猪胰岛的分离与纯化

目的 探索大规模猪胰岛细胞分离纯化的方法.方法 联合器官切取,胶原酶P主胰管灌注,COBE2991细胞分离机及HCA-Ficoil纯化猪胰岛细胞.通过双硫腙(DTZ)染色,倒置显微镜下计数胰岛细胞的数量和纯度,胰岛素释放试验检测胰岛细胞的分泌功能.结果 消化后平均每条胰腺可平均获得(275 000±20 895)胰岛细胞当量(IEQ),纯化后平均为(230 350±26 679)IEQ,平均每克胰腺组织可获得(2710±229)IEQ,纯化后胰岛细胞平均纯度为(50.2±1.95)%.纯化后的胰岛细胞对胰岛素释放刺激反应良好,高糖(16.7 mmol/L)时胰岛素的释放量为低糖(3.3 mmol/L)时的4.74倍(P≤0.001).结论 成功建立了猪胰岛细胞分离、纯化的方法,纯化的猪胰岛细胞具有良好的生物活性.     

胰岛分离、纯化制备的改进

目的 探讨大型哺乳动物胰岛机械化大量分离、纯化的方法,为人类胰岛移植物的大量制备摸索创造条件.方法 应用改进的机械化胰岛分离、纯化系统,用HCA和UW液顺序原位灌洗犬胰腺,主副胰管插管,4℃胶原酶-V(1.5 g/L)+胰酶抑制剂pefabloc(0.4 mmol/L)灌注后,Ricordi-Chamber消化罐消化,4℃COBE2991连续密度梯度离心纯化,测定胰岛当量(IEQ)、胰岛纯度及存活率、胰岛素及C-肽的释放量、培养24 h后光镜及电镜观察.结果 胰腺消化时间为(25.0±6.0)min,胰岛外分泌腺包裹率为(9.4±2.4)%,消化后胰岛收获量为(17.2±3.6)×104IEQ/每个胰腺,纯化后胰岛收获量为(8.3±2.0)×104IEQ/每个胰腺,胰岛纯度为(89.7±3.5)%.纯化胰岛体外低糖与高糖刺激下胰岛索分泌量及C-肽的释放量良好,培养24 h后形态结构及功能正常.结论 本实验室改进的胰岛机械分离方法及各设备运行可靠,获得的胰岛形态功能良好,可望用于临床人类胰岛的大量制备.     

几种小鼠胰岛的分离纯化方法比较

目的探讨几种小鼠胰岛的分离纯化方法及进行纯化后胰岛的计数和完整性的分析。方法选用ICR小鼠,采用不同胶原酶消化方法和不同的胶原酶种类,并用Ficoll-PaqueTMPLUS密度梯度离心法对胰岛进行纯化,并对获得的胰岛进行DTZ染色计数和计算当量,扫描电镜考察胰岛的完整性。结果采用胶原酶V和P胰管灌注、内外消化结合,消化需时较短,胶原酶V和P在胰岛纯化前后每只小鼠收获的胰岛细胞数量和当量无差别(P>0.05);扫描电镜结果显示消化较好的胰岛表面被膜完整,消化过度的胰岛导致被膜不完整,易致外层细胞损伤。结论小鼠逆行胰管灌注胶原酶、内外消化相结合的胰岛消化方法所需时间较短,但同时要注意防止消化过度。     

