人TIMP-2重组腺病毒载体的构建及其在血管平滑肌细胞中的表达

目的:构建含人TI MP-2(hTI MP-2)基因的重组腺病毒载体并在大鼠主动脉平滑肌细胞中表达,为血管疾病基因治疗提供实验基础。方法:利用基因重组技术将腺病毒骨架质粒pAdEasy-1以及线性化的重组穿梭质粒pTrack-CMV-hTI MP-2共转化BJ5183受体菌,并在其中发生同源重组,利用重组前后抗性的改变筛选出重组子;重组腺病毒质粒AdhTI MP-2经过293细胞的包装,扩增和纯化后,测定病毒滴度。腺病毒体外感染大鼠主动脉平滑肌细胞,一步法提取细胞总RNA,RT-PCR检测hTI MP-2的mRNA。并收集细胞上清,进行Western blot检测TI MP-2蛋白。结果:得到了携带hTI MP-2基因的重组腺病毒,纯化后滴度为4×1011efu/ml,在感染VSMC后24h即检测到hTI MP-2的表达。结论:成功地构建了携带hTI MP-2的重组腺病毒载体并体外转染VSMC,为下一步应用于血管疾病基因治疗提供了基础。     

腺病毒介导野生型PTEN基因对乳腺癌MDA MB 468细胞周期的影响

摘要：目的 探讨腺病毒介导野生型PTEN基因对乳腺癌MDA MB 468细胞周期时相的影响。方法 构建含PTEN目的基因的腺病毒穿梭质粒pAdTrack CMV PTEN与骨架质粒pAdEasy 1，将重组腺病毒pAd PTEN制备成纯化高效的含绿色荧光蛋白（GFP）的PTEN腺病毒，体外转染人乳腺癌MDA MB 468后，检测PTEN表达后对乳腺癌MDA MB 468细胞周期的影响。结果 携带PTEN基因重组腺病毒载体成功构建，检测病毒滴度为2.5×1010pfu/mL。表明重组腺病毒已含有PTEN蛋白。流式细胞术显示对照组处于G1期的细胞占41.18%，PTEN蛋白表达后处于G1期的细胞占56.47%。结论 利用腺病毒载体系统AdEasy 1可快速构建表达PTEN基因的腺病毒载体，可高效制备均一的高滴度重组病毒；野生型PTEN基因转染使乳腺癌细胞周期停滞于G1期。     

大鼠PnNOS基因修饰脂肪源性干细胞的构建

目的 构建过表达大鼠阴茎神经源性一氧化氮合酶（PnNOS）基因大鼠脂肪源性干细胞（ADSCs）,为基因修饰ADSCs移植治疗勃起功能障碍（ED）大鼠模型提供种子细胞.方法 获取大鼠PnNOS基因,并与线性化的腺病毒真核表达载体pDC315-EGFP定向克隆连接.构建正确的pDC315-PnNOS-EGFP穿梭质粒转染293 T细胞,采用Western Blot检测PnNOS基因在293 T细胞中的表达情况.利用AdMax腺病毒包装系统包装产生重组腺病毒,包装后扩增纯化并测定病毒滴度.PnNOS基因重组腺病毒转染大鼠ADSCs,观察GFP表达情况和Western Blot检测PnNOS基因表达情况.结果 经PCR鉴定、限制性酶切分析、测序鉴定和目的质粒Western Blot表达检测鉴定,证实pDC315-PnNOS-EGFP重组腺病毒载体构建成功.重组腺病毒包装成功且病毒滴度为5.0×109 PFU/ml.Western Blot于大鼠ADSCs中检测到约161 KDa大小条带,其与PnNOS蛋白分子量大小基本相一致.结论 大鼠PnNOS基因修饰的ADSCs构建成功,从而为基因修饰ADSCs移植治疗ED提供了良好的基因工程细胞.     

