integrin α6和MKK4基因在卵巢癌不同淋巴结转移能力细胞系中的表达及其意义

目的：探讨integrin α6、MKK4基因在卵巢癌不同淋巴结转移能力细胞亚系的表达及其意义。方法：采用TRIZOL一步法抽提卵巢癌不同淋巴结转移能力细胞的总RNA,逆转录合成CDNA,应用RT-PCR技术检测integrina6、MKK4基因在不同细胞亚系表达的灰度值,应用实时荧光定量PCR技术对integrin α6、MKK4基因在不同细胞亚系的表达进行定量分析。结果：integrin α6基因在SKOV3、SKOV3-PM2、SKOV3-PM3细胞系中的表达呈上升趋势,但在SKOV3-PM4细胞系中的表达低于SKOV3细胞系。MKK4基因表达量在SKOV3、SKOV3-PM2、SKOV3-PM3、SKOV3-PM4细胞系呈逐渐下降趋势。结论：integrin α6表达上调和MKK4表达下调与卵巢癌的淋巴结定向高转移过程密切相关,出现integrin α6在SKOV3-PM4细胞系表达的下调的现象,可能存在整合素各亚单位之间的相互调控。     

检测整合素αvβ3评估瘤细胞对腺病毒载体的易感性

目的：通过检测肿瘤细胞整合素αvβ3的表达,评估对腺病毒的易感性。方法：应用免疫组织化学法及原位杂交法,检测肿瘤细胞整合素αvβ3的表达。同时用携带LacZ的病毒载体（AdCMVLacZ）感染这些肿瘤细胞,通过X-gal染色,计算其转导率。结果：不同 的细胞系中腺病毒的转导率不同。整合素αvβ3及其mRNA在A549 及M21细胞内表达较多,在Hela及M21-L细胞无表达。整合素αvβ3表达高的瘤细胞,其腺病毒转导率也高。结论：瘤细胞的整合素αvβ3水平与腺病毒转导率有关,可用于评估肿瘤细胞对腺病毒载体的易感性。      

重组AAV1-Luc病毒制备及体外转导培养细胞的特性

目的制备携带萤火虫荧光素酶基因的重组AAV1病毒（rAAV1-Luc）,研究该病毒体外转导培养细胞的特性。方法通过“1株载体细胞/1株辅助病毒”的双因素包装策略制备出rAAV1-Luc,研究该重组病毒体外转导哺乳动物细胞的量效关系,肝素对转导的拮抗作用、rAAV1的竞争抑制作用及丁酸钠对表达水平的增强作用。结果在一定范围内,随着rAAV1-Luc感染细胞的感染复数（MOI,即每个细胞感染的病毒基因组数）值增高,荧光素酶的表达水平也增高;但更高的MOI（〉10^7）反而使荧光素酶的表达水平下降。肝素不能特异性阻断rAAV1介导的荧光素酶表达。携带不同基因的2种rAAV1病毒相互具有明显的竞争抑制作用。丁酸钠可显著增强rAAV1介导的荧光素酶表达水平。结论该研究揭示了rAAV1载体一定的细胞转导特性,对应用rAAV1载体介导基因转移研究具有指导意义。     

整合素β1基因shRNA慢病毒载体构建及其对乳腺癌他莫昔芬耐药细胞迁移和侵袭能力的影响

目的：构建整合素β1（integrin β1,ITGβ1）基因干扰慢病毒载体,并研究ITGβ1对他莫昔芬耐药乳腺癌MCF-7细胞（Tamoxifen-resistant MCF-7,MCF-7R）的迁移和侵袭能力的影响。方法：针对ITGβ1靶序列设计合成4条shRNA序列及1条阴性对照序列NC,构建ITGβ1RNAi慢病毒载体及对照病毒载体,并分别进行病毒包装,通过内源性筛靶,选取干扰效果最好的组病毒感染MCF-7R细胞,Western blot检测ITGβ1蛋白的表达水平;Transwell实验检测细胞迁移和侵袭能力。结果：成功构建4条慢病毒干扰重组质粒,并分别包装成病毒,通过内源性筛靶挑选出干扰效果最佳的慢病毒组,感染MCF-7R细胞后ITGβ1基因的蛋白表达量较NC组明显降低（P=0.000）,且细胞的迁移和侵袭能力明显弱于NC组（P=0.000）。结论：慢病毒介导的shRNA可有效地干扰MCF-7R细胞中ITGβ1的表达,并且抑制MCF-7R细胞的迁移和侵袭能力。     