机械分离成人尸体胰腺提高胰岛数量和质量

目的通过机械分离胰腺的方法,提高成人胰岛数量和质量。方法切取8个成人尸体胰腺,并保存于4℃UW液中。首先经主胰管插管灌注复合胶原酶溶液,使胰腺膨胀,然后将胰腺剪切成5~8块,放入Ricordi室中,于37℃下机械震荡消化。收集的胰岛溶于100mlUW液中,放置30min。先将Ficoll液泵入COBE2991细胞分离仪中,然后泵入UW/胰岛制剂,最后加入M199液冲洗。收集纯化的胰岛,计算胰岛当量(IEQ)和纯度,检测胰岛的活性。结果热缺血时间为0~1min,冷缺血时间0.5~4h。纯化前收集的胰岛数量平均为(376.7±50.2)×103IEQ,纯化后为(378.6±56.7)×103IEQ;胰岛收获率平均为(86.8±9.4)%,镶嵌的胰岛平均占(11.0±2.7)%;收获的胰岛纯度平均为(88.3±5.1)%;经AO-PI染色后检测胰岛的活度为(91.3±7.8)%。胰岛素分泌指数平均为5.87±0.81。结论通过机械分离胰腺的方法,可以获得更多的胰岛数量和更高的胰岛活性,适合用于临床移植。     

实验大鼠胰岛分离移植技术方法的比较分析

目的 探索高效的大鼠胰岛分离移植技术方法.方法 应用Wistai-Furth大鼠,于体内或体外胶原酶经胰管灌注膨化胰腺,联合不同密度Ficoll液或Histopaque液纯化胰岛细胞,评估胰岛的数量、纯度、胰岛当量以及肾被膜下胰岛移植的有效性.结果 体外经胰管灌注膨化胰腺结合Histopaque液纯化提取胰岛的数量、纯度和胰岛当量值均显著高于体内灌注组各数值(P＜0.01),其提取时间无显著差别.1000个胰岛细胞移植进入左肾被膜下,有效的逆转了糖尿病大鼠高血糖,其远期糖耐受结果优于500和800个胰岛细胞移植组.结论 体外灌注膨化消化胰腺结合Histopaque液纯化胰岛的分离方法是一种满意的分离技术.1000个胰岛细胞是保证肾被膜下胰岛移植成功的最低有效数量.     

乌司他丁对犬胰岛的保护作用

目的判定在机械分离纯化犬胰岛过程中,胰腺原位灌洗时应用乌司他丁(UTI)的保护作用,观察胰岛的获取产量。方法将20只犬随机分为两组,每组10只。HC-A组:胰腺经腹主动脉原位灌洗时用冷HC-A液2500ml;HC-A UTI组:胰腺原位灌洗时,HC-A液中加用UTI10000U/kg。两组均经主胰管用4℃胶原酶V控压灌注,在RicordiChamber系统中消化,用Ficoll连续梯度离心纯化。记录消化时间、胰岛外分泌腺包裹率、胰岛纯度、纯化后胰岛在体外用低糖与高糖刺激下胰岛素分泌量、C-肽的释放量及收获的胰岛当量(IEQ),并对纯化后的胰岛进行光镜及电镜观察。结果HC-A UTI组与HC-A组在胰腺消化时间、胰岛外分泌腺包裹率、胰岛纯度、纯化后的胰岛在体外经低糖与高糖刺激下胰岛素分泌量、C-肽的释放量以及胰岛的形态和结构上比较,差异均无统计学意义(P>0.05)。HC-A组收获胰岛[(3.42±1.47)×104]IEQ,HC-A UTI组收获胰岛[(6.17±2.86)×104]IEQ,两组差异有统计学意义(P<0.05)。结论在犬胰岛分离和纯化过程中,胰腺获取原位灌洗液中加用UTI能保护胰腺组织,增加胰岛产量。     