大鼠VEGF腺病毒基因转染系统的构建及鉴定

目的:探讨构建携带大鼠血管内皮细胞生长因子(VEGF)的重组腺病毒载体方法，为后续的基因转染研究作准备。方法:采用RT-PCR扩增的方法获取鼠源性的VEGF,并克隆入穿梭质粒pDC316。构建的质粒pDC316-VEGF经酶切及测序鉴定正确后，通过lipofectamine2000的介导与腺病毒包装质粒pBHGE3共转染至人胚肾细胞HEK293，经同源重组后获得携带鼠VEGF的重组腺病毒VDC316-VEGF。应用PCR鉴定重组腺病毒，空斑传代纯化病毒并反复冻融扩增病毒。以50%组织培养感染剂量法(TCID50)测定病毒滴度。结果:PCR鉴定证实重组腺病毒含有鼠VEGF,病毒滴度为3×109pfu/mL。结论:成功构建的携带大鼠VEGF的重组腺病毒载体能在HEK293细胞内扩增获得足够高的病毒滴度,可作为后续基因治疗研究工作中可靠的基因转染工具。     

绿色荧光蛋白和Nel1型蛋白基因共表达腺病毒载体的构建及体外转染大鼠BMSCs的初步实验研究

目的构建同时表达绿色荧光蛋白(green fluorescent protein,GFP)报告基因和人类Nel1型蛋白[homo sapiens NEL-like1,NELL1)]基因的重组腺病毒载体pAdxsi-GFP-NELL1,并转染大鼠BMSCs,观察其表达情况,为进一步研究NELL1蛋白的成骨作用提供理论基础。方法设计特异性引物从NELL1质粒中扩增NELL1,将测序正确的片段用XhoI/BglII酶切处理,定向插入至pShuttle-GFP-CMV(-)TEMP重组穿梭载体,然后将验证正确的重组穿梭质粒中的插入片段转移至pAdxsi载体构建重组腺病毒载体质粒,用PacI限制性内切酶线性化后转染HEK293细胞,扩增纯化重组腺病毒,半数组织培养感染量法测定重组腺病毒滴度。培养大鼠BMSCs,采用流式细胞仪鉴定表面标志物,并行成骨、成脂诱导鉴定。用构建的pAdxsi-GFP-NELL1及空病毒pAdxsi-GFP(作为对照)转染鉴定正确的BMSCs,RT-PCR检测NELL1的表达,免疫荧光检测GFP基因及NELL1的表达情况,细胞计数试剂盒(cell counting kit-8,CCK-8)法检测对细胞增殖的影响。结果成功构建同时表达NELL1和GFP基因的重组腺病毒载体(pAdxsi-GFP-NELL1),纯化后获得滴度达1×1011pfu/mL的重组腺病毒。成功分离获得大鼠BMSCs并传代、纯化,经流式细胞仪鉴定表面标志物及成骨、成脂诱导鉴定为BMSCs。pAdxsi-GFP-NELL1转染BMSCs后,RT-PCR检测示NELL1mRNA阳性表达,荧光显微镜观察示细胞爬片GFP阳性表达,NELL1抗体免疫荧光观察示NELL1蛋白阳性表达。CCK-8法鉴定显示转染后对BMSCs生长无明显影响。结论构建并纯化后的重组腺病毒载体(pAdxsi-GFP-NELL1)可高效转染大鼠BMSCs,并稳定表达NELL1和GFP两种基因,为进一步研究新的成骨基因NELL1的作用,追踪其在体内、体外表达情况提供了新工具。     

人血管内皮细胞生长因子重组腺病毒载体的构建、鉴定及在骨髓间充质干细胞的表达

目的构建人血管内皮细胞生长因子(VEGF)165腺病毒表达载体,体外转染大鼠骨髓间充质干细胞研究其相关特性。方法利用细菌内同源重组技术快速构建Ad-VEGF165腺病毒重组质粒,经酶切及测序鉴定正确后转染人胚肾细胞HEK293包装成为重组Ad-VEGF165腺病毒,并进行电镜观察及滴度测定。感染大鼠骨髓间充质干细胞观察VEGF165基因的转录和表达。结果酶切鉴定及基因测序证实重组腺病毒质粒含有hVEGF165cDNA,电镜显示包装细胞中有大量病毒颗粒存在,测定包装的病毒滴度为6.3×1010TCID50/ml。逆转录-聚合酶链反应(RT-PCR)、免疫组织化学及免疫印迹检测骨髓间充质干细胞内有VEGF165的转录和表达。结论构建的VEGF腺病毒表达载体可有效感染骨髓间充质干细胞,并在体外高效表达,为将表达VEGF基因的骨髓间充质干细胞用于基因治疗提供了实验依据。     

