RNA干扰技术抑制糖皮质激素受体在大鼠肺泡巨噬细胞表达及活性的研究

目的利用RNA干扰技术,建立糖皮质激素受体(glucocorticoid receptor,GR)基因阻断的大鼠肺泡巨噬细胞模型.方法构建2个针对GR的RNAi表达载体,分别命名为psilencer 3.1-GR1和psilencer 3.1-GR2,经测序确认后,转染大鼠肺泡巨噬细胞.RT-PCR、Western Blot分别检测GR mRNA水平和蛋白水平表达变化;相对荧光素酶活性法检测GR转录激活功能的变化.结果经测序证实:成功构建2个针对GR的RNAi表达载体(psilencer 3.1-GR1和2);转染psilencer 3.1-GR2可明显降低细胞内GR mRNA的丰度及GR蛋白表达,细胞GR转录激活活性明显降低;转染psilencer 3.1-GR1与对照组相比无显著变化.结论成功地构建并筛选出一个特异而高效地阻断GR表达及功能的质粒表达载体,为进一步研究糖皮质激素受体的功能提供新方法.     

RNA干扰技术对人U937巨噬细胞株糖皮质激素受体表达及功能的抑制效应

目的利用载体介导的RNA干扰技术,建立糖皮质激素受体(GR)基因阻断的人U937巨噬细胞株模型.方法构建两个针对GR的RNAi重组表达载体,分别命名为pSilencer 3.1-GR1和pSilencer 3.1-GR2,经测序确认后,转染人U937巨噬细胞株.RT-PCR、Western blot分别检测糖皮质激素受体mRNA水平和蛋白水平表达变化;相对荧光素酶活性法检测地塞米松作用后GR转录激活功能的变化.结果经测序证实:成功构建两个针对GR的RNAi表达载体(pSilencer 3.1-GR1和pSilencer 3.1-GR2);转染pSilencer 3.1-GR2可明显降低细胞内GR mRNA的丰度及GR蛋白表达,细胞内GR转录激活功能明显降低;转染pSilencer 3.1-GR1与对照组相比无显著变化.结论成功地构建并筛选出一个高效且特异阻断GR表达及功能的RNAi质粒表达载体,为进一步研究糖皮质激素受体的功能提供新方法.     

JAB1表达下调对LPS诱导炎性因子TNF-α和IL-6的影响

目的 初步探讨JAB1基因表达下调对炎症反应的影响.方法 构建特异性针对小鼠JAB1基因的RNA干扰真核表达载体,转染到RAW264.7细胞中降低其JAB1基因的表达,观察LPS刺激后培养上清中炎性因子TNF-α和IL-6的浓度变化.结果 成功得到小鼠JAB1基因的RNA干扰真核表达载体(pPUR/U6-siJAB1),将其转染RAW264.7细胞后使JAB1的蛋白表达水平显著下降,经LPS刺激后培养上清中炎性因子TNF-α和IL-6的浓度较正常细胞明显降低.结论 小鼠JAB1基因的低表达使RAW264.7细胞在经LPS诱导后的炎性因子产生降低,推测其在炎症反应中对致炎因素的影响更大.     

一氧化氮调节脂多糖致炎大鼠肺泡巨噬细胞糖皮质激素受体功能的研究

目的研究肺泡巨噬细胞在脂多糖(LPS)作用条件下一氧化氮供体(SNP)对糖皮质激素受体(GR)功能的调节作用.方法将GR荧光表达质粒pGFP-GR转染大鼠肺泡巨噬细胞,经LPS和SNP作用后,荧光显微镜观察质粒表达产物GFP-GR核移位情况;相对荧光素酶法检测GR转录激活活性;EMSA检测检测细胞NF-κB活性.结果 500 μmol/L SNP作用2 h出现GFP-GR核移位,同时GR转录激活活性显著增强,NF-κB活性被显著抑制.运用GR特异性拮抗剂RU486后,NF-κB活性抑制现象消失.结论肺泡巨噬细胞在致炎因子作用条件下,一氧化氮供体(500 μmol/L SNP )通过活化GR发挥抗炎活性.     

缺失激素结合区的人糖皮质激素受体突变体的研究

目的：构建缺失激素结合区（LBD）的人糖皮质激素受体突变体表达载体，并研究其表达和功能。方法：运用RT-nested PCR扩增人糖皮质激素受体（CR）基因不含编码LBD的cDNA序列，将PCR产物定向克隆至pEGFP-N2质粒载体，测序筛选编码正确的重组质粒；荧光显微镜观察GR突变体重组质粒转染人巨噬细胞株U937后的表达情况；相对荧光素酶法检测转染后细胞GR转录激活活性。结果：扩增出长1601bp的cDNA序列，测序证实：克隆片段与Genebank该基因序列同源性为100％；重组质粒表达产物定位于细胞核；转染组细胞GR转录激活活性增强。结论：成功构建缺失LBD的GR突变体表达载体，该突变体具有不依赖激素的GR转录激活活性。     

中药成分CDP逆转高甲基化状态的GSTP1基因启动子低转录活性的研究

目的 研究中药成分CDP逆转高甲基化状态的GSTP1基因启动子低转录活性的作用.方法 构建含完全甲基化GSTP1启动子区的重组质粒pGL3-GSTP1prom并转染MCF-7细胞,分别用不同剂量的CDP和5-aza-C作用细胞,检测表达的萤光素酶活性.观察GSTP1启动子转录活性的变化. 结果 GSTP1启动子区被完全甲基化后转录活性显著降低;CDP能够逆转高甲基化状态下GSTP1基因启动子区的低转录活性且呈剂量依赖关系.结论 中药成分CDP能够逆转高甲基化状态的GSTP1 基因启动子区的低转录活性.     

