生长抑素与三苯氧胺协同抗乳腺癌的体外实验研究

目的 研究生长抑素类似物奥曲肽（OCT）和三苯氧胺（TAM）联合抗乳腺癌的作用。方法 在体外培养条件下,分别或联合用OCT、TAM作用于雌激素受全（ER）阳性（MCF－7）和阴性（MDA－MB－435S）人乳腺癌细胞株,应用MTT比色法分析细胞生长抑制作用,流式细胞术测定细胞周期分布和凋亡率,透射电镜观察细胞超微结构。结果 OCT和TAM均可抑制MCF－7细胞生长、阻滞细胞周期于G0／G1期,并可诱导细胞凋亡;当两药联合应用时,上述作用得到显著加强,凋亡率明显上升。OCT和TAM对MDA－MB－435S细胞也有弱的抑制作用,并阻滞细胞周期于不同时相,而未发现明显的凋亡诱导,但两药联合应用,可诱导22.7%的细胞凋亡。结论 OCT和TAM联合应用对ER阳性和ER阴性乳腺癌细胞均具有协同抑制生长和诱导凋亡作用,可能提高乳腺癌的临床疗效。     

5氮2脱氧胞苷与三苯氧胺协同抗雌激素受体α阴性乳腺癌的体外实验研究

目的观察5氮2脱氧胞苷（5-aza—CdR）对雌激素受体α（ERα）阴性人类乳腺癌细胞株MDA—MB-231和MDA—MB-435ERα基因诱导表达作用;5-aza—CdR联合三苯氧胺（TAM）对ERα阴性乳腺癌细胞的体外抑制作用。方法应用甲基化特异性PCR（MSP）检测MDA—MB-231和MDA—MB-435细胞和20例ERα阴性乳腺癌组织ERα基因核心启动子区CpG岛甲基化情况;5-aza—CdR处理MDA—MB-231和MDA—MB-435细胞,逆转录PCR（RT—PCR）检测ERαmRNA表达;5-aza—CdR、TAM分别或联合作用于MDA—MB-231和MDA—MB-435细胞,M1T比色法分析细胞生长抑制作用,流式细胞仪测定细胞周期分布和凋亡率。结果适当浓度的5-aza—CdR能诱导MDA—MB-231和MDA—MB一435细胞表达ERctmRNA,抑制细胞生长、阻滞细胞周期于G0／G1期,诱导细胞凋亡;TAM对MDA—MB-231和MDA—MB-435细胞生长、细胞周期无影响;而两药联合时能显著抑制细胞生长,诱导细胞凋亡,凋亡率分别为48、8％和53、1％。结论5-aza—CdR能诱导ERα阴性乳腺癌表达ERctmRNA,恢复ERα阴性乳腺癌细胞对TAM的敏感性,联合TAM能协同抑制ERα阴性乳腺癌细胞生长,诱导细胞凋亡,从而为ERα阴性乳腺癌开辟新的内分泌治疗途径提供实验依据。     

丝裂霉素C与C2-神经酰胺联合应用治疗膀胱癌

目的 观察丝裂霉素C（MMC）与C2-神经酰胺（C2-cer）联合应用对人膀胱癌细胞的作用效果，并探讨其机制。方法 不同浓度MMC与C2-cer单独及联合作用于人膀胱癌BIU-87细胞后，应用噻唑蓝（MTT）比色法检测细胞生长抑制率，计算合用指数（CI），流式细胞仪（FCM）检测BIU-87细胞凋亡率，吖啶橙（AO）荧光染色观察凋亡形态学变化，Western blot检测细胞色素C在细胞内分布变化，并检测Caspase-3活性改变。结果 单独应用时MMC与C2-cer的中效浓度分别是159和28μmol／L，联合用药时下降为55和11μmol／L，CI=0．74。MMC与C2-cer单独及联合应用均可导致BIU-87细胞出现凋亡的形态学变化。两种药物联合应用时的凋亡率高于各自单用（P＜0．05）。线粒体细胞色素C含量在MMC与C2-cer单独及联合应用时均较对照组减少，联合用药时减少最为明显，细胞质内细胞色素C含量在联合用药时增加也最为明显（P＜0．05）。Caspase-3活性在MMC与C2-cer单独及联合应用时均较对照组升高，联合用药时升高最为明显（P＜0．05）。结论 MMC与C2-cer联合应用可以通过共同诱导细胞凋亡，协同抑制膀胱癌细胞生长。线粒体细胞色素C释放和Caspase-3活性变化可能发挥重要作用。     