ANG-1基因重组腺病毒载体的构建及其转染骨髓问充质干细胞的实验研究

目的构建携带大鼠血管生成素．1（angiopoietin-1,ANG．1）基因的重组腺病毒载体,并检测其转染对大鼠骨髓间充质干细胞（bone marrow mesenchymal stemcells,BMSCs）活力的影响。方法RT-PCR法获取大鼠ANG-1基因并亚克隆至穿梭质粒pAdTrack-CMV,经测序无误后与骨架质粒pAdEasy-1在BJ5183中同源重组。重组质粒pAdEasy-1-ANG-1经鉴定后转染293细胞进行病毒包装扩增。体外转染BMSCs,检测转染后BMSCs中ANG-1的表达。MTT法评估常氧及缺氧环境下ANG-1对BMSCs的影响。结果重组腺病毒载体pAdEasy-1-ANG-1经测序及酶切鉴定正确;BMSCs经转染ANG-1基因后表达ANG-1。MTT法检测提示常氧及缺氧条件下转染前后BMSCs活性的差异均无统计学意义（缺氧组与缺氧下转染组相比,P〉0-05;常氧组与常氧下转染组相比,P〉0-05）。结论成功构建大鼠ANG-1基因重组腺病毒载体,且其转染在体外不影响BMSCs的活性。     

N-甲基-D-天冬氨酸受体2B亚基基因重组腺病毒镇痛疫苗的构建

目的 构建携带N-甲基-D-天冬氨酸受体2B亚基(NR2B)基因的重组腺病毒镇痛疫苗.方法 以携带NR2B基因的腺病毒穿梭质粒pDC515-NR2B和腺病毒骨架质粒pBHGfrt(del)E1,3FLP共转染腺病毒包装细胞293细胞,连续观察细胞的形态学.取形成病毒空斑的293细胞,分别采用PCR、RT-PCR及Western blotting的方法从基因水平、转录水平和蛋白水平对病毒空斑筛选和鉴定,对鉴定正确的病毒空斑进行扩增和纯化.结果 转染后12 d可观察到细胞病变效应,15 d后形成病毒空斑.NR2B基因不仅正确插入重组腺病毒载体,而且可在293细胞正确表达NR2B蛋白.鉴定正确的病毒空斑命名为NR2B基因重组腺病毒5型镇痛疫苗,滴度为5×1011 VP/ml,纯度100%.结论 成功地构建了NR2B基因重组腺病毒镇痛疫苗,可用于疫苗镇痛的研究.     

重组Akt腺病毒的构建及其在肝硬化大鼠肝脏中的表达

目的:探讨持续活化Akt且带有HA标签(myr-HA-Akt)基因的重组腺病毒在肝硬化大鼠肝脏中的表达特性。方法:酶切正向插入目的基因的真核表达载体pcDNA3.1-myr-HA-Akt,获得myr-HA-Akt cDNA后，将其定向克隆到穿梭质粒pDC316中,然后将重组质粒与病毒骨架质粒pBHGloxΔE1,3Cre共转染293 细胞,获得复制缺陷型重组腺病毒Ad-myr-HA-Akt,并进行扩增、纯化。通过观察腺病毒感染293细胞后是否出现细胞病变效应;PCR和基因测序方法对所构建病毒进行鉴定，并采用TCID50法检测病毒滴度。自尾静脉注射重组腺病毒Ad-myr-HA-Akt感染肝硬化模型大鼠。免疫印迹法检测大鼠肝组织内Akt 和p-Akt蛋白的表达。结果:感染的293 细胞出现明显的细胞病变效应，PCR产物电泳证实重组腺病毒的存在，基因测序证实myr-HA-Akt的cDNA正确插入穿梭质粒且与pBHGloxΔE1,3Cre正确同源重组;病毒滴度为5.5×1011 vp/mL。从蛋白水平证实感染病毒的肝硬化模型大鼠有外源性Akt基因的表达。结论:构建的重组腺病毒Ad-myr-HA-Akt能有效地感染肝硬化模型大鼠,并可在模型大鼠中正确转录和翻译，为进一步研究腺病毒介导的Akt基因对肝硬化的治疗奠定了实验基础。     

