bcl-2 mRNA、baX mRNA表达在三氧化二砷诱导肝癌细胞凋亡中的意义

目的 分析三氧化二砷(As2O3)诱导肝癌细胞凋亡中凋亡相关基因bcl—2 mRNA、bax mRNA表达的意义。方法 采用电镜、流式细胞仪、电泳及半定量RT—PCR方法检测不同剂量三氧化二砷诱导人肝癌细胞株bel—7402后凋亡的出现及bcl-2mRNA、bax mRNA的表达。结果 0．5にmol/L As2O3处理肝癌细胞未见凋亡，8μmol/L As2O3诱导肝癌细胞出现明显凋亡。对照组和药物组未检测到bcl-2 mRNA的表达，随浓度增加，bax mRNA的表达上调。结论 As2O3通过诱导凋亡抑制肝癌细胞株bel-7402的增殖，凋亡诱导基因bax mRNA的表达上调与此过程有关。bcl—2mR—NA表达与此过程无关。     

Ki-67基因siRNA表达质粒的构建、鉴定及功能研究

目的构建肿瘤增殖基因Ki-67小干扰RNA(siRNA)表达质粒，研究其对人肾癌细胞Ki-67基因表达的阻抑作用，探索肾癌基因治疗新的途径。方法设计有小发夹机构的2条Ki-67 siRNA对应模板DNA序列，煺火处理后克隆至pSliencer 3．1质粒，构建重组质粒pSliencer-Ki-67。将pSliencer-Ki-67转染人肾癌786—0细胞，采用逆转录-聚合酶链反应(RT-PCR)、Western blot检测Ki-67表达。结果酶切及测序证实质粒pSliencer-Ki-67构建成功。其可显著抑制786-0细胞Ki-67 mRNA、Ki-67蛋白表达。结论成功构建的Ki-67 siRNA表达质粒pSliencer-Ki-67能抑制人肾癌786-0细胞Ki-67基因表达。     

三氧化二砷抗肝癌作用及其肾毒性作用的实验研究

目的：研究三氧化二砷（As2O3）抗肝癌作用及其对肾脏的毒副作用并探讨其作用机制。方法：二乙基亚硝胺灌胃制备大鼠肝癌模型。以As2O3或顺铂注射于大鼠腹腔，第7、14、28天获取肝癌结节，光、电镜下观察肝癌细胞形态学变化，流式细胞仪检测凋亡及细胞动力学变化。第28天获取肾脏，光镜下观察肾脏组织形态学变化，免疫组织化学SP法检测bcl-2、增殖细胞核抗原（PCNA）表达变化。结果：As2O3诱导大鼠肝癌细胞凋亡，出现典型形态学改变；引起肝癌细胞凋亡率上开，中剂量组（1mg/kg体重）显著上升，明显高于顺铂组（P＝0．000）。顺铂组大鼠肾脏（4／7）镜下出现肾小管上皮细胞浊肿、变性，集合管内蛋白管型出现，而砷剂组无明显改变（P＝0．013）；砷剂组肾上管上皮细胞bcl-2表达增加（P＝0．005），PCNA标记指数无明显改变，顺铂组PCNA LI明显升高（P＝0．001）。结论：As2O3可诱导大鼠肝癌细胞凋亡，且优于顺铂；与顺铂相比，无明显肾毒性。     

