肠癌SW480细胞增殖过程中APC蛋白的表达变化

目的 观察藤黄酸对人结肠癌SW480细胞增殖和APC蛋白表达的影响,探讨其可能抗肿瘤的作用机制.方法 以不同浓度(0、0.5、1、4μg/ml)的藤黄酸干预体外培养的人结肠癌细胞株SW480,分别于24、36、48 h后,应用MTT法检测细胞的增殖活性,流式细胞仪检测细胞周期分布和细胞凋亡率,Western-blot分析APC蛋白的变化.结果 1 μg/ml以上浓度的藤黄酸可以显著抑制人结肠癌细胞株SW480细胞增殖,且随浓度增加效果增强,随着时间增加抑制率显著增加(P＜0.01).藤黄酸浓度达到4 μg/ml时,C2/M期细胞达到54.74％;藤黄酸浓度为0、0.5、1、4μg/ml时SW480细胞的凋亡率分别为(0.17±0.08)％、(0.85±0.19)％、(7.12±1.21)％、(15.87±1.59)％.用不同浓度的藤黄酸处理SW480细胞株24 h,APC蛋白的表达随药物浓度的升高而增加(P＜0.01).结论 藤黄酸能够抑制人结肠癌细胞株SW480增殖并诱导其凋亡,干扰SW480细胞周期使其阻断于G2/M期,高表达APC蛋白有可能是藤黄酸抗癌作用的重要机制.     

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。     

多基因启动子甲基化参与的乳腺癌MCF-7对他莫昔芬敏感性探究

目的 探讨多基因启动子甲基化参与的乳腺癌MCF-7对他莫昔芬敏感性的关系.方法 用MTT法对比乳腺癌MCF-7细胞株与耐药株生长增殖差异;流式细胞检测亲本与耐药株细胞凋亡之间的差异;RT-PCR及Real-Time PCR对比两细胞株相关基因mRNA表达差异;甲基化特异性PCR(MSP)检测4个候选基因(DAPK、MGMT、P53、RASSF1A)在亲本及耐药株中的甲基化状态.结果 Real-Time PCR可见MCF-7/TAM细胞中抑癌基因DAPK、RASSF1A水平低于亲本株(P＜0.05);而MGMT、P53、DNMT1、DNM3B的mRNA表达水平却较亲本细胞株升高(P＜0.05);基因DNMT3A的mRNA表达水平较低,在亲本及耐药株中均未检出.MSP显示,在MCF-7细胞中,DAPK、P53呈非甲基化状态,而对他莫昔芬细胞株这2个基因为甲基化状态.结合Real-Time PCR检测结果,基因甲基化状态与甲基转移酶(DNMT1、DNMT3B)表达呈正相关性;除P53基因外,基因mRNA表达水平与甲基化状态呈负相关性.结论 相关基因表达差异及启动子的甲基化状态可能与乳腺癌细胞株对他莫昔芬敏感性相关,为下一步进行临床评估和机制探讨提供了依据.     

稳定低表达DNA甲基转移酶3b基因对膀胱癌细胞生长和凋亡的影响

目的探讨稳定低表达DNA 甲基转移酶3b（DNMT3b）基因对人膀胱癌细胞生长和凋亡的影响。方法包装表达
DNMT3b siRNA和阴性对照siRNA的慢病毒,通过慢病毒感染的方法获得稳定低表达DNMT3b的膀胱癌细胞BIU-87及对照
细胞。MTT和流式细胞仪检测DNMT3b稳定低表达对细胞增殖和凋亡的影响;体内裸鼠成瘤实验观察DNMT3b稳定低表达
对肿瘤的抑制效果;荧光定量PCR和Western blot检测DNMT3b稳定低表达对细胞生长和凋亡相关基因表达的影响;甲基化特
异性PCR检测对细胞生长和凋亡相关基因甲基化的影响。结果荧光定量PCR和Western blot检测结果显示,DNMT3b mRNA
和蛋白水平在BIU-87稳定细胞株中稳定低表达;DNMT3b稳定低表达可以抑制BIU-87细胞的生长,并且抑制裸鼠皮下瘤体的
生长;DNMT3b稳定低表达可以促进BIU-87细胞的凋亡;DNMT3b稳定低表达可以增加凋亡及细胞生长相关基因DAPK,Bax
和RASSF1A mRNA和蛋白水平的表达;DNMT3b稳定低表达可以减少凋亡及细胞生长相关基因DAPK,Bax和RASSF1A启
动子区域的甲基化水平。结论DNMT3b稳定低表达可能通过调控凋亡相关基因以及细胞生长相关基因启动子区域的甲基化
水平来影响基因的表达,从而抑制BIU-87细胞的生长,诱导细胞凋亡。
     