雌激素受体在人乳腺癌MCF-7细胞阿霉素敏感株和耐药株中的变化及意义

目的研究MCF 7人乳腺癌细胞阿霉素敏感株 (MCF 7)和耐药株 (MCF 7/ADR)细胞中雌激素受体 (estrogenreceptor,ER)表达的变化与其对细胞生物学特性的影响。方法采用Westernblot法检测MCF 7细胞和MCF 7/ADR细胞中雌激素受体表达 ,RT PCR检测 2株细胞ERmRNA的表达水平 ,MTT法检测细胞增殖及细胞对雌激素 (estrogen ,E2 )和屈洛昔芬 (droloxifene ,Dro)的敏感性 ,流式细胞仪检测细胞周期变化。结果经Westernblot检测 ,MCF 7细胞ER为阳性 ,而MCF 7/ADR细胞ER为阴性 ,采用RT PCR可检测到MCF 7细胞中ER的mRNA ,而在MCF 7/ADR细胞则检测不到。经MTT分析 ,与MCF 7相比 ,MCF 7/ADR细胞生长速度减慢 ,细胞多分布于G0 /G1期。E2 在 1× 10 -12mol/L的浓度时开始促进MCF 7细胞的生长 ,到达 1× 10 -9mol/L时促生长作用进入平台期 ;而任何浓度的E2 对MCF 7/ADR细胞均无促生长作用。Dro的浓度达到 10 μmol/L的时候 ,开始出现对MCF 7细胞生长的浓度依赖性抑制 ,当Dro浓度达到 2 0 μmol/L时对MCF 7/ADR细胞的生长出现明显抑制 ,且高于对MCF 7细胞的抑制。结论MCF 7/ADR细胞雌激素受体表达缺失 ,缺失发生在mRNA水平以上 ,细胞生长速度减慢 ,同时细胞失去对雌激素的依赖性。     

联合应用胸苷激酶基因和胞嘧啶脱氨酶基因对MCF-7细胞的杀伤作用

目的研究双自杀基因(胞嘧啶脱氨酶基因CD和胸苷激酶基因HSV-tk)的应用对乳腺癌细胞的杀伤作用和旁观者效应。方法脂质体法将CD和HSV-tk双自杀基因转染入PA317细胞，用其病毒上清转染乳腺癌细胞MCF-7，G418筛选出阳性细胞MCF-7／CD tk，然后加入不同浓度的5-氟胞嘧啶(5-Fc)或／和丙氧鸟苷(GCV)，MTT法观察其杀伤作用；将不同比例的MCF-7／CD tk和MCF-7细胞混合，联合应用5-Fc和GCV杀伤，观察其旁观者效应。结果单独应用5-Fc或GCV均能对MCF一7／CD tk细胞产生明显的杀伤效应，联合应用可以在较小的剂量下产生更大的杀伤作用；当阳性细胞比例达到20％时即可对半数混合细胞产生杀伤作用。结论双自杀基因的应用对乳腺癌细胞具有明显的杀伤作用．其旁观者效应明显。     

表皮生长因子对ER阴性乳腺癌细胞株细胞周期的影响

目的　研究表皮生长因子 (epidermalgrowthfactor,EGF)对雌激素受体 (ER)阴性乳腺癌细胞株MDA MB 4 35S细胞周期的影响。方法　采用Westernblot检测MDA MB 4 35S细胞周期蛋白D1(cyclinD1 )的表达 ,用流式细胞技术检测细胞周期的变化。结果　EGF促cyclinD1 表达作用显著 ,蛋白激酶C抑制剂Go6 .976能抑制核转录因子κB(NF κB)的激活 ,并能抑制EGF的促cyclinD1 表达作用 ;EGF组G0/ G1 期 6 9 .36 %,S期 2 2 . 77%,细胞增殖指数 (PI) 0 . 31 ,与对照组比较差异有统计学意义 (P <0. 0 5 ) ,加用Go6 976后 ,G0 /G1 期明显上升达 91. 5 4 %,S期比例降至 7. 81 %,PI0. 0 9,与EGF组比较差异有统计学意义 (P <0 .0 5 )。结论　EGF促进MDA MB 4 35S的cyclinD1 表达 ,使肿瘤细胞进入DNA合成期。Go6 976可以抑制NF κB的活性。     

胰腺癌的辅助化疗

胰腺癌的发病率近年来在世界范围内有逐年增高的趋势 ,虽然手术是治疗胰腺癌的根本手段 ,但目前胰腺癌手术切除率仅为 10 %～ 15 % ,因此围手术期化疗、放疗等综合治疗尤为重要。目前使用的化疗药物中 5 氟脲嘧啶 (5 Fu)和丝裂霉素(MMC)的抗胰腺癌作用较为肯定 ,广为临床应用。目前各化疗方案仍主要以 5 Fu为基础 ,最常用的方案有 :5 Fu 阿霉素(ADM ) MMC ;5 Fu 顺铂 (DDP) ;5 Fu 健择 (GEM )等。近期对胰腺癌患者应用新一代细胞毒性药物健择的临床研究十分流行 ,Burris等针对晚期胰腺癌患者应用健择和 5 Fu进行了Ⅲ期…     