三氧化二砷对小鼠肝脏祖细胞生物学特性的影响

目的 观察三氧化二砷(As2O3)对小鼠肝脏祖细胞(AHPCs)增殖及代谢的影响.方法 应用改良Seglen二步法原位灌注结合机械离心获取AHPCs,体外培养并持续观察超过40 d.应用免疫荧光技术对AHPC及其形成的克隆进行白蛋白、甲胎蛋白(AFP)和细胞角蛋白19 (CK19)染色.取原代培养12d的细胞随机分为3组:对照组(不含As2O3)、低浓度组(含2μmol/L As2O3)和高浓度组(含4 μmol/L As2 O3).干预48 h后,采用免疫荧光法观察细胞核增殖抗原(Ki-67)阳性细胞所占的比例,逆转录-聚合酶链反应(RT-PCR)技术检测Ki-67基因的表达,Western blot技术检测增殖细胞核抗原(PCNA)蛋白的表达.酶联免疫吸附试验(ELISA)检测细胞上清液中尿素浓度的变化.结果 体外培养的AHPC可持续扩增超过40 d,培养第1天强阳性表达白蛋白,培养第5天细胞克隆开始表达AFP,第35天表达CK19.阴性对照组所含Ki-67阳性细胞的比例较高(25.1±2.8)％,而As2O3(2μmol/L)组[(18.8±1.0)％]和As2O3(4μmol/L)组[(11.8±2.0)％]的Ki-67阳性细胞比例逐渐降低.Ki-67 mRNA的表达量在As2 O3(2μmol/L)组(0.823 ±0.012)明显下降,并以As2O3(4μmol/L)组(0.309 ±0.002)降低最为明显.随着As2O3浓度的增加,PCNA表达逐渐下降,并呈剂量-效应关系.阴性对照组细胞代谢产物尿素浓度较高[(0.586 ±0.056) mmol/L]随着As2O3浓度增加As2O3(2μmol/L)组[(0.506±0.058) mmol/L]与As2O3 (4 μmol/L)组[(0.410±0.045) mmol/L]尿素浓度逐渐下降.结论 成功建立AHPC体外培养模型.As2O3能抑制原代AHPC的增殖,并降低其代谢能力.     

三氧化二砷对前列腺癌DU-145细胞周期阻滞及凋亡的影响

目的探讨三氧化二砷(As2O3)对前列腺癌非雄激素依赖细胞系DU-145细胞的增殖抑制作用及对细胞周期和凋亡的影响.方法应用体外细胞生长抑制试验(MTT比色法)研究As2O3对DU-145细胞生长的影响,流式细胞仪检测细胞周期分布情况及凋亡情况,琼脂糖凝胶电泳检测细胞DNA的变化.结果As2O3对DU-145细胞的增殖有明显的抑制作用,随着药物浓度增加和作用时间延长,凋亡细胞明显增加,出现G2/M期阻滞和DNA的断裂.结论As2O3可明显抑制前列腺癌DU-145细胞的增殖,诱导细胞凋亡,抑制细胞周期.     

三氧化二砷抑制人结肠癌细胞生长的实验研究

目的探讨三氧化二砷(As2O3)抗人结肠癌细胞SW480生长及其机制。方法采用MTT法和流式细胞术对细胞生长、凋亡、细胞周期、增殖细胞核抗原(PCNA)、Fas、Bcl-2表达率及细胞内钙离子(IECa2+)含量进行分析。结果As2O3可显著抑制SW480细胞生长并诱导凋亡,且呈现剂量和时间依赖关系,其24、48和72h的半数抑制浓度(IC50)分别为9.71、8.78和7.34μmol/L。As2O3作用的SW480细胞周期阻滞在G2/M期,Fas和IECa2+含量增加、PCNA表达下降(P<0.01),但Bcl-2表达无变化(P>0.05)。结论As2O3可有效抑制SW480生长并诱导凋亡,其机制可能与上调Fas和IECa2+含量、降低PCNA表达及阻滞细胞周期有关。     

