特异性阻断MecR1-MecI-MecA通路逆转耐甲氧西林金黄色葡萄球菌耐药性

目的:研究互补于MRSA的耐药基因mecR1 mRNA的PS-ODNs6088对MRSA耐药性的影响.方法:利用电穿孔的方法将PS-ODNs6088导入MRSA内,平板克隆形成实验计数菌落数(CFU);微量法测定细菌的生长曲线;RT-PCR法检测目的基因mecRl和mecA的表达变化.结果:PS-ODNs6088能明显抑制MRSA的生长,PS-ODNs6088各剂量组MRSA的CFU与空白对照组比较明显减少(P＜0.05),且具有剂量依赖性,而随机对照组的菌落数却无明显减少;PS-ODNs6088能显著抑制MRSA mecR1和mecA的表达.结论:反义硫代寡核苷酸能特异的与mecRl mRNA结合并阻断其表达,从而阻断了MRSA耐药基因表达信号通路,减少MRSA的菌落数,抑制其生长,提示反义寡核苷酸能有效的逆转MRSA对β-内酰胺类抗生素的耐药性.利用反义技术阻断细菌的耐药信号通路可能成为解决细菌耐药问题的一种新策略.     

多重耐药鲍曼不动杆菌gyrA基因高效反义肽核酸序列筛选及其体外抗菌活性观察

目的 筛选出能与多重耐药鲍曼不动杆菌gyrA基因的mRNA紧密结合的反义肽核酸序列,评估其体外抗菌活性.方法 利用Mfold、RNA Structure 4.6两种计算机软件对多重耐药鲍曼不动杆菌gyrA基因的mRNA进行二级结构分析计算,根据最低自由能原则在其mRNA局部杂交松弛区设计反义寡核苷酸,与体外转录的地高辛标记的gyrAmRNA进行斑点杂交,根据杂交信号的强弱筛选出与gyrA mRNA紧密结合的反义寡核苷酸,根据其序列合成肽核酸(pep-tide nucleic acid,PNA),另加设1条与靶序列有6个碱基错配的PNA作为阴性对照,2条PNA分别连接穿膜肽形成肽-肽核酸(peptide-PNA,PPNA).测定经不同浓度PPNA处理的细菌光密度[D(600)]值并进行平板菌落计数,观察其对细菌生长的抑制作用,采用RT-PCR检测gyrA mRNA的表达水平.结果 斑点杂交结果显示,计算机软件设计的10条反义寡核苷酸探针中有5条与gyrA mRNA显示出杂交信号,其中1条信号最强,能够与gyrA mRNA稳定结合,将其以肽-肽核酸的形式合成处理细菌,结果表明其在5 μmol/L浓度可完全抑制gyrA mRNA的表达,并抑制鲍曼不动杆菌的生长,在10μmol/L浓度具有杀菌活性,而具有错配碱基的PPNA对细菌生长无明显抑制作用.结论计算机辅助设计联合斑点杂交可在体外模拟体内环境对寡核苷酸与靶基因结合的有效性进行验证,实现在体外高通量筛选高效反义序列.经筛选得到的靶向gyrA基因反义肽-肽核酸可在体外高效抑制多重耐药鲍曼不动杆菌生长.     

ino1反义寡核苷酸对结核分枝杆菌的抑制作用

目的选取合适的靶点，探讨利用反义技术抑制结核杆菌的新方法。方法选取分枝杆菌ino1基因为靶基因，设计并合成3段反义寡核苷酸，将不同浓度的反义寡核苷酸加入细菌的培养液中，观察ino1的mRNA表达情况，结核杆菌分枝硫醇的水平以及结核杆菌的生长情况。结果当反义寡核苷酸的浓度达到20μmol/L时，可以特异性地抑制ino1的表达，此浓度水平时ino1 mRNA的量和对照组的比值为0.67，40μmol/L时的比值则为0.45。分枝硫醇的合成以及分枝杆菌的生长也相应地受到抑制。反义寡核苷酸的浓度为20μmol/L时，分枝杆菌浓度的对数值下降1.3，40μmol/L时下降1.7。结论利用反义技术抑制分枝杆菌是完全可行的，并且ino1可以作为反义寡核苷酸的靶基因。     