雌二醇、三苯氧胺对乳腺癌和子宫内膜细胞增殖的影响

目的　通过雌二醇 (E2 )、三苯氧胺 (TAM )作用于乳癌及子宫内膜细胞 ,研究这两种因素对雌激素受体 (ER)阳性细胞的增殖作用 ,探讨乳癌TAM耐受的产生与细胞来源的关系。方法1× 1 0 - 6 mol/LTAM、1× 1 0 - 8mol/LE2 作用于乳癌及子宫内膜细胞 ,利用3H TdR示踪细胞增殖动力学。结果　TAM对子宫内膜细胞增殖 (6 .32 % )的抑制作用与对照组 (6 .40 % )比较差异无显著性 (P <0 .0 1 ) ,TAM对乳癌细胞增殖 (45 .84% )的抑制作用与对照组 (52 .72 % )比较差异有显著性 (P <0 .0 1 ) ,但其对TAM耐受的乳癌细胞增殖 (9.64 % )与对照组 (6 .32 % )比较有显著刺激作用 ;E2 对 3种细胞增殖的刺激作用与对照组比较差异都有显著性。E2 与TAM联合作用于子宫内膜及乳癌细胞与单独使用E2 时相比 ,表现为抑制细胞增殖的作用 ;但它们联合作用于TAM耐受乳癌细胞时表现出比E2 更大的刺激细胞增殖的作用。结论　E2 对 3种来源细胞都表现为刺激增殖的作用 ,但TAM抑制乳癌细胞增殖的作用则依赖E2 的浓度水平而不同     

雌激素水平对三苯氧胺抑制乳腺癌细胞的体外研究

目的探讨雌激素水平对三苯氧胺(tamoxifen,TAM)抑制乳腺癌细胞MCF-7(ER阳性)生长的影响。方法应用不同浓度的雌二醇(estradiol,E2)和10μmol/L浓度的TAM作用于MCF-7细胞,应用四甲基偶氮唑蓝(MTT)比色法分析其对细胞生长的抑制作用。结果用1×10-12mol/L的E2开始刺激MCF-7细胞生长,到1×10-9mol/L时细胞生长达到平台期。10μmol/L的TAM可以明显抑制细胞生长,不同浓度的E2可以影响TAM对MCF-7细胞生长的抑制作用。当E2浓度达到1×10-8mol/L时,TAM对细胞生长的抑制作用最弱。结论E2是乳腺癌的危险因素,肿瘤细胞周围E2的浓度可以影响TAM的疗效。     

亚甲蓝对兔肝缺血再灌注损伤的影响

目的 探讨亚甲蓝对兔肝缺血再灌注损伤的影响.方法 健康成年新西兰大白兔24只,雌雄不拘,体重2.0～2.3 kg,随机分为3组(n=8):假手术组(S组)、肝缺血再灌注组(I/R组)和亚甲蓝组(MB组).I/R组及MB组采用夹闭肝左外叶、中叶、右中叶及方形叶肝动脉分支40min再灌注60 min的方法制备肝缺血再灌注模型,S组仅游离相应血管.MB组于再灌注前20 min经耳缘静脉注射亚甲蓝5 mg/kg(用生理盐水稀释至5 ml),S组及I/R组给予等容量生理盐水.于缺血前即刻(T1)、缺血20 min(T2)、加min(T3)、再灌注1 min(T4)、再灌注5 min(T5)、30 min(T6)、60 min(T7)时记录MAP和HR.于T1,5-7时取股动脉血样1 ml,测定血清TNF-α及IL-6的浓度.分别于T1,6,7时取股动脉血样1.5 ml,测定血浆ALT及AST的活性.于T7时测定肝左叶组织SOD活性及MDA含量,光镜下观察肝组织病理学结果.结果 与S组比较,I/R组T4-7,时MAP降低,T7时HR降低,肝组织SOD活性降低,MDA含量升高,I/R组及MB组T3-5时血清TNF-α和IL-6浓度升高,T6,7时血浆ALT和AST活性升高(P<0.05或0.01);与I/R组比较,MB组T4-7时MAP升高,肝组织SOD活性升高,MDA含量降低,T3-5时血清TNF-α和IL-6浓度降低,T6,7时血浆ALT和AST活性降低(P<0.05或0.01).MB组肝组织损伤较I/R组减轻.结论 亚甲蓝可维持血液动力学稳定,减轻兔肝缺血再灌注损伤.     

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.     

A comparison of the inhibitory effects of four volatile anaesthetics on the metabolism of chlorzoxazone, a substrate for CYP2E1, in rabbits

Background: Halothane inhibits in vitro and in vivo activity of cytochrome P-450 (CYP) 2E1. There are several fluorinated volatile anaesthetics besides halothane, and most of them are defluorinated by CYP2E1. It is unclear whether other fluorinated anaesthetics inhibit the in vivo activity of CYP2E1.
Methods: We compared the inhibitory effects of therapeutic concentrations of four inhalational anaesthetics, halothane, enflurane, isoflurane, and sevoflurane, on chlorzoxazone metabolism in rabbits receiving artificial ventilation.
Results: All four inhalational anaesthetics decreased arterial blood pressure and increased plasma chlorzoxazone concentration. However, no significant differences in the plasma chlorzoxazone concentration were found between the four anaesthetics. The estimated chlorzoxazone clearance increased after beginning inhalation with all four agents, but no significant difference in clearance was noted between agents.
Conclusions: At therapeutic concentrations, the in vivo inhibitory effect on chlorzoxazone metabolism was similar for all four inhalational anaesthetics examined, even though their chemical characteristics and extent of hepatic metabolism differ considerably.     

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.