大鼠肺泡巨噬细胞Bcl-2相关抗凋亡蛋白-1表达变化及其调节糖皮质激素受体功能的研究

目的：在模拟大鼠肺泡巨噬细胞炎症合并糖皮质激素治疗的条件下，阐明Bcl-2相关抗凋亡蛋白-1（BAG-1）的表达变化及其对糖皮质激素受体（GR）功能的调节作用。方法：Western blot检测脂多糖（LPS）和地塞米松（Dex）作用大鼠肺泡巨噬细胞BAG-1表达变化；免疫共沉淀法检测核蛋白中BAG-1与GR相互结合作用；相对荧光素酶法检测GR转录激活活性。结果：LPS和Dex作用后，细胞总蛋白中BAG-1L表达增加，BAG-1S表达无变化；核蛋白中只能检测到BAG-1L，表达量在LPS作用24h内逐渐增加；细胞核内BAG-1L与GR在LPS和Dex作用后存在着相互结合，同时GR转录激活活性逐渐降低，其变化与核蛋白中BAG-1L表达变化呈负相关。结论：LPS和Dex作用大鼠肺泡巨噬细胞BAG-1L表达增加，与GR结合后共同转核，并抑制GR活性。该机制可能是感染条件下糖皮质激素抵抗的重要因素。     

HIV-1 Ta填核表达载体的构建及在Huh-7细胞中表达

目的构建1型人类免疫缺陷病毒(HIV-1)反式激活因子(Tat)基因真核表达质粒,并研究该质粒在真核细胞中的有效表达。方法以含有HIV-1全长基因的质粒pNL4-3为模板,通过PCR扩增HIV-1tat第一外显子,应用基因工程技术将扩增的基因片段插入真核表达质粒pCDNA3.1( ),构建重组真核表达质粒pCDNA3.1-tat,经限制性内切酶酶切分析及测序鉴定正确后,应用脂质体转染技术转染入人体肝癌细胞系Huh-7细胞。RT-PCR检测其基因转录情况,Westernblot鉴定其是否能够表达相应的目的蛋白质。结果成功扩增了Tat基因,酶切和测序证明正确构建了重组真核表达质粒pCDNA3.1-tat,RT-PCR和Westernblot方法证实该质粒能在Huh-7真核细胞中有效表达HIV-1Tat目的蛋白质。结论成功构建了HIV-1Tat基因的真核表达质粒载体,并在Huh-7细胞中表达,为在Huh-7细胞模型中研究Tat的功能奠定了基础。     

人糖皮质激素受体变异体cDNA序列克隆和表达及其活性研究

目的构建人糖皮质激素受体变异体表达载体,研究其表达和功能.方法运用RT-nested PCR扩增糖皮质激素受体不含有编码LBD(ligand binding domain)的cDNA序列,将PCR产物定向克隆至pEGFP-N2质粒载体,测序筛选重组质粒;荧光显微镜观察重组质粒转染表达情况;相对荧光素酶法检测GR转录激活活性.结果扩增出长1 601 bp的cDNA序列,测序证实:克隆片段与Genbank该基因序列同源性为100%;重组质粒表达产物定位于细胞核;转染组细胞转录激活活性增强.结论成功构建糖皮质激素受体变异体的表达载体,该变异体具有不依赖激素的转录激活活性.     

IFN-γ和TNF-α对乙型肝炎病毒转录后调节机制的影响研究

目的探讨IFN-γ和TNF-α对乙型肝炎病毒转录后调节机制的影响.方法在体外构建含有HPRE片段的pDM138质粒(含有CAT报告基因),应用脂质体介导方法转染HepG2细胞,观察细胞因子对重组质粒载体CAT报告基因活性表达的影响,CAT基因的活性的检测采用ELISA检测试剂盒.结果成功构建了含有HPRE片段的重组质粒载体.转染重组质粒的HepG2细胞CAT活性表达明显增强,而转染空载体的HepG2细胞仅有本底表达,分别为896.5±213.6 和36.7±11.3 pg/ml.IFN-γ和TNF-α对转染重组质粒的HepG2细胞CAT活性表达的影响均具有浓度依赖性,而对空载体CAT活性的表达无影响.转染重组质粒的HepG2细胞在未加入细胞因子时,其CAT表达活性为896.5±213.6 pg/ml;与IFN-γ、TNF-α及IFN-γ+TNF-α孵育后,其CAT表达活性分别为324.4±56.8, 395.8±82.3, 175.3±35.6 pg/ml,与未加入时比较, t=5.19,4.39,6.68,P<0.01,差异有显著性.结论 IFN-γ和TNF-α可能通过抑制HPRE的功能,调控乙型肝炎病毒基因的表达.     

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.