非甾体类抗炎药NS398诱导肾癌细胞凋亡的作用及其机制

目的观察COX-2选择性抑制剂NS398的对肾癌细胞增殖和凋亡作用的影响及其作用机制。方法采用标准的细胞培养方法对人肾癌786-0细胞进行培养，将NS398分别以25、50、100、150、200μmol／L的剂量加入细胞中作用24及48h后，应用MTT法检测NS398对肾癌细胞增殖的影响；将NS398以50、100及200μmol／L的浓度作用于肾癌786-0细胞24h后，用流式细胞仪测定细胞凋亡的情况；将NS398分别以25、50、100、150、200txmol／L的剂量加入细胞中作用24h后，应用RT．PCR和Western blotting分别检测肾癌786-0细胞中COX-2和bcl-2的mRNA及其蛋白表达的情况。结果NS398对肾癌786-0细胞具有较强的抑制作用，且这种抑制作用随浓度和时间的增加而增大，呈浓度依赖关系（P〈0．05）；NS398作用肾癌786-0细胞24h后，在细胞周期G0／G1期前出现明显的亚二倍体凋亡峰，随着浓度升高凋亡峰亦越来越增高（P〈0．05）；RT-PCR和Western Blot结果表明，不同浓度NS398作用下的肾癌786-0细胞中，COX-2和bcl-2无论在mRNA水平还是在蛋白水平均明显减弱，且呈剂量梯度下降。结论肾癌786-0细胞中存在着COX-2的过表达，选择性COX-2抑制剂NS398通过诱导凋亡来抑制肾癌786-0细胞的增殖；其机制可能是通过抑制COX-2的表达，导致bcl-2抗凋亡活性的降低来完成的。     

三氧化二砷对激素非依赖性前列腺癌PC-3细胞株Bc1-2、Bax表达的影响

目的了解三氧化二砷对激素非依赖性前列腺癌PC-3细胞株细胞增殖、细胞凋亡及bcl-2、bax基因表达的影响。方法分别以1、2、3、6、10μmol／L的三氧化二砷作用于PC-3细胞，48h后对细胞增殖活性、细胞凋亡及细胞bcl-2、bax基因表达的变化进行检测。结果各浓度的三氧化二砷均可抑制PC-3细胞增殖，并具有剂量依赖性。3、6、10μmol／L的三氧化二砷还可诱导PC-3细胞凋亡，计算细胞凋亡率分别为11．8％、12．7％、29．6％。逆转录聚合酶链反应（RT-PCR）检测PC-3细胞中bcl-2、bax表达的相对强度，发现bcl-2的表达强度随三氧化二砷浓度的升高而逐渐降低（P〈0．01），而bax表达强度变化不明显（P〉0．05）。结论三氧化二砷可有效抑制PC-3细胞增殖，诱导PC-3细胞凋亡，这一过程与三氧化二砷抑制PC-3细胞中bcl-2的表达有关。     

Bcl-2反义寡核苷酸诱导增生性瘢痕细胞凋亡的研究

目的：探讨反义寡核苷酸(ASODN)对bcl-2基因的表达调控及诱导增生性瘢痕成纤维细胞凋亡的作用。方法：采用RT-PCR法和流式细胞术检测ASODN对bcl-2蛋白和mRNA表达量的调控；通过MTT法比较两条人工合成ASODN直接作用及脂质体D0’FAt，转染对增生性瘢痕成纤维细胞的抑制效果；用相差显微镜、电子显微镜观察bcl-2ASODN诱导增生性瘢痕成纤维细胞凋亡效果。结果：ASODN降低bcl-2蛋白和mRNA表达；两条ASODN抑制增生性瘢痕成纤维细胞增殖的作用呈浓度和时间依赖性，且靶位点于mRNA蛋白编码区(ASODN2)和以DOTAP为载体的ASODN对成纤维细胞的增殖抑制效果最佳。Bcl-2ASODN作用于成纤维细胞后，成纤维细胞缩小、凋亡小体和染色质浓缩等特征性改变出现。结论：bcl-2ASODN能抑制bcl-2mRNA和蛋白表达，诱导成纤维细胞凋亡。     

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.     