Effects of Integrins and Integrin αvβ3 Inhibitor on Angiogenesis in Cerebral Ischemic Stroke

Summary： Integrins such as αvβ3, α5β31 play a key role in angiogenesis regulation, invasion and metastasis, inflammation, wound healing, etc. The up-regulation of integrin αvβ3 after cerebral ischemic stroke can promote angiogenesis, which in turn improves functional recovery. In addition, the integrin αvβ3 inhibitor can block the blood-brain barrier （BBB） leakage induced by vascular endothelial growth factor （VEGF） and also can reduce inflammatory reaction, decrease the deposition of fibrinogen. Other studies showed that integrin αvβ3 is not essential in revascularization. Therefore, the effect of integrin αvβ3 in the whole process of brain function recovery merits further study.     

慢病毒介导的GSK-3β基因稳定沉默表达的SH-SY5Y细胞系建立

目的构建靶向糖原合成激酶3β(GSK-3β)基因的RNA干扰慢病毒载体,建立其对GSK-3β基因沉默表达的人神经母细胞瘤细胞(SH-SY5Y)的细胞系模型。方法根据GSK-3βmRNA序列设计合成靶向目的基因的短发夹状RNA(shRNA)序列并构建慢病毒表达载体,经酶切和测序鉴定,将慢病毒重组载体与包装载体共转染293T细胞,获得纯化浓缩病毒液,侵染SH-SY5Y细胞,采用绿色荧光示踪观察细胞的转染情况,设为慢病毒干扰组(实验组)、阴性对照组(Lenti-NC组)、空白对照组(Blank组),利用qRT-PCR和Western blot法检测GSK-3βmRNA和蛋白表达水平。结果成功构建了靶向GSK-3β的shRNA慢病毒,重组慢病毒转染人SH-SY5Y细胞,72 h后观察转染效率达90%以上,与对照组比较,靶基因变化结果显示,实验组中GSK-3βmRNA和蛋白的表达均显著降低(均P<0.01)。结论有效建立慢病毒介导的GSK-3β基因稳定沉默表达的SH-SY5Y细胞模型,为体外评价研究GSK-3β沉默后药物或行为治疗AD提供试验工具细胞模型。     

腺病毒介导的LacZ基因在乳腺癌细胞中的表达

目的：观察腺病毒介导的基因转移在乳腺癌细胞的转导效率及腺病毒转导对细胞生长的影响。方法：腺病毒介导的基因转导。结果：在病毒的重复感染率（MOI）为500时细胞的转导效率达到100％；不同数量的病毒转导乳腺癌细胞对癌细胞的生长没有明显影响。低温冻存可使病毒的感染性明显下降。结论：腺病毒介导的基因转移在乳腺癌细胞中有高的转导效率，而病毒载体本身对细胞的生长没有影响，可用作乳癌特异药物前体酶激活基因治疗的基因转移方法。     

