RNA干扰下调人胆管癌细胞系Hucct-1乳酸脱氢酶-A表达的体外研究

目的:体外实验观察乳酸脱氢酶-A(lactate dehydrogenase-A,LDHA)短发夹RNA对胆管癌细胞系Hucct1 LDHA表达的抑制效果,筛选出针对LDHA基因有效的干扰靶点,并检测LDHA表达下调后,细胞增殖活力的改变情况?方法:设计合成干扰LDHA表达的寡核苷酸片段,经退火?连接等步骤克隆到线性化pGPU6/GFP/Neo真核表达载体,转化入DH-5α大肠杆菌中扩增,经质粒抽提纯化后测序鉴定?重组质粒用脂质体法转染胆管癌细胞系,荧光显微镜观察转染效率;并分别利用RT-PCR及Western blot技术检测LDHA mRNA及蛋白表达,验证短发夹RNA的干扰效果?将干扰效率最高的质粒转染Hucct-1,MTT法检测干扰组较对照组细胞增殖的变化?结果:成功构建了靶向LDHA基因的3个shRNA质粒表达载体,脂质体法平均转染效率为(80 ± 5)%?荧光定量RT-PCR和Western blot显示瞬时转染si-LDHA-1?si-LDHA-3的Hucct1细胞LDHA mRNA和蛋白表达均较阴性对照组有不同程度的下降,其中si-LDHA-3干扰效果尤为明显?MTT法检测显示转染组Hucct1细胞较阴性对照组增殖减慢,转染后96 h两组差异具有统计学差异(P < 0.05)?结论:重组质粒能有效干扰胆管癌细胞Hucct1中LDHA基因的表达,经瞬时转染筛选出了有效干扰靶位,下调LDHA的表达,明显抑制胆管癌细胞系Hucct-1的增殖活力?     

Livin shRNA表达载体的构建及其稳定转染宫颈癌siha细胞系的建立

目的 构建Livin shRNA真核表达载体,获得干扰质粒稳定转染的宫颈癌siha细胞系.方法 合成Livin基因干扰序列并定向插入到RNA干扰(RNAi)真核表达载体pGenesil-1质粒,通过测序进行鉴定.干扰质粒经脂质体Lipofectamine 2000介导转染siha细胞.经G418持续压力选择和有限稀释法获得稳定转染的细胞系.RT-PCR检测所筛选克隆Livin mRNA的转录水平.结果 测序证明Livin干扰序列及读码框完全止确,干扰质粒稳定转染的siha细胞在倒置荧光显微镜下呈明亮绿色荧光.RT-PCR结果显示Livin shRNA序列对Livin mRNA有较好的抑制效果.结论 成功构建了Livin shRNA真核表达载体,Livin shRNA质粒稳定转染的siha细胞系的建芷为进一步研究Livin在官颈癌细胞中的作用奠定了基础.     

CTGF特异性siRNA慢病毒表达载体的构建及鉴定

目的:构建CTGF特异性siRNA慢病毒表达载体并观察其介导的RNA干扰(RNA interference,RNAi)对人肝癌细胞CTGF(connective tissue growth factor,CTGF)表达的影响?方法:针对已经筛选确定的人CTGF基因RNAi有效靶序列,合成靶序列的Oligo DNA,退火形成双链DNA,与Plk0.1-GFP-SP6载体连接产生shRNA 慢病毒载体,经PCR筛选阳性克隆进行DNA测序鉴定?用脂质体转染法将质粒共转染293T 细胞,将包装产生的慢病毒颗粒感染HepG2细胞,Real-time PCR?Western-blot检测CTGF mRNA和蛋白的表达?结果:PCR与DNA测序证实合成的含CTGF shRNA慢病毒载体寡核苷酸链插入正确?经RNA干扰后的靶细胞CTGF mRNA以及蛋白表达水平明显降低?结论:成功构建人CTGF基因RNA干扰慢病毒载体,体外感染HepG2细胞可有效降低CTGF mRNA和蛋白的表达,为以CTGF基因为靶点的肝癌基因治疗研究奠定了基础?     