大鼠胰岛的分离纯化方法改进与功能鉴定

目的 通过改进胰腺消化和分离的技术条件,提高成年大鼠胰岛分离纯化产率和质量. 方法 用胶原酶Ⅺ液灌注消化成年SD大鼠胰腺,对胰岛分离纯化方法加以改进:以 4 种比重的 Euro- Ficoll (F1∶D=1.132,F2∶D=1.108,F4∶D=1.069) 和 Hank's 液(F5∶D=1.023) 不连续密度梯度离心,以离心半径 15 cm,2 000 r/min 于4℃缓慢升降离心 20 min,收集位于F1 和 F2界面的胰岛.双硫腙特异染色法鉴定胰岛纯度;二醋酸酯荧光素/碘化丙啶染色法计算胰岛成活率;放射免疫分析法检测葡萄糖刺激的胰岛素分泌量,计算刺激指数.将胰岛当量(islets equivalent quantity,IEQ) 为 1000 的胰岛移植于同品系糖尿病大鼠肾包膜下,9d 内隔日观察动物血糖的变化,评价胰岛功能.比较分离条件优化前后收获胰岛的产率和质量. 结果 改进纯化方法后每只大鼠胰岛收获量为(920±122) IEQ,胰岛纯度> 90%,胰岛细胞成活率为 91%±2%.胰岛细胞功能良好,在低糖和高糖刺激后培养液中胰岛素浓度分别为(18.25±0.32) mU/L 和(36.70±3.57)mU/L,刺激指数为 2.01±0.15.1000 IEQ 胰岛移植于糖尿病大鼠肾包膜下,观察期内可维持动物血糖水平正常. 结论 改进后的胶原酶灌注消化和不连续梯度离心方法提高了胰岛的产率,保证了胰岛的高纯度及高成活率.     

一种快速分离纯化小鼠胰岛的方法

目的 介绍一种快速分离纯化小鼠胰岛的方法及进行纯化后胰岛的活性、完整性和胰岛内结构的质量分析.方法 雄性ICR小鼠,采用2 mg/mL胶原酶V灌注和消化,并用Hanks液快速洗涤,用Histopaque（R）-1077和Histopaque（R）-1119密度梯度离心对胰岛进行纯化,用手工方法进行胰岛挑选,DTZ、FD-PI染色鉴定胰岛纯度及其活性,透射电镜观察胰岛内的结构.结果 胰岛开始消化至手工挑选前过程耗时＜ 25 min,每只小鼠得到胰岛数量为：128±36,当量为：145±42,纯度＞90％.透射电镜显示胰岛内部血管仍有损伤.结论 采用此方法可快速得到数量较多、结构较完整的小鼠胰岛,且活性高,为进一步进行胰岛的体外质量研究及体内移植奠定了基础.     

采用改良的胰腺三管插管法分离成人胰岛的实验研究

目的 探讨胰腺插管方式对成人胰岛分离及纯化的影响.方法 共对17例成人胰腺进行了胰岛的分离和纯化.采用改进的腹部器官联合快速切取技术获取胰腺,分别采用标准法(3例)、单管法(11例)和三管法(3例)对胰腺进行灌注.标准法是将胰腺从胰颈处完全切断,沿主胰管分别向胰头和胰尾插管,主胰管人十二指肠处予以结扎.单管法为采用加长插管自主胰管插入,直至胰尾.三管法是在胰颈背侧切开胰腺至主胰管,经主胰管分别向胰头和胰尾方向插管,在主胰管进入十二指肠处插第3根插管.采用胶原酶LibarseHI消化,Ficoll连续密度梯度离心法纯化.双硫腙染色,鉴定胰岛的纯度,并计算胰岛当量(IEQ).丫啶橙/溴乙啶荧光染色,计数活细胞百分率.体外葡萄糖刺激试验鉴定胰岛功能.结果 标准法的灌注量平均为0.71 ml/g胰腺,单管法的灌注量平均为0.96 ml/g胰腺,三管法的灌注量平均为1.24 ml/g胰腺,明显多于前两种方法(P＜0.05).标准法的胰岛收获量平均为1914 IEQ/g胰腺,单管法为2270 IEQ/g胰腺,三管法为2514 IEQ/g胰腺,单管法和三管法明显高于标准法(P＜0.05);其胰岛纯度/活性分别为74 ％/79.3％、75.6 ％/79.4％和78.3 ％/84.0％,三者间的差异无统计学意义.标准法所获得的胰岛胰岛素释放指数平均为3.46,单管法为4.74,三管法为5.27,单管法和三管法明显高于标准法(P＜0.05).结论不同的插管灌注方式对成人的胰岛分离有一定影响,三管法有利于提高胰腺灌注量,增加胰岛的收获量.     