Livin反义寡核苷酸对肺腺癌A549细胞增殖的影响

目的：探讨Livin基因反义寡核苷酸（antisense oligodeoxynucleotides,ASODN）对肺腺癌A549细胞增殖的影响。方法：用阳离子脂质体介导Livin ASODN转染至A549细胞,反转录-聚合酶链反应（RT—PCR）检测Livin基因mRNA的表达;MTT法检测转染细胞的生长抑制情况。结果：转染ASODN后A549细胞的Livin基因mRNA的表达明显减少,Livin基因ASODN显著抑制A549细胞增殖,并呈剂量依赖性,与对照的错义寡核苷酸（missense oligodeoxynucleotides,MSODN）比较差异有统计学意义（P〈0．05）。结论：Livin反义寡核苷酸能明显抑制A549细胞Livin基因mRNA的表达,抑制细胞增殖,Livin基因有望成为肺癌基因治疗的新靶点。     

靶向人Ku70 mRNA反义核苷酸的优化设计与筛选

目的：联合应用RNAdraw和Sfold软件设计并筛选低毒且高效抑制Hela细胞Ku70 mRNA表达的反义寡核苷酸。方法：从GenBank获得人Ku70 mRNA全序列，联合应用RNAdraw与Sfold软件，根据最小自由能原理设计多条20 nt的反义寡核苷酸，脂质体转染后MTT法检测其非序列特异性细胞毒性，选择毒性最小者并转染Hela细胞，RT-PCR检测转染前后Ku70 mRNA的表达水平并进行比较。结果：靶向Ku70 mRNA设计6条20 nt的反义寡核苷酸，其中NO.1 序列非特异性毒性最低，对Hela细胞的生长抑制率（50、100和200 nmol•L^-1浓度组分别为4.8%、9.5%和10.2%）与脂质体组比较差异无显著性(P>0.05)，用毒性最低序列转染Hela细胞后使Ku70 mRNA表达水平显著降低(P<0.01)，条带强度和面积与未转染组比较分别下降81.6%和77.6%。结论：联合应用计算机软件RNAdraw和Sfold对设计反义核酸有较大的指导意义，能实现较高的命中率，设计的反义寡核苷酸能有效抑制Hela 细胞Ku70mRNA的表达。     

经小脑延髓池局部应用反义寡核苷酸对脑组织TNF-α表达的影响

目的：观察TNF－α反义寡核苷酸对TNF－α的阻断作用。方法：用DNA合成仪合成17聚体的TNF－α正义寡核苷酸和反义寡核苷酸。24只犬随机分为对照组和实验组（包括人工脑脊液组、正义链组和反义链组）。实验组动物用已建成的颅脑爆炸伤模型装置致伤,伤前经小脑－延髓池分别注入人工脑脊液、TNF－α正义寡核苷酸和TNF－α反义寡核苷酸,比较3组动物脑组织TNF－αmRNA表达的变化。结果：与人工脑脊液组和正义链组相比,伤前经小脑－延髓池脑内局部应用TNF－α反义寡核苷酸组,颅脑爆炸伤后脑组织内TNF-αmRNA表达量明显减少（P＜0．01）。结论：经小脑延髓池局部应用反义寡核苷酸可有效阻断颅脑爆炸伤后脑组织TNF－α的表达。     

Survivin反义核酸对乳腺癌细胞增殖的影响

目的：探讨Survivin反义核酸对乳腺癌细胞增殖的影响。方法：本实验分6组，分别以脂质体介导反义寡核苷酸、无义寡核苷酸及RPMI 1640培养液作为空白的对照组，转染人乳腺癌MCF-7细胞系，采用Realtime RT-PCR，Western blot方法检测各组MCF-7细胞Survivin蛋白及mRNA的表达情况，测定转染后细胞贴壁率变化，研究Survivin反义寡核苷酸对MCF-7细胞的生长抑制作用。结果：脂质体介导Survivin反义寡核苷酸转染人乳腺癌MCF-7细胞后，细胞内Survivin蛋白及mRNA表达显著降低，通过Realtime RT-PCR检测MCF-7细胞经作用后其Survivin mRNA起始拷贝数明显下降；同时细胞贴壁率降低达32．96％，细胞生长抑制率最高达73．62％，ASOND／LiP组与NODN／LiP组和LiP组差异有显著性（P＜0．05）。结论：脂质体介导转染的反义寡核苷酸可在Survivin蛋白及mRNA水平有效地降低乳腺癌MCF-7细胞内Survivin的表达，并能够有效抑制乳腺癌细胞的增殖活性，提示Survivin靶向治疗可能成为乳腺癌综合治疗的一种重要方法。     

