以L929细胞为滋养层的尿道黏膜上皮细胞体外培养

目的 探索尿道黏膜上皮细胞体外培养的技术和方法，为进一步采用组织工程技术构建尿道黏膜组织奠定基础，并为尿道黏膜的生理、药理、毒理学及微生态研究提供实验模型。方法 取刚离乳的雄性新西兰幼兔尿道黏膜组织，分别以DispaseⅠ消化液和混合消化液消化成单细胞悬液，以差速贴壁法排除成纤维细胞，接种后以L929细胞为滋养层细胞进行培养，定期换液，细胞生长、增殖至80％～90％融合时传代。细胞进行常规HE染色、流式细胞仪检测，以扫描电镜、透射电镜观察其超微结构。再分别设立实验组(n=20)、阳性对照组(正常尿道黏膜组织石蜡切片，n=20)及阴性对照组(成纤维细胞铺片，n=20)行免疫组织化学染色。结果 原代培养10天左右细胞逐渐生长融合成片，如铺路石状，细胞大小均一。上皮细胞为二倍体细胞，生长期内均为单一的上皮细胞，无成纤维细胞混杂生长，细胞可传11～13代，成活50～60天。结论 新西兰幼兔尿道黏膜上皮细胞可在体外进行培养，在一定时间内保持增殖活力，为构建组织工程化尿道奠定了基础，且为尿道黏膜的体外研究提供了实验模型。     

尿道移行上皮细胞与生物可降解尿道支架体外复合培养的研究

目的：研究体外培养的兔尿道上皮细胞在生物可降解性网状尿道支架上的贴附和生长增殖情况,观察其对尿道上皮细胞形态和功能的影响,利用组织工程技术培养种植细胞的尿道内支架。方法：应用机械分离与酶消化法分离培养兔尿道移行上皮细胞,并在体外行原代培养与扩增后制成细胞悬液接种在网状尿道支架上,形成尿道移行上皮细胞-支架复合物。采用免疫组织化学、荧光染色法鉴定尿道上皮细胞及其活性;采用倒置显微镜、扫描电镜观察尿道上皮细胞在支架表面吸附与生长状态。结果：网状尿道支架具有良好的生物相容性,能使尿道移行上皮细胞增殖,不影响其活性。尿道移行上皮细胞在尿道支架上贴附生长良好,1～2天后完全贴壁,3～7天细胞生长增殖活跃,支架网眼内充满上皮细胞,长期培养仍保持尿道移行上皮细胞特性,扫描电镜可见上皮细胞与网状支架贴附紧密,适度伸展并有基质分泌。结论：网状尿道支架适合尿道移行上皮细胞黏附生长．可作为尿道组织工程的细胞载体,利用组织工程方法可获得适于移植尿道细胞的组织工程化尿道。     

兔包皮上皮细胞的分离培养和鉴定

目的:收集兔的小块包皮组织,分离培养上皮细胞,并传代增殖,为组织工程化尿道构建提供细胞来源.方法:取雄性家兔包皮小块组织,酶消化成单细胞悬液,接种后静置培养、传代.并定期观察细胞形态变化及生长增殖情况,免疫组化染色,细胞计数及MTT测定以判断细胞增殖能力.结果:分离消化后的上皮细胞生长良好,经免疫组化测定为正常上皮细胞,并传代3代,细胞数量达到植入生物支架并生长的需要.结论:家兔的包皮上皮细胞可在体外培养增殖并传代,并达到相应细胞数量.     

人体尿道粘膜上皮细胞的连续培养

目的：探讨人尿道粘膜上皮细胞体外培养技术，为构建组织工程化尿道提供种子细胞。方法：取尿道下裂患者尿道粘膜，经中性蛋白酶和胰蛋白酶消化，获取粘膜上皮细胞接种于含8％胎牛血清培养基中连续培养，观察细胞形态变化及生、增殖过程。结果：体外培养细胞长满后呈上皮细胞特有的铺路石样外观，体外可连续传9-10代，生长期50-60d。细胞角质蛋白免疫组织化学染色阳性，透射电镜下可见上皮细胞间特有的桥粒结构。结论：尿道粘膜上皮细胞可在体外连续培养，4代以内的培养细胞可用于组织工程化尿道的构建。     

尿道移行上皮细胞与生物可降解尿道支架体外复合培养的研究

目的:研究体外培养的兔尿道上皮细胞在生物可降解性网状尿道支架上的贴附和生长增殖情况,观察其对尿道上皮细胞形态和功能的影响,利用组织工程技术培养种植细胞的尿道内支架.方法:应用机械分离与酶消化法分离培养兔尿道移行上皮细胞,并在体外行原代培养与扩增后制成细胞悬液,接种在网状尿道支架上,形成尿道移行上皮细胞-支架复合物.应用免疫组织化学、荧光染色法鉴定尿道上皮细胞及其活性,并用倒置显微镜、扫描电镜观察尿道上皮细胞在支架表面吸附与生长状态.结果:网状尿道支架具有良好的生物相容性,能使尿道移行上皮细胞增殖,不影响其活性.尿道移行上皮细胞在尿道支架上贴附生长良好,1～2天后完全贴壁,3～7天细胞生长增殖活跃,支架网眼内充满上皮细胞;长期培养仍保持尿道移行上皮细胞特性,扫描电镜可见上皮细胞与网状支架紧密贴附,适度伸展并有基质分泌.结论:网状尿道支架适合尿道移行上皮细胞黏附生长,可作为尿道组织工程的细胞载体,利用组织工程方法可获得适于移植尿道细胞的组织工程化尿道.     

