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。     

尿道粘膜上皮细胞的分离培养和鉴定

目的：为采用组织工程技术构建尿道粘膜组织提供实验依据。方法：取雄性新西兰幼兔尿道粘膜组织小块,酶消化成单细胞悬液,接种后静置培养、传代。动态观察细胞形态变化及生长增殖情况。细胞进行常规组化染色、免疫组化染色及流式细胞仪检查,并观察超微结构。结果：整个上皮细胞生长期内无成纤维细胞混杂生长,均为单一的上皮细胞,并证实为二倍体细胞。细胞可传11－13代,成活50－60d。结论：新西兰幼兔尿道粘膜上皮细胞可在体外培养,在一定时间内保持增殖能力。     

皮肤烧伤增强小鼠肠道上皮细胞的凋亡

已有研究表明皮肤烧伤后肠道粘膜上皮细胞增殖加强,但粘膜重量下降,因而烧伤后肠道粘膜上皮细胞死亡更为显著。通过利用小鼠烧伤模型,体外观察烧伤后肠道粘膜上皮和细胞凋亡和增殖的变化,旨在验证烧伤小鼠肠道粘膜上皮和细胞凋亡增强的假设。 实验用63只成年雄性C57BL6小鼠,3只作为正常对照,余随机分为假伤组和烧伤组,均麻醉、剔毛、置于致伤模具中,利用蒸汽     

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。     

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。     

组织工程尿道粘膜的体外构建

目的:利用组织工程方法在体外构建形成尿道粘膜结构。方法:酶消化法获得猪膀胱尿路上皮细胞(UC),以免疫荧光和RT-PCR方法进行UC鉴定。制备膀胱脱细胞基质移植物(BAMG)作为支架材料,以苏木精-伊红染色、Masson染色、免疫组化和扫描电镜进行评价。经过体外培养和扩增后,将P3代UC接种在BAMG上。结果:BAMG支架上的UC经过体外培养1周后即可形成沿基膜排列的复层上皮结构。扫描电镜显示细胞-材料复合良好,免疫组化检测支架上的UC广谱角蛋白表达阳性。结论:运用组织工程方法能够在体外快速进行尿道粘膜上皮结构的初步构建,为进一步临床修复诸如尿道下裂、尿道狭窄等尿道缺损奠定技术基础。     

人类肥大乳房乳腺上皮细胞的原代培养

目的 探讨人类肥大乳房乳腺上皮细胞的原代培养方法.方法 采用胶原酶消化培养法,在体外进行人类肥大乳房乳腺上皮细胞的分离培养,用倒置显微镜观察、细胞爬片、HE染色和细胞角蛋白免疫组织化学染色的方法对分离培养的细胞进行形态学观察和鉴定.结果 倒置显微镜下观察细胞形态呈鹅卵石型或多角型,部分形态不规则,增殖的过程中形成岛屿状闭合型的细胞群,细胞之间连接紧密.细胞HE染色可见细胞胞质呈粉红色或浅紫色,细胞核呈蓝紫色,圆形或椭圆形,核中可见深蓝色的染色体.免疫组化鉴定结果显示,培养的细胞胞浆内可见棕黄色的细胞角蛋白着色,表达上皮细胞特异的细胞角蛋白18.结论 应用酶消化法和条件培养液可以获得纯度较高的人类肥大乳房乳腺上皮细胞.     

猪气管上皮细胞的分离、培养和鉴定

目的 探索猪气管上皮细胞体外适宜的生长、增殖条件,以期为组织工程化气管内壁上皮化提供种子细胞.方法 无菌切取猪颈段气管,细菌蛋白酶消化.以明胶作为铺层,接种、培养.待细胞贴壁后,进行光镜观察、广谱角蛋白免疫组化检测、免疫荧光鉴定.结果 光镜观察可见细胞呈铺路石样生长.角蛋白检测和免疫荧光检测均呈阳性,而阴性对照组和空白对照组呈阴性.结论 以明胶作为铺层可以培养出活力较好、纯度高的气管上皮细胞.     

人肛瘘管壁上皮细胞的体外分离培养和鉴定

目的:寻找建立人肛瘘管壁上皮细胞体外分离、培养及鉴定细胞起源的技术方法.方法:通过组织块法分离并原代培养人肛瘘管壁组织上皮细胞,观察上皮细胞形态并对培养细胞进行细胞化学鉴定.结果:分离后的上皮细胞在24～36 h贴壁,在7～9 d进入对数期生长期.细胞鉴定角蛋白免疫细胞化学染色为阳性,波形蛋白免疫细胞化学染色为阴性.结论:该方法获得的肛瘘管壁上皮细胞能够在体外稳定培养,可以为细胞分子水平上研究肛瘘瘘管愈合机制提供可靠的细胞模型.     

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 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.     

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.     

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.     

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.     

The analgesic effect of racemic ketamine in patients with chronic ischemic pain due to lower extremity arteriosclerosis obliterans

Background : Ketamine in sub-dissociative doses has been shown to have analgesic and phantom-Limb pain, where conventional treatment has often failed. Chronic ischemic pain due to lower extremity arteriosclerosis obliterans often responds poorly to analgesics, and the pain-generating mechanisms are not well understood.
Methods : Eight patients with rest pain in the lower extremity due to arteriosclerosis obliterans were given sub-dissociative doses of 0.15, 0.30, or 0.45 mg/kg racemic ketamine and morphine 10 mg as a 5-min infusion on four separate days in a cross-over, double-blind, randomised protocol. Plasma levels of (S)- and (R)-ketamine and their nor-metabolites were analysed with an enantioselective high-performance liquid chromatography (HPLC) method. Pain levels were evaluated with a visual analogue scale (VAS).
Results : Individual pain levels were highly variable during and after all the infusions but the pooled pain levels showed a dose-dependent analgesic effect of ketamine with a transient but complete pain relief in all patients at the highest dose (0.45 mg/ kg). Side-effects, mainly disturbed cognition and perception, were pronounced and dose-dependent. Morphine 10 mg had an analgesic peak at 20 min and 5/8 patients had complete pain relief. The remaining 3 patients also had high baseline pain scores, indicating a higher analgesic potency for the 0.30 and 0.45 mg/ kg ketamine doses than for morphine 10 mg.
Conclusion : We have demonstrated a potent dose-dependent analgesic effect of racemic ketamine in clinical ischemic pain. Due to a narrow therapeutic window, this analgesic effect is probably best utilised in combination with other analgesics.