Skp2 shRNA表达质粒的构建及其对肺癌细胞生长的影响

目的 构建Skp2基因的RNAi真核表达质粒,观察其对SIC-A-1肺癌细胞内Skp2基因表达及细胞生长的影响.方法 根据GenBank中Skp2cDNA序列设计针对Skp2基因的shRNA序列,合成序列并克隆人pGenSil-1质粒中,构建skp2 pshRNA重组质粒.脂质体介导重组质粒转染SPC-A-1肺癌细胞.RT-PCR检测肺癌细胞Skp2 mRNA的表达,Western blot检测Skp2蛋白表达.MTT检测各纽细胞生长情况,流式细胞仪检测细胞周期改变.结果 重组质粒经酶切鉴定和测序,证实表达干扰质粒构建成功.转染重组质粒的肺癌细胞Skp2 mRNA和蛋白的表达明显下降(P<0.05),细胞生长增殖受到抑制,阻滞在G0/G1期的细胞比例增加,而进入S期的细胞比例明显下降(P<0.05).结论 构建的Skp2 shRNA表达质粒能有效下调Skp2基因的mRNA及蛋白的表达、抑制细胞的生长增殖.     

重组抗HER2 ScFv/tbid基因的表达及其对乳腺癌SKBr-3细胞的促凋亡作用

目的:观察构建的重组抗HER2 ScFv /tbid基因在SKBr-3细胞中的表达及其对转染的SKBr-3细胞的作用. 方法:用限制性内切酶双酶切已构建的pcDNA-bid质粒,获得带有PE转膜结构域和tbid的重组基因,将所获基因插入到真核表达载体pCMV单链抗体基因ScFv23e的下游,转染SKBr-3细胞. 间接免疫荧光法检测目的蛋白的表达,MTT法检测目的基因转染后细胞的增殖情况. 结果:转染SKBr-3细胞后,检测出目的蛋白的表达. MTT实验发现细胞的增殖被明显抑制. 间接免疫荧光双标记染色检测tBid的表达,并可观察到Cyt c在细胞质中存在, SKBr-3细胞出现凋亡. 结论:重组抗HER2 ScFv /tBid分子可以在转染的SKBr-3细胞中表达,并且可抑制转染细胞的生长,诱导细胞发生凋亡.     

转录因子E2F-1 siRNA表达质粒的构建及对胃癌MGC803细胞的沉默效应

目的:通过构建人E2F-1基因的小干扰RNA(siRNA)表达质粒,探讨其对胃癌MGC803细胞中E2F-1基因表达的影响.方法:将设计合成的具有短发夹结构的人E2F-1基因siRNA寡核苷酸链退火形成双链,连接人经BamH I和Hind Ⅲ双酶切后的pSilencer4.1-CMV neo表达质粒,并做酶切及测序鉴定.用脂质体把重组质粒转染至细胞,采用实时定量PCR(real-time quantitative PCR)及蛋白印迹(western blotting)技术分别检测E2F-I mRNA及蛋白表达.结果:经酶切及测序鉴定,成功构建了E2F-l siRNA表达质粒;它可显著抑制MGC803细胞中E2F-1基因mRNA及蛋白表达,与未转染组及阴性对照组细胞比较,分别降低了86.4%、86.1%和81,5%、79.6%.结论:pSilencer4.1一E2F-l重组质粒能抑制人胃癌MGC803细胞E2F-1基因的表达,为进一步E2F-1基因的功能研究及胃癌治疗研究提供了实验基础.     

靶向bcl-2基因转录shRNA重组质粒的构建和筛选

目的 构建能高效抑制肝星状细胞(HSCs)bcl-2表达的小发夹RNA(shRNA)重组质粒.方法 利用RNA干扰技术,设计有小发夹结构的3条DNA序列,经退火形成互补双链,克隆至转录载体psiRNA-hHlneo上构建重组质粒,予以酶切和测序鉴定.重组质粒转染HSC-T6细胞株,运用实时荧光定量PCR和Western blotting进行筛选鉴定.结果 酶切及测序鉴定表明,成功构建重组质粒shRNA1、shRNA2和shRNA3.实时荧光定量PCR和Western blotting检测结果显示,shRNA1和shRNA2转染后显著抑制HSC-T6中bcl-2 mRNA和蛋白表达(P＜0.05),其中siRNA1的bcl-2表达抑制效率＞80%.结论 构建的重组质粒shRNA1能有效抑制HSCs中bcl-2的表达.实验为进一步探索肝纤维化基因治疗的新途径奠定了基础.     

Geldanamycin对HER2/neu高表达人乳腺癌细胞系SKBr3增殖及迁移能力的影响

目的 探讨Hsp90抑制剂苯醌安莎霉素类抗生素(GA)对HER2/neu高表达人乳腺癌细胞系SKBr3增殖及迁移能力的影响.方法 用Western蛋白印迹法检测GA介导的HER2/neu酪氨酸激酶的降解;MTT法分析GA对肿瘤细胞存活率的影响;流式细胞仪测定GA对肿瘤细胞周期的影响;RT-PCR和real-time PCR分析GA对cyclin D1表达的影响以及分析GA对肿瘤细胞迁移能力的干预作用.结果 GA以剂量/时间依赖性的方式降解了HER2/neu酪氨酸激酶的表达并抑制了SKBr3乳腺癌细胞的增殖,这种增殖抑制效应表现在:GA干预后乳腺癌细胞的存活率明显降低;GA阻断了乳腺癌细胞细胞周期的进展,使细胞周期停滞于G1期,而这两种表现与cyclin D1表达量的减低有着明显的关系.GA的干预也同时降低了肿瘤细胞的迁移能力.结论本研究证实GA能够降解HER2/neu酪氨酸激酶并抑制HER2/neu高表达人乳腺癌细胞系SKBr3的增殖和迁移能力.     