A Teaspoon Pump for Pumping Blood with High Hydraulic Efficiency and Low Hemolysis Potential

Abstract: Virtually all blood pumps contain some kind of rubbing, sliding, closely moving machinery surfaces that are exposed to the blood being pumped. These valves, internal bearings, magnetic bearing position sensors, and shaft seals cause most of the problems with blood pumps. The original teaspoon pump design prevented the rubbing, sliding machinery surfaces from contacting the blood. However, the hydraulic efficiency was low because the blood was able to "slip around" the rotating impeller so that the blood itself never rotated fast enough to develop adequate pressure. An improved teaspoon blood pump has been designed and tested and has shown acceptable hydraulic performance and low hemolysis potential. The new pump uses a nonrotating "swinging" hose as the pump impeller. The fluid enters the pump through the center of the swinging hose; therefore, there can be no fluid slip between the revolving blood and the revolving impeller. The new pump uses an impeller that is comparable to a flexible garden hose. If the free end of the hose were swung around in a circle like half of a jump rope, the fluid inside the hose would rotate and develop pressure even though the hose impeller itself did not "rotate"; therefore, no rotating shaft seal or internal bearings are required.     

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.     

Is the recovery profile of mivacurium independent of the rate of decay of its plasma concentration in patients with normal plasma cholinesterase activity?

Background: The duration of action of muscle relaxants is poorly correlated to the rate of decay of their plasma concentration. The plasma concentration of mivacurium may rapidly decrease below its active concentration because of the extensive hydrolysis of mivacurium. By inflating a tourniquet on one upper limb for 3 min after the administration of atracurium, mivacurium or vecuronium, we studied the influence of the initial decline of their plasma concentration on their effect. Methods: In 50 patients anaesthetised with thiopental, isoflurane and fentanyl, the effect of bolus doses of 0.15 or 0.25 mg . kg^?1 mivacurium (MIV 15, MIV 25), 0.3 or 0.5 mg . kg^?1 atracurium (ATR 30, ATR 50) and 0.06 or 0.1 mg . kg^?1 vecuronium (VEC 06, VEC 10) were measured on both arms (evoked response of the adductor pollicis to train-of-four stimulation every 12 s), a tourniquet being applied on one arm just before and during 3 min after the muscle relaxant bolus. Results: Tourniquet inflation of 3 min almost abolished the neuromuscular effect of mivacurium. In the vecuronium groups and in the ATR 50 group, tourniquet inflation did not modify the maximum degree of depression of the twitch response. Also, the duration of action of vecuronium was unaffected by the tourniquet. In the ATR 30 group, times to return of the twitch response to 25% (duration 25%) and 75% (duration 75%) of control response were significantly shorter in the cuffed arm, 23 min vs 27 min, and 41 min vs 45 min, respectively. In the ATR 50 group, only duration 25% was significantly shorter in the cuffed arm (41 min vs 45 min). Conclusion: The results suggest that the rate of decline of the plasma concentration of mivacurium is so rapid, that a very low and almost clinically ineffective concentration is present as soon as 3 min after its administration. The results also indicate that the recovery from a mivacurium-induced neuromuscular blockade is not influenced by the rate of decay of its plasma concentration in patients with genotypically normal plasma cholinesterase.     

Membranes in Artificial Organs

Abstract: Membrane processes play a pivotal and enabling role in modern replacement therapy for acute and chronic organ failure and in the management of immunologic diseases. In fact, virtually all contemporary extracorporeal blood purification methods employ membrane devices, and the next generation of artificial organs and tissue engineering therapies are almost certain to be similarly grounded in membrane technology. In this short essay, we comment on the similarities and differences among synthetic membranes and their natural counterparts and also provide a critical overview of the demographics and technology of hemodialysis, hemofiltration, apheresis, oxygenation, and emerging membrane technologies and applications.     

Tissue perfusion in relation to cardiac output during continuous positive-pressure ventilation and administration of propranolol or verapamil