应用AdEasy腺病毒载体系统构建人骨形成蛋白2基因重组腺病毒

目的利用AdEasy腺病毒载体系统构建人骨形成蛋白2(BMP2)基因重组腺病毒并在293E细胞中扩增制备重组病毒。方法自人骨形成蛋白2真核表达载体pcDNA3-hBMP2中酶切出hBMP2基因，插入pAdtrackCMV中构建成腺病毒穿梭质粒pAdtrackCMV—hBMP2，经酶切线性化后，采用电穿孔转化到事先电转化腺病毒骨架质粒pAdEasy的BJ5183大肠杆菌电感受态细菌中，挑选同源重组质粒，酶切线性化重组质粒并转染293E细胞包装成重组病毒颗粒，荧光显微镜观察绿色荧光表达。重组病毒上清感染293细胞，荧光显微镜观察绿色荧光表达。结果经限制性内切酶检测和GFP表达证实成功地构建了携带hBMP2基因的重组腺病毒载体并制备出高滴度重组病毒。结论成功地构建了携带hBMP2基因的重组腺病毒载体，为进一步研究rBMP2基因治疗奠定了基础。     

Vasopressor hormone response following mesenteric traction during major abdominal surgery

Background : We investigated the vasopressor hormone response following mesenteric traction (MT) with hypotension due to prostacyclin (PGI₂) release in patients undergoing abdominal surgery with a combined general and epidural anesthesia. Methods : In a prospective, randomized, placebo-controlled study we administered 400 mg ibuprofen (i.v.) in 42 patients scheduled for abdominal surgery. General anesthesia was combined with epidural anesthesia (T4-L1). Before as well as 5, 15, 30, 45, and 90 min after MT we recorded plasma osmolality, hemodynamics and measured 6-keto-PGFlα (stabile metabolite of PGI₂), TXB₂ (stabile metabolite of thromboxane A₂) active renin, and arginine vasopressin (AVP) plasma concentrations by radioimmunoassay. Catecholamine levels were assessed by high-pressure liquid chromatography (HPLC) with electrochemical detection. Results : Following MT, arterial hypotension occurred along with a substantial PGI₂ release. This was completely abolished by ibuprofen administration. Although plasma levels of 6-keto-PGF_1α (1133 (708) vs. 60 (3) ng/L, median (median absolute deviation), P=0.0001, placebo vs. ibuprofen) remained significantly elevated, blood pressure was restored within 30 min after MT in the placebo group. At the same point in time plasma concentrations of TXB² (164 (87) vs. 58 (1) ng/L, P=0.0001), epinephrine (46 (33) vs. 14 (6) ng/L, P=0.001), AVP (41 ± (18) vs. 12 (7) ng/L, P=0.0004), and active renin (27 (12) vs. 12 (4) ng/L, P = 0.001) were significantly higher in placebo-treated patients. Conclusion : Under combined general and epidural anesthesia arterial hypotension following MT due to endogenous PGI₂ release is associated with enhanced release of AVP, active renin, epinephrine and thromboxane A₂, presumably contributing to hemodynamic stability within 30 min after MT.     

Microspheres Based Detoxification System: A New Method in Convective Blood Purification

Abstract: A variety of protein-bound or hydrophobic substances, accumulating as a result of pathologic conditions such as exogenous or endogenous intoxications, are removed poorly by conventional detoxification methods because of low accessibility (hemodialysis), insufficient adsorption capabilities (hemosorption), low efficiency (peritoneal dialysis), or economic limitations (high-volume plasmapheresis). Combining advantages of existing methods with microspheric technology, a module-based system was designed. Major operating parameters of the latter can be modified to allow for adjustment to individual clinical situations. An extracorporeal blood circuit including a plasmafilter is combined with a secondary high-velocity plasma circuit driven by a centrifugal pump. Different microspheric adsorbers can be combined in one circuit or applied in sequence. Thus, a prolonged treatment can be tailored using specially designed selective adsorber materials. Comparing this system with existing methods (high-flux hemodialysis, molecular adsorbent recycling system), results from our in vitro studies and animal experiments demonstrate the superior efficiency of substance removal.     