Stat3信号传导通路对结肠癌细胞G1～S期的调控

目的：探讨Stat3信号传导通路对结肠癌细胞G1-S期调控的可能机制。方法：应用阳离子脂质体介导Stat3反义寡核苷酸转染人结肠癌SW480与HCT116细胞，阻断其Stat3通路；MTT法检测细胞增殖状态；流式细胞术检测细胞周期；Western blot检测Stat3、磷酸化Stat3、G1期Cyclins、CDKs、p21、p27的表达。结果：转染Stat3反义寡核苷酸后结肠癌细胞增殖受抑制，Stat3、磷酸化Stat3、G1期Cyclins、CDKs表达水平下降，p21与p27表达水平上升。结论：Stat3信号传导通路可能通过调节CDK/Cyclin复合物与CK1成员之间的平衡而调节结肠癌细胞G1-S期转换。     

血管内皮生长因子反义寡核苷酸体外抑制皮肤血管瘤内皮细胞的增殖

目的 观察血管内皮生长因子（VEGF）反义寡核苷酸对皮肤血管瘤内皮细胞体外增殖及生物学特性的影响,并探讨其机制。方法 体外培养皮肤血管瘤内皮细胞;脂质体介导VEGF反义寡核苷酸进行转染;MTT法观察其对内皮细胞生长的抑制;免疫细胞化学技术检测VEGF的蛋白表达;流式细胞仪分析细胞周期变化。结果 VEGF反义寡核苷酸作用后的内皮细胞生长明显受到抑制,作用后48hVEGF蛋白表达明显下降,细胞周期中S期细胞数明显减少,并于72h后抑制效应逐渐减弱。结论 VEGF反义寡核苷酸能显著抑制皮肤血管瘤内皮细胞的生长,其机制是抑制细胞的增殖,而不是促进其凋亡。     

脂质体介导的99mTc标记c-myc mRNA反义寡核苷酸的反义作用效果的研究

目的探讨脂质体介导的99mTc标记癌基因c-myc mRNA的反义寡核苷酸能否抑制癌细胞的生长和癌蛋白的表达.方法合成15 bp的癌基因c-myc mRNA的反义、正义及无义寡核苷酸,用99mTc标记后(简称99mTc-DNA),一部分用脂质体包裹,以不同放射性强度的99mTc-DNA及脂质体包裹的99mTc-DNA转染细胞,于转染后不同时间测定细胞摄取率,在转染后18 h测定细胞返流比率,用四唑盐比色实验检测反义抑制细胞的生长状况,用流式细胞术测定癌蛋白的表达.结果在1～5 h内,细胞的摄取率随时间的延长而增加,脂质体包裹的99mTc-DNA的细胞摄取率明显高于未用脂质体包裹的99mTc-DNA.四唑盐比色实验,随着脂质体介导的99mTc-反义DNA剂量的增加,细胞数量逐渐减少,而正义、无义寡聚核苷酸组,细胞数量则没有减少的趋势.细胞的返流比率,反义、正义、无义寡核苷酸分别为39.51%、44.12%、63.92%.测定癌蛋白的荧光强度,反义寡核苷酸组2.9860±0.3733、正义寡核苷酸组4.2600±0.2218、无义寡核苷酸组5.2620±0.8562,反义寡聚核苷酸组的荧光强度明显低于正义和无义寡聚核苷酸组.结论脂质体介导的99mTc标记的反义寡核苷酸能抑制癌细胞的生长和癌蛋白的表达.     

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.