串联表达大鼠心肌细胞Kir2.1 shRNA载体的构建及其体外效应

目的 构建串联表达5个针对大鼠kir2.1基因shRNA的真核表达载体,观察其对该基因表达的影响。方法 选择5个针对大鼠kir2．1基因的RNA干扰位点,分别设计合成相应的5对寡核苷酸链,形成双链后分别连入带有u6启动子的相应载体,反复酶切连接将表达5个kir2．1 shRNA的目的基因依次连接入载体pEGFP6—1,构建成串联真核表达载体pEGFP6—1Kir2．1,将其转染培养的大鼠心肌细胞,RT-PCR和Western印迹检测其对Kir2．1mRNA转录和蛋白表达的影响。结果 成功构建串联表达5个大鼠Kir2．1基因shRNA的真核表达载体,该载体对大鼠心肌细胞Kir2．1mRNA转录的抑制率为83．5％,对心肌细胞Kir2．1蛋白表达的抑制率为68．1％。结论 串联表达多个大鼠心肌细胞Kir2．1基因shRNA真核表达载体介导的RNA干扰可以特异性抑制该基因的表达,可能成为制造生物起搏器的一种新方法。     

靶向心肌细胞TLR4基因小干扰RNA载体构建

目的:设计合成有效靶向Toll样受体4(TLR4)基因小干扰RNA(siRNA)表达载体,且挑选TLR4基因稳定沉默心肌细胞。方法:依据RNA干扰(RNAi)序列设计原则,以TLR4基因为靶基因,合成3对小发夹RNA(shRNA)寡核苷酸链,经退火、与线性化pSilence 2.1-U6neo质粒连接、酶切、测序并鉴定。脂质体法转染心肌细胞,新霉素(G418)加压筛选并收集稳定表达质粒的心肌细胞,并观察光镜下心肌细胞形态。RT-PCR及Western Blot法检测收集的心肌细胞TLR4mRNA和蛋白的表达,确定siRNA对TLR4的抑制效率。结果:测序鉴定插入发夹样序列正确,成功构建了TLR4基因siRNA表达载体;并获得了稳定沉默TLR4的心肌细胞。未见细胞毒性形态学改变。多种方法均证实siRNA作用于TLR4基因后,心肌细胞TLR4 mRNA和蛋白表达均被抑制,其中pSilence2.1-siTLR4-1抑制作用最强。结论:成功构建了靶向TLR4基因siRNA表达载体,并有效抑制心肌细胞TLR4表达。     

自噬基因Beclin 1 shRNA表达质粒的构建及其对宫颈癌细胞HeLa生长的作用

目的 构建自噬基因Beclin 1小发夹RNA(short hairpin RNA,shRNA)表达质粒,探讨Beclin 1抑制后对宫颈癌HeLa细胞生长的作用.方法 根据人Beclin 1 mRNA编码序列,设计RNA干扰靶点,构建Beclin 1 shRNA表达质粒,脂质体法转染人宫颈癌HeLa细胞,通过荧光定量PCR(RT-PCR)和Western blot检测其对HeLa细胞自噬基因Beclin 1 mRNA及蛋白表达的影响.MTT法分析其对细胞增殖、流式细胞仪检测其对细胞周期和细胞凋亡的影响.结果 构建的shRNA表达载体可以使HeLa细胞中自噬基因Beclin 1的mRNA及其蛋白含量降低,转染质粒的细胞生长增殖速度加快,凋亡率降低.结论 成功构建了针对自噬基因Beclin 1的shRNA表达载体,通过转染HeLa细胞,可有效抑制细胞中Beclin 1的表达,并促进细胞生长,抑制凋亡.     

