SPRY1基因与角质形成细胞衰老的相关性研究

目的：探讨软脂酰化磷蛋白Sprouty1（SPRY1）基因在不同年龄段人体皮肤角质形成细胞和人永生化角质形成细胞（HaCaT细胞）衰老中的表达及意义。方法：收集不同年龄段正常皮肤标本120例,分为少年儿童组（＜18岁）、青年组（18～44岁）、中年组（45～59岁）、老年组（≥60岁）,各30例。通过免疫组化、Real-time PCR、Western blot技术检测SPRY1在各年龄段皮肤角质形成细胞中的定位及表达差异。用200 μmol/L H2O2处理HaCaT细胞构建细胞老化组模型。Real-time PCR技术和Western blot技术检测SPRY1在正常组和衰老组HaCaT细胞中的表达。结果：SPRY1的表达主要分布在皮肤角质形成细胞的棘层及颗粒层,呈细胞质分布;SPRY1 mRNA及蛋白在人皮肤角质形成细胞中的表达随着年龄段的增长呈逐渐增高的趋势,差异具有统计学意义（P＜0.05）。HaCaT细胞经H2O2处理后发生了老化。 SPRY1 mRNA及蛋白在老化后的HaCaT细胞中表达上调,差异具有统计学意义（P＜0.05）。结论：SPRY1基因在人体衰老的皮肤角质形成细胞及老化HaCaT细胞中表达增加,提示SPRY1基因参与角质形成细胞衰老调控。     

人表皮角质形成细胞Toll样受体表达

目的 研究人表皮角质形成细胞Toll样受体表达谱.方法 培养人永生化角质形成细胞系HaCaT细胞和正常人表皮角质形成细胞(NHEK),采用分散酶分离表皮.逆转录-聚合酶链反应(RT-PCR)法分别检测10种Toll样受体mRNA表达,流式细胞仪检测HaCaT细胞和NHEK表面Toll样受体2和4蛋白的表达.结果 HaCaT细胞、NHEK以及分离的表皮均有全部10种Toll样受体mRNA表达,其表达强弱各不相同.流式细胞仪检测显示HaCaT细胞和NHEK表面有Toll样受体4蛋白的表达,而Toll样受体2无明显表达.结论 人表皮角质形成细胞中有Toll样受体1～10 mRNA的组成性表达.     

体外培养人皮肤鳞癌A431细胞Smad4的表达下调

目的研究体外培养的人皮肤鳞癌细胞中Smad4的表达情况。方法采用反转录-实时荧光定量聚合酶链式反应及Western blotting,分别检测体外培养的人皮肤鳞癌A431细胞与人永生化角质形成细胞(HaCaT细胞)中Smad4 mRNA及其蛋白的表达水平。结果在mRNA表达水平上,A431细胞与对照HaCaT细胞相比,其2-△△CT值大于2,在蛋白表达水平上,两者差异有统计学意义(t=2.536,P<0.05),提示人皮肤鳞癌A431细胞Smad4的mRNA及其蛋白的表达水平均显著低于对照的HaCaT细胞。结论Smad4表达下调可能是鳞癌A431细胞所固有的转化生长因子β信号转导通路的异常变化之一。     

黄芩甙对角质形成细胞产生IL-8和增殖活性的影响

目的 研究黄芩甙对角质形成细胞产生IL-8和增殖活性的影响，探讨黄芩甙治疗银屑病机制。方法 采用四甲基偶氮盐比色法（MTT）观察黄芩甙对角质形成细胞株HaCaT细胞增殖的影响，实时定量RT—PCR方法检测黄芩甙对HaCaT角质形成细胞表达IL-8 mRNA的影响；用酶联免疫吸附方法（ELISA）检测HaCaT细胞分泌至培养液中IL-8的量。结果 100μg／mL黄芩甙可抑制HacaT细胞增殖；10—100μg／mL黄芩甙均可抑制HaCaT角质形成细胞表达IL-8mRNA和分泌IL-8。结论 黄芩甙可抑制体外培养的HaCaT细胞增殖和表达IL-8mRNA和蛋白，发挥抗炎和治疗银屑病的作用。     