Bariatric Surgery in Some "Central and East-European (former Eastern bloc)"Countries - Current Status and Prediction for the Next Millennium

Background: Obesity is increasing globallly, including in the formerly "Eastern Bloc" countries. Methods: A survey was made of obesity and bariatric surgery. Results: In the 8 East and Central European countries studied, with total population 300 million, roughly 43% of the population was overweight (BMI 25-30), 23% obese (BMI > 30), with about 15 million people morbidly obese (BMI > 40). From 0-10 morbidly obese individuals/100,000/year undergo bariatric surgery. Conclusion: Most countries were found to provide inadequate treatment for obesity.The majority of the morbidly obese are not treated effectively. However, health-care awareness of obesity and bariatric surgeons are slowly increasing.     

Synergistic interaction between the effects of propofol and midazolam with fentanyl on phrenic nerve activity in rabbits

Background: It has been shown that the depressive effects of both propofol and midazolam on consciousness are synergistic with opioids, but the nature of their interactions on other physiological systems, e. g. respiration, has not been fully investigated. The present study examined the effect of propofol and midazolam alone and in combination with fentanyl on phrenic nerve activity (PNA) and whether such interactions are additive or synergistic. Methods: PNA was recorded in 27 anaesthetised and artificially ventilated rabbits. In three groups, propofol, fentanyl and midazolam were administered intravenously in incremental doses to construct dose-response curves for the depressant effects of each one on PNA. In another two groups, the effect of pretreatment with either fentanyl 1 μg · kg^?1 i. v. or midazolam 0.05 mg · kg^?1 i. v. on the effects of propofol and fentanyl respectively on PNA were studied. Results: Propofol and fentanyl caused a dose-dependent depression of PNA with complete abolition at the highest total doses of 16 mg · kg^?1 i. v. and 32 μg · kg^?1 i. v., respectively. In contrast, midazolam in incremental doses to a total of 0.8 mg · kg^?1 reduced mean PNA by 63%, but approximately 12% of PNA remained at a total dose as high as 6.4 mg · kg^?1. The mean ED₅₀s, calculated from dose-response curves, were 5.4 mg · kg^?1, 3.9 μg · kg^?1 and 0.4 mg · kg^?1 for propofol, fentanyl and midazolam, respectively. Initial doses of either fentanyl 1 μg · kg^?1 i. v. or midazolam 0.05 mg · kg^?1 i. v. acted synergistically with subsequent doses of either propofol or fentanyl to abolish PNA at total doses of 8 mg · kg^?1 and 8 μg · kg^?1, respectively. Conclusion: Fentanyl has a synergistic interaction with both propofol and midazolam on PNA and hence potentially on respiration.     

Influence of dopexamine hydrochloride on haemodynamics and regulators of circulation in patients undergoing major abdominal surgery

Background: Catecholaminergic support is often used to improve haemodynamics in patients undergoing major abdominal surgery. Dopexamine is a synthetic vasoactive catecholamine with beneficial microcirculatory properties. Methods: The influence of perioperative administration of dopexamine on cardiorespiratory data and important regulators of macro- and microcirculation were studied in 30 patients undergoing Whipple pancreaticduodenectomy. The patients received randomized and blinded either 2 μg · kg^?1 · min^?1 of dopexamine (n=15) or placebo (n=15, control group). The infusion was started after induction of anaesthesia and continued until the morning of the first postoperative day. Endothelin-1 (ET-1), vasopressin, atrial natriuretic peptide (ANP), and catecholamine plasma levels were measured from arterial blood samples. Measurements were carried out after induction of anaesthesia, 2 h after onset of surgery, at the end of surgery, 2 h after surgery, and on the morning of the first postoperative day. Results: Cardiac index (CI) increased significantly in the dopexamine group (from 2.61±0.41 to 4.57±0.78 1 · min^?1 · m^?2) and remained elevated until the morning of the first postoperative day. Oxygen delivery index (DO₂I) and oxygen consumption index (VO₂I) were also significantly increased in the dopexamine group (DO₂I: from 416±91 to 717±110 ml/m² · m²; VO₂I: from 98±25 to 157±22 ml/m² · m²), being significantly higher than in the control group. pHi remained stable only in the dopexamine patients, indicating adequate splanchnic perfusion. Vasopressive regulators of circulation increased significantly only in the untreated control patients (vasopressin: from 4.37±1.1 to 35.9±12.1 pg/ml; ET-1: from 2.88±0.91 to 6.91±1.20 pg/ml). Conclusion: Patients undergoing major abdominal surgery may profit from prophylactic perioperative administration of dopexamine hydrochloride in the form of improved haemodynamics and oxygenation as well as beneficial influence on important regulators of organ blood flow.     