Background : Our objective was to determine whether administration of propranolol or verapamil modifies the hemodynamic adaptation to continuous positive-pressure ventilation (CPPV), in particular the regional distribution of cardiac output (CO).
Methods : General hemodynamics and regional blood flows assessed by microsphere technique (15 (μm) were recorded in 16 anesthetized pigs during spontaneous breathing (SB) and CPPV with 8 cm H₂O end-expiratory pressure (CPPV8) before and after intravenous administration of propranolol (0.3 mg · kg⁻¹ followed by 0.15 mg · kg⁻¹ · h⁻¹, n=8) or verapamil (0.1 mg · kg⁻¹ followed by 0.3 mg · kg⁻¹ · h⁻¹, n=8).
Results : CPPV8 depressed CO by 25% without shifts in its relative distribution with the exception of a noteworthy increase in adrenal perfusion. Propranolol increased arterial blood pressure, and due to a fall in heart rate, CO dropped by 25%. The kidneys and, to a lesser extent, the splanchic region and central nervous system received increased fractions of the remaining CO at the expense of skeletal muscle flow. Similar patterns were seen during SB and CPPV8 such that the combination of propranolol and CPPV8 depressed CO by 50%. The circulatory effects of verapamil were less evident but myocardial perfusion tended to increase.
Conclusions : The combination of propranolol or verapamil with CPPV does not result in any specific hemodynamic interaction in anesthetized pigs, except that the combined effect of propranolol and CPPV may severely reduce CO.     

Volatile anaesthetics reduce adhesion of blood platelets under low-flow conditions in the coronary system of isolated guinea pig hearts

Background : Inhibitory effects of volatile anaesthetics on platelet aggregation have been demonstrated in several studies. However, the influence of volatile anaesthetics on intracoronary platelet adhesion has not been elucidated so far.
Methods : Isolated hearts of guinea pigs were perfused with buffer in the absence or presence of volatile anaesthetics (0.5 and 1 MAC) at constant coronary flow rates of 5 ml/min for 25 min, then 1 ml/min for 30 min and again 5 ml/min for 10 min. Before, during and after low-flow perfusion, a bolus of human platelets was applied into the coronary system. To simulate thrombogenic conditions, 0.3 U/ml human thrombin was infused during low-flow perfusion and reperfusion. The number of platelets sequestered to the endothelium was calculated from the difference between coronary in- and output of platelets. The myocardial production of lactate and consumption of pyruvate and coronary perfusion pressure were also determined.
Results : At a flow rate of 5 ml/min only about 3% of the applied platelets did not emerge from the coronary system, in any group. In contrast, 13.1±1.2% (mean±SEM) of infused platelets became adherent in low-flow perfusion in the control group without anaesthetic. The adherence was reduced with each 1 MAC isoflurane (to 6.2±1.2%), sevoflurane (to 4.4±0.9%) or halothane (to 3.2±1.5%) (each P <0.05 vs. control). Volatile anaesthetic, 0.5 MAC, did not inhibit platelet adhesion to a statistically significant extent in any case. Perfusion pressure and metabolic parameters were not statistically different between the control and the hearts exposed to anaesthetics.
Conclusion : Volatile anaesthetics in a concentration of 1 MAC can reduce the adhesion of platelets in the coronary system under reduced flow conditions. This action does not arise from vasodilation or inhibition of ischaemic stress.     

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.     

Dilemma of Membrane Biocompatibility and Reuse

Abstract: Numerous articles have been published on the multiple use of dialyzers and on the effect of different reprocessing chemicals and techniques on the dialyzer biocompatibility and performance. The results often appear contradictory, especially those comparing standard biocompatibility parameters. Despite this confusion, a discerning review of the published works allows certain limited conclusions to be drawn. Reprocessing of used hemodialyzers changes the biocompatibility profile of a dialyzer as defined by the parameters complement activation. leukopenia, and cytokine release. The effect of reprocessing depends on the chemicals and reprocessing technique applied and also on the type of membrane polymer being subjected to the reprocessing procedure. Reports of pyrogenic reactions indicate that the flux of the membrane also influences how suitable it is for safe reuse. An increased risk of allergic and pyrogenic reactions appears to be associated with dialyzer reuse. Furthermore, there has been a lack of investigations into the immunologic effect of the layer of adsorbed and chemically altered proteins that remains on the inner surface of reprocessed dialyzers. We conclude that the clinical benefit of dialyzer reuse cannot be generally accepted from a biocompatibility point of view.     

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.