靶向CCR7基因shRNA表达载体的构建及鉴定

目的设计并构建靶向CCR7基因shRNA真核表达质粒,并检测其干扰效果。方法以CCR7为靶基因设计具有短发夹结构的模板寡核苷酸,退火形成互补双链结构,再克隆至pGC-silencer-CRM30载体,构建的3条重组短发夹shRNA表达载体分别命名为pGC-silencer-CRM30-CCR7A、-CCR7B和-CCR7C。采用测序法鉴定寡核苷酸序列。将构建好的真核表达载体转染人结肠癌SW480细胞,荧光显微镜观察转染效果,RT-PCR法检测CCR7干扰效率。结果重组质粒测序结果与Genebank中的CCR7 cDNA序列相符,转染SW480细胞后,荧光显微镜下可观察到绿色荧光蛋白,RT-PCR结果表明,pGC-silencer-CRM30-CCR7C干扰效果最强。结论靶向CCR7基因shRNA表达载体构建无误,为进一步探讨趋化因子受体CCR7在胃肠道恶性肿瘤生物学行为中的作用奠定了基础。     

靶向CGI-100基因shRNA干扰载体的构建与鉴定

目的：构建靶向CGI-100基因的RNAi表达载体并鉴定其抑制效率。方法：设计及化学合成3对靶向CGI-100基因的短发夹结构寡核苷酸,经退火成双链DNA片段,将其与经限制性内切酶BamHI和州ndⅢ双酶切的PGenesil-1质粒连接,构建表达shRNA的重组质粒,用脂质体2000转染到K562细胞中进行表达,经RT-PCR和斑点杂交检测转染前后CGI-100基因表达的变化。结果：经DNA测序证实3对靶向CGI-100基因的RNA干扰表达载体PGenesil-1-CGI-100构建成功,其中第1对PGenesil-1-CGI-100在mRNA水平抑制白血病K562细胞中CGI-100基因表达的效率最高,达54％。结论：成功构建针对CGI-100基因的RNA干扰的表达载体,并筛选出1对抑制效率较高的重组干扰载体,为进一步研究K562细胞中CGI-100基因的功能奠定基础。     

人组织因子基因RNA干扰载体的构建与鉴定

目的 构建人组织因子基因RNA干扰载体,为达到阻止凝血过程的启动打下基础.方法 利用Invitrogen公司在线软件设计人组织因子基因(NM 001993)干扰片段,合成shRNA序列,再制备双链寡核苷酸(ds oligo),退火后形成的ds oligo双链克隆到pENTRTM/U6载体的粘性末端,连接产物转化到感受态细胞,增菌,质粒DNA小提、测序.将构建的载体转染到脐静脉内皮细胞,应用RT-PCR和免疫荧光验证载体对目的基因的干扰情况.结果 测序证实阳性克隆为pENTRTM/U6-TF-shRNA,转染到脐静脉内皮细胞后组织因子的表达受到明显抑制.结论 成功构建出凝血途径的人组织因子基因RNA干扰载体,为研究组织因子基因RNA干扰载体在凝血疾病中的应用提供了稳定的转染细胞载体.     

人Smad3的shRNA表达载体构建和鉴定

目的：构建人Smad3的shRNA表达载体，运用RNAi下调Smad3基因在肿瘤细胞系中的表达，观察对肿瘤细胞生长的影响．方法：化学合成针对人smad3基因的dsRNA，瞬时转染入Hela细胞，检测RNA干扰效果，筛选有效干扰靶位点；设计并合成针对人smad3基因的siRNA寡核苷酸链，经退火形成双链后克隆入质粒载体pSilencer^TM3．1-H1hygro，转染入Hela细胞，用hygromycinB筛选稳定单克隆细胞株，对细胞株行RT-PCR和Western Blot检测，观察siRNA对靶基因的抑制效果，进一步研究下调目的分子表达水平以后肿瘤细胞生长的变化．结果：瞬时转染筛选到了有效的RNA干扰靶位点，构建载体后，建立稳定转染的单克隆细胞株，smad3mRNA和蛋白质水平均显著下调，导致肿瘤细胞生长抑制．结论：成功构建了针对人Smad3高效、特异、作用持久的shRNA表达载体，转染细胞后可下调Smad3的表达，为进一步研究smad3的功能和肿瘤的基因治疗奠定了基础．     

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.     

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.