Systemic analgesia adapted to the children's condition

A concept of balanced analgesia using nonsteroidal anti-inflammatory drugs (NSAIDs), paracetamol (acetaminophen), opioids, and corticosteroids can also be used in patients with pre-existing illnesses. NSAIDs are the most effective treatment for acute pain of moderate intensity in children; however, these drugs should be avoided in patients at increased risk for serious side effects, e.g. patients with renal impairment, bleeding tendency, or extreme prematurity. NSAIDs can be given with minimal risks to the younger child with mild to moderate asthma, and, in these patients, the use of steroids can be encouraged; in addition to their antiemetic and analgesic action, a beneficial effect on asthma symptoms can be expected. In the non-intubated child with cerebral trauma, exaggerated sedation caused by opioids and increased bleeding tendency caused by NSAIDs must be avoided. In neonates and small infants, the oral administration of sucrose or glucose is helpful to minimize pain reaction during short uncomfortable interventions.     

Intraoperative autotransfusion is associated with modest reduction of allogeneic transfusion in prosthetic hip surgery

Background: The efficacy of intraoperative salvage and washing of wound blood and the predictors of allogeneic red cell transfusions in prosthetic hip surgery are insufficiently known.
Methods: In 96 patients, undergoing primary or revision surgery, salvaged and washed red cells and, if necessary, allogeneic blood were used to keep haematocrit not lower than 33%. The bleeding of red cells during hospital stay was calculated from the red cell balance. The preoperative red cell reserve (millilitres of red cells in excess of a haematocrit of 33%) was estimated and the difference between this volume and the total bleeding of red cells was retrospectively used to classify patients with regard to the need for red cells. Stepwise regression analysis was used to define patient-related variables associated with allogeneic blood transfusion.
Results: Preoperative knowledge of the type of operation (primary, revision), the preoperative red cell reserve, and the body mass could predict roughly half of the need for banked blood (r²=0.45). Only one-third of the total bleeding of red cells was retransfused. For complete avoidance of allogeneic blood, autotransfusion was most effective in patients with a moderate need (0–4 u). However, 32% of such patients required allogeneic blood.
Conclusions: Autotransfusion has a limited efficacy to decrease the need for allogeneic blood, and other blood-saving methods should be added for this purpose. It is difficult to predict the need for allogeneic blood preoperatively.     

缺氧对肾小管上皮细胞分泌外泌体的影响

目的    观察缺氧对肾小管上皮细胞分泌外泌体的影响,探讨外泌体在缺氧致肾脏损伤中的作用及机制。 方法    （1）常氧（21% O₂）及缺氧（1% O₂）分别处理大鼠肾小管上皮细胞（NRK-52E）48 h,收集细胞上清液并使用高速梯度离心法分离外泌体。采用透射电镜、纳米示踪分析、Western印迹、蛋白浓度定量鉴定并比较两组外泌体的基本特性。（2）在共培养实验中,以不同浓度（1、10、50、100、300 mg/L）的常氧外泌体、缺氧外泌体分别干预脂多糖（LPS）诱导的大鼠原代腹腔巨噬细胞,使用实时荧光定量PCR与酶联免疫吸附试验（ELISA）法分别检测巨噬细胞白细胞介素6（IL-6）、肿瘤坏死因子α（TNF-α）、诱导型氮氧化物合酶（iNOS）水平;使用Western印迹法检测巨噬细胞磷酸化（p）STAT/STAT及细胞因子信号传导抑制蛋白1（SOCS1）的蛋白表达;最后,使用实时荧光定量PCR法检测常氧外泌体与缺氧外泌体中炎性反应相关微RNA（microRNA,miR）的表达差异。 结果    （1）离心得到的囊泡具有外泌体典型的结构,粒径小于150 nm,表达外泌体标志蛋白CD63,说明分离得到外泌体。缺氧对肾小管上皮细胞分泌的外泌体形态、粒径分布比例无明显影响,但提高了外泌体的分泌量。（2）缺氧外泌体相比于常氧外泌体促进了LPS诱导的M1型巨噬细胞IL-6、TNF-α、iNOS 的表达和分泌（均P＜0.01）,同时提高STAT的磷酸化水平并减少SOCS1的蛋白表达（均P＜0.01）;对炎性反应相关microRNA检测发现缺氧外泌体中miR-155、miR-27a表达量较常氧外泌体明显升高（P＜0.05）。 结论    缺氧可改变外泌体的生物学功能,表现为协同促进LPS诱导的M1型巨噬细胞的表型转化,这可能是慢性肾脏病微炎性反应状态持续的原因之一。     