靶向非病毒载体一次及持续介导Ⅰ型单纯疱疹病毒胸腺嘧啶核苷激酶基因转导卵巢癌细胞的比较

目的比较靶向非病毒载体GE7系统介导Ⅰ型单纯疱疹病毒胸腺嘧啶核苷激酶(HSV1-tk)基因一次及持续转导卵巢癌细胞的转导效率及杀伤效应。方法靶向非病毒载体系统GE7分别与标志基因β-半乳糖苷酶(β-gal)基因和治疗基因HSV1-tk基因构成载体复合物,一次及持续体外转导卵巢癌细胞CaOV3,通过5-溴-4-氯-3-吲哚-β-D-半乳糖吡喃糖甙(X-gaI)染色比较GE7系统一次及持续转导外源基因的转导效率。将丙氧鸟苷(GCV)加入一次及持续转导HSV1-tk基因的卵巢癌细胞,通过细胞生长抑制曲线、流式细胞分析等,观察GCV对一次及持续转导GE7/HSV1-tk的卵巢癌细胞的杀伤作用。结果X-gal染色显示,GE7-次转导外源基因的平均转导效率可达80%,持续转导达85%。当GCV为1μg/mL时,GE7/HSV1-tk一次转导的生长抑制率达82%,凋亡指数为15,而持续转导可达90%,凋亡指数为30。在相同的GCV浓度下,持续转导GE7/pCMV-tk卵巢癌细胞生长抑制率显著高于一次转导(P<0.05)。结论GE7载体系统持续转导卵巢癌细胞较一次转导的平均转导效率高,持续转导的GE7/HSV1-tk/GCV基因治疗系统杀伤作用更大。     

慢病毒介导Smad7基因对角膜基质细胞增殖与纤维增生的抑制作用

【摘要】 目的 探讨慢病毒介导的Smad7基因转导能否抑制角膜基质细胞的增殖和纤维增生反应。方法 培养SD大鼠的角膜基质细胞,慢病毒载体介导转染Smad7基因或空质粒基因,分别建立Smad7阳性细胞克隆及空质粒阳性细胞克隆。角膜基质细胞进行分组：①正常细胞组：正常基质细胞,不加TGFβ2;②TGFβ2对照组：正常基质细胞加TGFβ2共孵育;③空质粒组：感染空质粒慢病毒的阳性细胞,加TGFβ2共孵育;④Smad7组：感染Smad7慢病毒的阳性细胞,加TGFβ2共孵育。TGFβ2的终浓度为10ng/ml。在TGFβ2刺激2h后Western blot检测TGFβ/Smad信号传导因子Smad2蛋白的磷酸化蛋白（phosphorylated Smad2,p-Smad2）。TGFβ2刺激48h后Western blot和real-time PCR检测α-SMA（α-smooth muscle actin）、Ki67、Ⅲ型胶原蛋白（Type Ⅲ collagen, ColⅢ）mRNA和蛋白的表达。MTT法检测基质细胞生长活性。结果 Smad7基因转导抑制了Smad2蛋白的磷酸化,减少了Ki67、α-SMA和胶原III mRNA和蛋白的表达,角膜基质细胞的生长活性也下降。结论 Smad7基因转导可阻断大鼠角膜基质细胞TGFβ信号传导通路,减少Smad2的磷酸化,阻止基质细胞的活化增殖,阻断基质细胞向肌纤维母细胞的转化,并且减少胶原蛋白III的合成,从而抑制角膜基质细胞增殖和纤维增生。     

携带靶向干扰VEGF基因shRNA重组腺相关病毒载体的构建和制备

目的：构建并制备携带靶向干扰VEGF165基因shRNA的重组腺相关病毒载体．方法：将PCR法扩增所得EGFP-U6-shRNA（VEGF165）片段插入载体质粒pSNAV2．0-lacz-α的EcoRI和SalI酶切位点，构建重组质粒pSNAV2．0-EGFP—U6-shRNA（VEGF165）．以HSV1-RapCap／△UL2为辅毒，包装rAAV2-EGFP-U6-shRNA（VEGF165）病毒．对所获病毒载体进行PCR，SDS-PAGE鉴定分析、滴度测定和EGFP的活性检测．结果：PCR，SDS-PAGE鉴定分析结果表明靶向干扰VEGF165基因的shRNA片段成功包装入重组AAV2载体，病毒纯度在98％以上．点杂交法测定重组病毒载体基因组滴度约为3×10^14v．g．（vector genomes）／L．EGFP活性检测显示重组AAV2感染细胞阳性率在30％以上．结论：成功制备了rAAV2-EGFP-U6-shRNA（VEGF165）病毒载体，为通过RNA干扰技术特异性抑制VEGF基因的表达来阐明VEGF在缺血性脑损伤后各种生物学事件中的作用奠定了实验基础．     

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.     

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…     

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.