新型胰岛分离液对小鼠胰岛分离效果的研究

目的探讨新型胰岛分离液(IPS液)在小鼠胰岛分离中的分离效率及胰岛保护作用。方法将消化后的小鼠胰腺按体积平均分为两组(IPS组和UW组),分别应用IPS-Optiprep液及UW-Optiprep液进行连续梯度密度离心分离胰岛,比较两组分离液的分离纯化效率和分离后的胰岛活性。将诱导成功的糖尿病小鼠随机分为3组:实验组(n=10),接受采用IPS-Optiprep液分离纯化的胰岛移植;对照组(n=10),接受采用UW-Optiprep液分离纯化的胰岛移植;假性移植组(n=5),仅给予手术但并不进行胰岛移植。分析3组小鼠术后血糖水平以及测实验组和对照组小鼠术后21 d的腹腔葡萄糖耐量试验的血糖水平。比较两种分离液的配制成本。结果与UW组相比,IPS组的IPS-Optiprep液可提供更高的胰岛当量(IEQ)、胰岛纯度、回收率及胰岛完整度。胰岛形态观察可见,IPS组胰岛被膜完整,直径明显大于UW组。UW组纯化后的胰岛活性率高于IPS组[(88±5)%比(84±3)%,P0.01]。与UW-Optiprep液相比,IPS-Optiprep液可获得相当的在体胰岛功能。IPS-Optiprep液可显著降低胰岛分离纯化成本。结论新型胰岛分离液IPS-Optiprep液在胰岛分离提纯中显示出较好的分离效率,增加了胰岛的纯度、完整度与回收率,并显著降低了纯化成本,但对胰岛细胞活性的保护作用稍逊,可能与胰岛的高完整度及IPS-Optiprep液中的内毒素有关。     

Reduction of Plasma Fibrinogen, Immunoglobulin G, and Immunoglobulin M Concentrations by Immunoadsorption Therapy with Tryptophan and Phenylalanine Adsorbents

Abstract Immunoadsorption (1A) therapy with tryptophan (TR-350) or phenylalanine (PH-350) adsorbents has been used to reduce the concentration of serum antibodies in human lymphocyte antigen (HLA)-immunized patients. Other forms of plasma purification have been reported to reduce the level of fibrinogen, which affects the blood properties. In this study we investigated the effects of IA therapy using both adsorbents on plasma fibrinogen and immunoglobulins G and M in 13 patients (8 patients were treated with TR-350, and 5 patients were treated with PH-350). During each session 1 plasma volume (2.8 ± 0.4 L of plasma) was processed through the immunocolumn and then returned to the patient together with the blood cells. Compared with the pretreatment values, the plasma fibrinogen, IgG, and IgM concentrations were significantly reduced after IA therapy (p < 0.01 for TR-350; p < 0.04 for PH-350). There was a positive correlation between the degree of reduction of plasma proteins and the number of IA treatments given. A nonpara-metric test (Wilcoxon's signed-rank test or the Mann-Whitney test) was used for statistical analysis. We conclude from our study that IA therapy effectively lowers the plasma levels of fibrinogen, IgG, and IgM and thus can be considered a valuable alternative to other blood purification methods.     

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.     

The multiply injured child

Blunt trauma is the principal cause of childhood death in many developed countries. This review outlines the differences between adults and children with respect to resuscitation and treatment of orthopaedic injuries in a child with polytrauma. Recent advances in techniques of fracture stabilization are reported.     

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.     

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.     