In Vitro Function and Durability Assessment of a Novel Polyurethane Heart Valve Prosthesis

Abstract While flexible-leaflet, central-flow prosthetic heart valves promise relief from anticoagulation therapy, they continue to be restricted by inadequate durability. In consequence, a novel trileaflet valve, made entirely from polyurethane, has been developed. A batch of 6 consecutively manufactured polyurethane valves was subjected to hydrodynamic function and accelerated fatigue testing. Computerized data acquisition and control systems have been introduced to improve valve testing methodologies. In terms of hydrodynamic function, the polyurethane valve demonstrates transvalvular pressure gradients similar to those for a bioprosthetic valve (Carpentier-Edwards) and levels of retrograde flow significantly less than those for either the bioprosthetic valve or a bileaflet mechanical valve (St Jude Medical). The equivalent of 10 years of cycling without failure has been exceeded by all 6 polyurethane valves in accelerated fatigue tests with 2 valves remaining intact after 674 million cycles (equivalent to approximately 17 years) in continuing tests. Highspeed photography revealed considerable differences in leaflet motion between valves cycled at accelerated and physiological rates.     

Negative pressure ventilation vs. spontaneous assisted ventilation during rigid bronchoscopy: A controlled randomised trial

Background: Ventilation during interventional rigid bronchoscopy (IRB) under general anaesthesia (jet ventilation, positive pressure ventilation and spontaneous assisted ventilation) may offer some difficulties. This study compares the effectiveness during IRB of intermittent negative pressure ventilation (INPV) and spontaneous assisted ventilation (SAV). Methods: Thirty-eight patients submitted to IRB were randomised into two groups: SAV or INPV. All patients received a total intravenous anaesthesia; INPV patients were paralysed. Pre-and intra-operative arterial blood gases and O₂ flow through a rigid bronchoscope were assessed. The endoscopist applying a subjective score evaluated the operating conditions. Results: Patients of the INPV group, as compared to the SAV group, required a lower dosage of fentanyl (2.6 ± 1.8 (μg · kg^?1· h^?1 vs. 6.6 ± 4.8 μg · kg^?1· h^?1), a lower O₂ supply (3.3 ± 2.8 1/min vs. 11.6 ± 3.4 1/min), a shorter recovery time (5.4 ± 2.9 min vs. 9.8 ± 7.1 min) and no manually assisted ventilation (0 ± 0 vs. 1 ± 1.1 nd?/procedure). Intraoperative PaCO₂ was higher in the SAV (8.1 ± 1.3 kPa) than in the INPV group (5.0 ± 1.6 kPa) and intraoperative pH differed in the two groups (7.26 ± 0.05, SAV vs. 7.47 ± 0.08, INPV). Operating conditions, as assessed by a subjective score, were considered better with INPV than with SAV (4.9 vs. 4.3). Conclusions: As compared to SAV, INPV in paralysed patients during IRB reduces administration of opioids, shortens recovery time, prevents respiratory acidosis, excludes the need for manually assisted ventilation, reduces 02 need and affords optimal surgical conditions. INPV appears a safe, non-invasive and effective ventilatory management during IRB.