阴道粘膜上皮细胞的分离培养和鉴定

目的 初步建立阴道粘膜上皮细胞体外培养方法,为阴道粘膜上皮研究提供实验模型.方法 取雌性新西兰大白兔阴道粘膜组织小块,胶原酶Ⅳ和胰蛋白酶联合消化分离法收集上皮细胞,接种于角朊细胞无血清培养液中静置培养、传代.动态观察细胞生长增殖情况,扫描和透射电镜观察超微结构,流式细胞仪测定细胞增殖周期,并进行免疫组织化学鉴定.结果 体外培养的阴道粘膜上皮细胞为二倍体细胞,增殖状态良好,细胞间可见桥粒连接,免疫组化角蛋白染色阳性.细胞的超微结构和免疫组化染色均具有上皮细胞特征.结论 在本实验条件下,体外培养的阴道粘膜上皮细胞具有较好的增殖能力,可作为阴道粘膜上皮研究的理想实验模型.     

膀胱黏膜上皮细胞体外培养的实验研究

目的探索组织工程化尿道黏膜类似组织体外培养条件.方法取雄性大耳白兔幼兔的膀胱壁组织,分别用Ⅱ型胶原酶和Dispase Ⅱ处理后,胰蛋白酶消化获得单细胞悬液,接种后用含有或不含有表皮生长因子的培养液培养并传代,动态观察细胞形态变化及生长增殖的不同情况,并对细胞进行超微结构观察.结果用Ⅱ型胶原酶处理的标本在有表皮生长因子存在的情况下,细胞生长良好,混杂少量的成纤维细胞,在体外可传10代以上,成活30天以上.结论Ⅱ型胶原酶处理的幼兔膀胱黏膜上皮细胞纯度较高,可在体外培养一定时间并保持增殖能力,表皮生长因子是必要条件.     

膀胱黏膜上皮细胞体外培养的实验研究

目的 探索组织工程化尿道黏膜类似组织体外培养条件。方法取雄性大耳白兔幼兔的膀胱壁组织，分别用Ⅱ型胶原酶和DispaseⅡ处理后，胰蛋白酶消化获得单细胞悬液，接种后用含有或不含有表皮生长因子的培养液培养并传代，动态观察细胞形态变化及生长增殖的不同情况．并对细胞进行超微结构观察。结果用Ⅱ型胶原酶处理的标本在有表皮生长因子存在的情况下，细胞生长良好，混杂少量的成纤维细胞，在体外可传10代以上，成活30天以上。结论Ⅱ型胶原酶处理的幼兔膀胱黏膜上皮细胞纯度较高，可在体外培养一定时间并保持增殖能力．表皮生长因子是必要条件。     

原位定量检测体外培养的尿道黏膜上皮细胞活性

目的解决在不透明载体上观察细胞生长状况的难题。方法取雄性新西兰幼兔1只,进行尿道黏膜上皮细胞的原代培养并传至第3代。将其接种于胶原-壳聚糖复合材料上,体外培养3、7、14和21d后,行6-羧基二乙酸甲酯荧光素与碘化丙啶荧光双染,并应用激光共聚焦细胞仪检测。结果尿道黏膜上皮细胞在胶原-壳聚糖复合材料载体上生长,增殖良好。培养3、7d,活细胞荧光强度值为(1.09±0.13)×108、(2.04±0.13)×108,培养14、21d活细胞荧光强度值分别为(0.55±0.09)×108、(0.47±0.03)×108,与培养3、7d比较差异均有统计学意义(P<0.05)。各时间点死细胞荧光强度值差异无统计学意义(P>0.05)。结论利用激光共聚焦细胞仪对不同波长荧光强度分别进行定量分析,可以在原位定量快速检测尿道黏膜上皮细胞活性,动态观察组织工程组织生长情况,解决无法在不透明载体上观察细胞生长状况的难题。     

膀胱移行上皮细胞的体外培养研究

目的：探索并建立大鼠膀胱移行上皮细胞的体外培养方法，为膀胱癌相关研究提供实验依据。方法：获取Wistar大鼠膀胱粘膜，采用Dispase酶消化法收集上皮细胞。动态观察细胞形态变化、生长增殖情况；对培养细胞进行细胞周期检测及超微结构观察，并行免疫组织化学鉴定。结果：培养细胞为二倍体移行上皮细胞，无成纤维细胞混杂生长。免疫组织化学证实角蛋白染色阳性。结论：膀胱移行上皮细胞体外培养为体外研究提供了理想的模型。     

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.     

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.     

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.     

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.     

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.     

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.     

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.