siRNA沉默膀胱癌T24细胞DNA甲基转移酶1(DNMT1)表达的研究

目的 研究siRNA对膀胱癌细胞T24 DNA甲基转移酶1(DNMT1)表达的沉默作用.方法 将筛选出的siRNA转染入膀胱癌T24细胞分为空白组、脂质体组、阴性对照组和siRNA组,分别运用RT-PCR、Western blot检测转染后不同时段DNMT1 mRNA水平、蛋白水平变化.结果 筛选出的siRNA转染膀胱癌T24细胞DNMT1 24 h开始出现mRNA减少,24 h、48 h、72 h分别降至(66.05±4.87)％、(39.36±4.53)％和(40.86±4.81)％(P＜0.05).转染后24 h、48 h、72 h蛋白水平分别降至(90.21±3.68)％、(71.45±3.44)％和(67.74±3.38)％(P＜0.05).结论 siRNA能有效地沉默膀胱癌细胞T24 DNMT1的表达.     

亚砷酸对人喉癌Hep-2细胞RECK基因表达及去甲基化的影响

目的:探讨不同浓度亚砷酸对人喉癌Hep-2细胞中伴Kazal域的富含半胱氨酸的逆转诱导蛋白(RECK)基因表达及去甲基化的影响。方法:分别将0.5、1.0、2.0、4.0μmoL/L的亚砷酸作用于体外培养的Hep-2细胞24～72 h,MTT法计算细胞生长抑制率,流式细胞术检测细胞周期,并用甲基特异性PCR检测RECK基因甲基化情况。RT-PCR和W estern b lot分别检测RECK mRNA和蛋白表达水平。结果:0.5～4.0μmoL/L亚砷酸能抑制Hep-2细胞增殖,使Hep-2细胞阻滞于S期和G2/M期;RECK基因甲基化水平随亚砷酸浓度增高而降低,非甲基化水平则逐渐增高;亚砷酸能增加RECK mRNA和蛋白表达。结论:亚砷酸能促进Hep-2细胞中RECK基因去甲基化并提高其表达水平,从而发挥抑制细胞增殖。     

MCF-7/Adr细胞mdr-1基因启动子甲基化和组蛋白乙酰化状态与多药耐药的关系

目的：分析MCF-7/Adr及MCF-7细胞mdr-1基因启动子甲基化和组蛋白乙酰化状态,初步探讨乳腺癌多药耐药的表观遗传机制。 方法：用甲基化敏感PCR技术检测两个细胞系mdr-1基因启动子甲基化状态。实时定量PCR技术检测DNA甲基转移酶（DNA methyltransferases,DNMTs) mRNA及组蛋白去乙酰化酶（histone deacetylases,HDACs) mRNA的表达。光密度值法检测组蛋白H3和H4乙酰化水平。 结果：MCF-7细胞mdr-1基因启动子呈现高甲基化,MCF-7/Adr细胞mdr-1基因启动子呈现低甲基化。与MCF-7细胞比较,MCF-7/Adr细胞DNMT1,DNMT3a及DNMT3b mRNA表达显著下降(P＜0.05)。MCF-7/Adr细胞组蛋白H3和H4乙酰化水平较MCF-7细胞明显升高(P＜0.01)。与MCF-7细胞比较,MCF-7/Adr细胞HDAC1,HDAC2,HDAC7及SIRT1 mRNA的表达显著下降(P＜0.01)。 结论：mdr-1基因启动子低甲基化、组蛋白H3和H4高乙酰化、DNMTs mRNA及HDACs mRNA低表达可能是介导MCF-7/Adr细胞MDR形成的重要表观遗传学因素。     

STAT3-siRNA对人结直肠癌细胞SW480的干涉作用

【目的】应用经小干扰RNA（shortinterfereRNA，smNA）表达盒介导的RNAj对结直肠癌细胞中的STAT3基因进行干涉作用以研究STAT3-siRNA对STAT3基因表达阳性的结直肠癌细胞凋亡的影响。【方法】用脂质体转染试剂将STAT3-siRNA表达盒（STAT3-siRNA expression cassettes，STAT3-SECs）体外转染至人结直肠癌SW480细胞及人成纤维细胞中。于48h后收集细胞，先经荧光染色方法观察细胞表象变化，再通过流式细胞仪检测人结直肠癌SW480细胞凋亡情况，最后分别提取细胞总RNA，用RT—PCR测定STAT3基因在mRNA水平的表达。【结果】SW480STAT3-SECs组的细胞可见凋亡小体，出现明显的凋亡现象，而其它实验组均未出现明显的凋亡现象。流式细胞术结果显示SW480STAT3-SECs组中凋亡细胞百分比由0．2％增加至26．0％；S期细胞比率由32．3％下降至9．2％。在mRNA水平上，SW480STAT3-SECs组细胞的STAT3基因表达减少了（62．16±12．50）％，显著低于SW480对照组（P〈0.01）。【结论】应用RNAi技术沉默STAT3基因可以降低人结直肠癌SW480细胞中STAT3的表达，诱导细胞的凋亡。     

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.