Intravenous endotoxin does not increase tissue extravasation of albumin in rats

Background : It is unclear whether activation of the inducible nitric oxide synthase (iNOS) increases or decreases the extravasation of plasma.
Methods : Chloralose anaesthetised male Wistar rats received E. coli lipopolysacharide (LPS), 3 mg kg^-1 i.v., or the corresponding volume of saline, 3 or 5 h before the end of the experiment. Mean arterial pressure (MAP) and heart rate (HR) were recorded. Tissue clearance of radio-labelled albumin, during the last 2 h of each experiment, was determined by a double-isotope method. In separate animals, the serum concentration of nitrite and nitrate was determined, 5 h after LPS or the solvent.
Main Results : LPS initially decreased MAP and lastingly increased HR. In the 3-h LPS animals (n=8), tissue plasma clearance was lower in the heart and calf muscle and increased only in diaphragm, compared to corresponding control animals (n=8). In the 5-h LPS rats, clearance was lowered (n=8) in the entire gastrointestinal tract and in testes, compared to controls (n=8). The serum nitrite/nitrate concentration was higher in animals given LPS (n=6) than in controls (n=6).
Conclusion : After LPS, tissue clearance of albumin was not increased in any major tissue, in spite of increased serum levels of NO end products. Apparently, after activation of iNOS, the augmented release of NO is not necessarily associated with increased albumin extravasation.     

Adrenaline markedly improves thoracic epidural analgesia produced by a low-dose infusion of bupivacaine, fentanyl and adrenaline after major surgery: A randomised, double-blind, cross-over study with and without adrenaline

Background: Basic pharmacological research indicates that there are synergistic antinociceptive effects at the spinal cord level between adrenaline, fentanyl and bupivacaine. Our clinical experience with such a mixture in a thoracic epidural infusion after major surgery confirms this. The objectives of the present study were to evaluate the effects on postoperative pain intensity, pain relief and side effects when removing adrenaline from this triple epidural mixture. Methods: A prospective, randomised, double-blind, cross-over study was carried out in 24 patients after major thoracic or abdominal surgery. Patients with only mild pain when coughing during a titrated thoracic epidural infusion of about 10 ml · h^?1 of bupivacaine 1 mg · ml^?1, fentanyl 2 μg · ml^?1, and adrenaline 2 μg · ml^?1 were included. On the 1^st and 2^nd postoperative days each patient was given a double-blind epidural infusion, at the same rate, with or without adrenaline. The effect was observed for 4 h or until pain when coughing became unacceptable in spite of a rescue analgesic procedure. Rescue analgesia consisted of up to two epidural bolus injections per hour and i.v. morphine if necessary. All patients received rectal paracetamol 1 g, every 8 h. Fentanyl serum concentrations were measured with a radioimmunoassay technique at the start and end of each study period. Main outcome measures were extent of sensory blockade and pain intensity at rest and when coughing, evaluated by a visual analogue scale, a verbal categorical rating scale, the Prince Henry Hospital pain score, and an overall quality of pain relief score. Results: The number of hypaesthetic dermatomal segments decreased (P <0.001) and pain intensity at rest and when coughing increased (P <0.001) when adrenaline was omitted from the triple epidural mixture. This change started within the first hour after removing adrenaline. After 3 h pain intensity when coughing had increased to unacceptable levels in spite of rescue analgesia (epidural bolus injections and i.v. morphine). Within 15–20 min after restarting the triple epidural mixture with adrenaline, pain intensity was again reduced to mild pain when coughing. Serum concentration of fentanyl doubled from 0.22 to 0.45 ng · ml^?1 (P <0.01), and there was more sedation during the period without adrenaline. Conclusions: Adrenaline increases sensory block and improves the pain-relieving effect of a mixture of bupivacaine and fentanyl infused epidurally at a thoracic level after major thoracic or abdominal surgery. Serum fentanyl concentrations doubled and sedation increased when adrenaline was removed from the epidural infusion, indicating more rapid vascular absorption and systemic effects of fentanyl.