针对HER2/neu的siRNA对非小细胞肺癌细胞生长的抑制作用

目的:构建针对HER2/neu的RNA干涉载体,观察对HER2/neu表达抑制及抑制后对非小细胞肺癌生长的影响. 方法:设计和合成长度为64nt的脱氧寡核苷酸链,退火互补成双链,克隆至pSUPER质粒载体,转化DH5α菌株,提取质粒,转染过表达HER2/neu的肺腺癌细胞系SPC-A-1,RT-PCR检测HER2/neu的mRNA表达,Western blot和间接免疫荧光法检测HER2/neu的蛋白表达,FCM分析细胞周期变化,MTT法观察细胞生长. 结果:肺腺癌细胞系SPC-A-1存在HER2/neu过表达;成功构建了HER2/neu特异性RNA干涉载体pSUPER-siHER2;瞬时转染SPC-A-1细胞,HER2/neu的mRNA及蛋白表达均降低;G1期细胞较亲代增加9.9%;肿瘤细胞增殖速度减慢(P＜0.05). 结论:成功构建了HER2/neu RNA干涉载体,转染后可有效降低肺腺癌细胞系SPC-A-1 HER2/neu的mRNA转录及蛋白水平表达,阻滞转染细胞于G1期,造成转染细胞生长速度减慢,这有望为肺癌基因治疗提供一种选择手段.     

干扰AFP蛋白表达的shRNA表达载体的构建及活性鉴定

目的:利用含报告基因EGFP的pGenesil-1质粒构建干扰AFP蛋白表达的短发夹RNA(short hairpin RNA,shRNA)表达的载体,并进行活性鉴定.方法:设计表达短发夹RNA的互补DNA序列,经退火成双链,克隆至带有U6启动子的质粒载体pGenesil-1中,构建重组质粒,转化大肠杆菌DH5α菌株,扩增,提取质粒,酶切鉴定后测序分析.构建正确的重组质粒经脂质体转染HepG2细胞,检测转染率和培养细胞上清的AFP含量变化,确定重组质粒的活性.结果:构建了靶向AFP蛋白的2个shRNA重组质粒载体pGenesil-1-siAFP1和pGenesil-1-siAFP2.酶切鉴定和测序分析,shRNA编码序列与设计的片段完全一致,经酶切凝胶电泳证实载体构建成功.重组质粒有效转染HepG2细胞并显著抑制AFP表达.结论:成功构建具有活性的、能够表达shRNA靶向干扰AFP蛋白的2个重组质粒载体.     

针对人caspase-8基因的siRNA的载体的构建及其表达

目的 构建针对人caspase-8 基因mRNA的siRNA表达载体,并观察其对转染细胞中caspase8的抑制作用.方法 化学合成针对caspase-8发夹状的RNA寡核苷酸,将其退火形成双链,连接入经HindⅢ和BglⅡ双酶切后的pSUPER真核表达载体.对重组质粒进行酶切分析和测序鉴定.通过脂质体介导,把重组质粒瞬时转染入HeLa细胞,RT-PCR测其对mRNA和蛋白表达的干涉效果.结果 成功地构建了针对人caspase-8基因的RNA干涉真核表达载体pSUPER-C1和pSUPER-C2,并在人HeLa细胞中有效发挥了对caspase8基因的干涉作用,而且pSUPER-C1对于caspase-8基因的抑制作用要优于pSUPER-C2.结论 成功地构建了针对人caspase-8基因的siRNA载体,转染HeLa细胞后可抑制caspase-8基因的表达.     

靶向bcl-2基因转录shRNA重组质粒的构建和筛选

目的构建能高效抑制肝星状细胞(HSCs)bcl-2表达的小发夹RNA(shRNA)重组质粒。方法利用RNA干扰技术,设计有小发夹结构的3条DNA序列,经退火形成互补双链,克隆至转录载体psiRNA-hH1neo上构建重组质粒,予以酶切和测序鉴定。重组质粒转染HSC-T6细胞株,运用实时荧光定量PCR和Western blotting进行筛选鉴定。结果酶切及测序鉴定表明,成功构建重组质粒shRNA1、shRNA2和shRNA3。实时荧光定量PCR和Western blotting检测结果显示,shRNA1和shRNA2转染后显著抑制HSC-T6中bcl-2mRNA和蛋白表达(P<0.05),其中siRNA1的bcl-2表达抑制效率>80%。结论构建的重组质粒shRNA1能有效抑制HSCs中bcl-2的表达。实验为进一步探索肝纤维化基因治疗的新途径奠定了基础。     

hTERT siRNA表达载体的构建及对转染的HeLa细胞生长抑制作用

目的:通过RNA干涉技术抑制肿瘤细胞端粒酶表达,探讨干涉后对肿瘤细胞生长的抑制作用. 方法:根据人端粒酶逆转录酶(hTERT)mRNA编码序列,设计RNA干涉靶点,构建siRNA(small interferencing RNA)表达载体,并转染HeLa细胞,通过RT-PCR法观察重组质粒转染肿瘤细胞后端粒酶mRNA、蛋白含量及细胞生长情况. 结果:构建的siRNA表达载体可以使HeLa细胞端粒酶逆转录酶mRNA及其蛋白含量降低,转染质粒的细胞生长增殖速度减慢. 结论:成功构建了针对人端粒酶逆转录酶的siRNA表达载体,通过转染HeLa细胞,可有效抑制细胞中端粒酶的表达,并引起细胞生长抑制.     

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.