他克莫司软膏对特应性皮炎皮损角质形成细胞Toll样受体2和4表达的影响

目的:探讨Toll样受体 (Toll-like receptor,TLR) 2和 TLR4在特应性皮炎(atopic dermatitis,AD)发病机制中的意义及外用他克莫司软膏(商品名,普特彼)对TLR2和 TLR4表达的影响.方法:采用免疫组织化学法检测9例中至重度AD患者急性发作期炎性皮损与正常对照皮肤角质形成细胞TLR2和 TLR4的表达, 以及外用他克莫司软膏单一疗法治疗AD 3周后TLR2和 TLR4表达的变化.结果:免疫组化检测显示在正常皮肤基底层角质形成细胞胞膜散在表达TLR2和TLR4.TLR2和TLR4在AD急性发作期炎性皮损角质形成细胞胞膜及胞浆过度表达,以细胞膜表达为主,主要定位于基底层至颗粒层;他克莫司治疗3周后改善特应性皮炎皮损角质形成细胞的TLR2和TLR4表达,主要定位于基底或基底上层角质形成细胞膜.结论:正常角质形成细胞表达的TLR2和TLR4是构成皮肤天然免疫系统的组成部分.AD角质形成细胞诱导性过度表达TLR2和TLR4与AD皮肤天然免疫炎症反应有关,外用他克莫司可通过直接或间接作用抑制角质形成细胞的TLR2和TLR4诱导性表达,从而抑制与TLR-细胞核因子κB(NFκB)信号转导和调控途径有关的AD皮肤天然免疫炎症反应.     

miR-21-3p靶向STAT3促进银屑病角质形成细胞增值的研究

目的 探讨miR-21-3p及其靶蛋白信号转导子和转录激活子3蛋白（STAT3）在寻常型银屑病发生过程中角质形成细胞的作用意义。方法 对皮肤组织中STAT3蛋白表达情况的检测选择免疫组织化学法,对健康人群皮肤组织及银屑病患者皮损组织miR-21-3p表达量的检测选择实时荧光定量PCR法,靶基因STAT3和miR-21-3p关系的检测选择双荧光素酶报告基因。化学合成miR-21-3p模拟物、抑制剂,瞬时转染HaCaT细胞,对不同分组细胞进行CCK8、流式细胞凋亡实验,探究分析miR-21-3p调控细胞表型主要作用。对于转染细胞中STAT3蛋白表达情况,选择Western Blot检测。结果 寻常型银屑病患者皮肤组织中miR-21-3p呈高表达（P＜0.05）。STAT3免疫组化阳性表达定位于细胞浆,在银屑病组患者皮损组织阳性表达率为82.22%（37/45）高于正常皮肤组织（P＜0.01）。双荧光素酶报告基因显示miR-21-3p与STAT3存在结合,呈靶向关系。miR-21-3p mimics促进HaCaT细胞增殖活性,抑制细胞凋亡;miR-21-3p inhibitor抑制HaCaT细胞的增殖能力,促使细胞趋向凋亡。miR-21-3p转染HaCaT过表达可对STAT3蛋白表达量产生促进,相反则降低（F=48.632, P＜0.01）。结论 miR-21-3p呈现正调控关系,能够靶向调控STAT3;两者共同作用参与并加重银屑病的病程,并抑制患者细胞凋亡,调节角质形成细胞增值活性     

IL-10对HaCaT细胞中VEGF-A表达的影响

目的 探讨白介素-10(IL-10)对皮肤角质形成(HaCaT)细胞中血管内皮生长因子A(VEGF-A)表达的影响及其可能机制.方法 培养HaCaT细胞,用不同剂量IL-10刺激后,通过荧光定量PCR(Real-time PCR)在mRNA水平上分析IL-10对HaCaT细胞中VEGF-A表达的影响;通过Western...     

短发夹状RNA靶向抑制HaCaT细胞Hax-1基因的表达研究

目的 观察短发夹状干扰RNA靶向抑制Hax-1基因诱导HaCaT细胞凋亡的作用.方法 构建特异性抑制Hax-1的shRNA表达载体并转染HaCaT细胞;分别采用半定量PCR以及Western blot从mRNA和蛋白水平检测干扰效果;流式细胞术检测HaCaT细胞的凋亡.结果 Hax-1基因mRNA水平以及蛋白水平的表达均得到显著抑制,HaCaT细胞凋亡率明显升高.结论 靶向Hax-1的shRNA显著抑制了Hax-1基因在HaCaT细胞中的表达,并明显诱导HaCaT细胞凋亡.     

骨连接素对角质形成细胞的增殖抑制作用

目的:观察骨连接素对体外培养人角质形成细胞(HaCaT)的细胞增殖、细胞周期及相关基因表达的影响,初步探讨骨连接素对人角质形成细胞的作用机制.方法:用不同浓度骨连接素处理HaCaT细胞,MTT法检测HaCaT细胞增殖;流式细胞术测定细胞周期;实时定量RT-PCR方法检测CCND1和PCNA mRNA表达水平的变化.结果:与未加入骨连接素的HaCaT细胞相比,5 μg/ml以上浓度骨连接素作用的细胞增殖率明显下降(P<0.05), 10 μg/ml骨连接素作用的细胞G0/G1期细胞明显增多.10 μg/ml以上浓度的骨连接素作用后,HaCaT细胞CCND1和PCNA的mRNA水平显著减弱(P<0.05,P<0.01).结论:骨连接素可能是通过抑制CCND1和PCNA的表达,从而发挥抑制细胞增殖的作用.     

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.