硝普钠灌注对移植小肠粘膜细胞凋亡的影响

目的:探讨外源性一氧化氮(nitric oxide,NO)供体硝普钠(sodium nitroprusside,SNP)对移植小肠粘膜细胞凋亡的影响。方法:64只220～300g雄性SD大鼠随机分成3组:A1组(n=8),仅行剖腹关腹手术;A2组(n=12):12对大鼠随机作为供受体行同种异体节段小肠移植,无SNP干预;A3组(n=16):16对大鼠随机作为供受体行同种异体节段小肠移植,SNP加入灌注液进行供肠灌注。采用前述3组动物模型再灌注5小时肠造口标本,TUNEL法检测小肠蜡块标本的细胞凋亡情况。结果:与A1组(3.86±4.74%)相比,A2(22.44±10.94%)、A3组(17.12±8.44%)小肠粘膜的细胞凋亡指数均有显著增高(P<0.05),A3组较A2组细胞凋亡指数显著降低(P<0.05)。结论:小肠移植导致小肠粘膜细胞凋亡增加,外源性NO供体SNP灌注能够显著降低植入小肠的细胞凋亡,从而可能减弱粘膜屏障的损伤。     

硝普钠灌注对移植小肠粘膜细胞凋亡的影响

目的：探讨外源性一氧化氮（nitricoxide,NO）供体硝普钠（sodiumnitroprusside,SNP）对移植小肠粘膜细胞凋亡的影响。方法：64只220-300g雄性SD大鼠随机分成3组：A1组（n=8）,仅行剖腹关腹手术;A2组（n=12）：12对大鼠随机作为供受体行同种异体节段小肠移植,无SNP干预;A3组（n=16）：16对大鼠随机作为供受体行同种异体节段小肠移植,SNP加入灌注液进行供肠灌注。采用前述3。组动物模型再灌注5小时肠造口标本,TUNEL法检测小肠蜡块标本的细胞凋亡情况。结果：与A1组（3．86±4．74％）相比,A2（22．44±10．94％）、A3组（17．12±8．44％）小肠粘膜的细胞凋亡指数均有显著增高（P〈0．05）,A3组较A2组细胞凋亡指数显著降低（P〈0．05）。结论：小肠移植导致小肠粘膜细胞凋亡增加,外源性NO供体SNP灌注能够显著降低植入小肠的细胞凋亡,从而可能减弱粘膜屏障的损伤。     

兔急性肠道缺血再灌注损伤模型的建立

目的:建立小肠急性缺血再灌注损伤模型,确定合适的肠系膜上动脉阻断时间.方法:将70只新西兰兔随机按不同的肠系膜缺血时间(0、15、30、45、60min)分为A、B、C、D、E5组,每组14只,取8只用于恢复血供2h后留取各组兔下腔静脉血标本及肠组织,检测血清中MDA含量的变化,光镜下观察小肠组织形态学变化并对小肠黏膜损伤程度进行评分.另6只用于术后24h、 48h及72h生存率的观察.结果:A、B、C组的术后生存率均>83.3%.C、D、E组的MDA含量及肠黏膜损伤评分与A组比较,差异均有显著性(F=12.13～280.24,p<0.01).结论:肠系膜缺血30min,再灌注2h是建立兔小肠急性再灌注损伤的合适时间.     

小鼠左肺原位移植模型的建立

目的通过显微外科技术建立小鼠原位肺移植模型,为肺移植研究提供动物模型。方法采用C57BL/6小鼠作为供、受体,行同基因小鼠原位左肺移植,使用Cuff套管法进行气管及血管吻合。术后7、14、21、28 d取移植肺及原肺,行HE染色,评价肺移植后效果。结果学习曲线后,共30例小鼠移植,手术成功率89%,小鼠成活率100%。供体手术时间:(35.2±9.81)min,受体手术时间:(24.6±7.42)min,冷缺血时间是:(46.6±8.92)min,热缺血时间是:(17.2±3.08)min。同基因移植物大体及病理无明显改变,病理显示与原肺无差别。结论本技术能够方便快捷建立小鼠肺移植模型,成功率高,可重复性强,符合原位肺移植临床生理,是研究肺移植发病机制和治疗的良好动物模型。     

Kyoto-1保存液保存的移植用肺Ⅱ型肺泡上皮细胞钠-钾泵的研究

目的：临床认为肺水肿已成肺移植过程中的最大障碍。近年来资料表明,过是肺泡液的吸收与Ⅱ型肺泡上皮细胞Na^ 的跨膜能力有关,保存中若能保持肺上Na^ -K^ -ATPase的活性,对成功的肺移植是非常必要的,本实验研究0℃、4℃Kyoto-1（NewET－K）保存液保存体肺脏,型Ⅱ肺泡上皮细胞Na^ -K^ -ATPase的活性变化,为Kyoto-1在临床上最大时限地保存供体肺提供基础理论依据。方法：取Wistar大白鼠,随机分为对照组和实验组,对照组取新鲜肺脏。实验组分别灌注0℃、4℃的Kyoto-1保存液,分别放入0℃、4℃冰箱中保存4h,24h,48h,进行光,电镜酶组织化学研究。使用图像分析系统进行定量测定。结果：4℃、24h实验组钠泵活性可以得到较好的保存（P＞0．05）。结论：钠泵可以作为检测供保存质量的重要指标。     

高效液相测定同型半胱氨酸方法的建立

为测量包括同型半胱氨酸在内的 1 8种氨基酸 ,用高效液相紫外检测法 ,在氨基酸混合标样中 ,加进Hcy标准品 ,仍用 4 5min程序分析 ,对AccqTag方法进行了修改 ,衍生温度改为常温 ,衍生后用醋酸酸化 ,并保存于 0～ 1℃ ,同型半胱氨酸出峰的时间为第 33min左右 ,结果得到了分离良好的 1 8种氨基酸图谱。     

同种异体肌腱不同时段深低温保存对肌腱愈合影响的实验研究

目的:探讨深低温(-80℃)保存异体肌腱的最佳时间段.方法:体重250-300g的雌雄不分的SD大鼠144只,随机分为Ⅰ、Ⅱ两组.每组72只.Ⅰ组为供腱组,提供144条供移植用的肌腱.Ⅱ组又随机分为A、B、C、D四组,每组18只,分别接受由Ⅰ组提供的经深低温保存1天、10天、1个月、3个月的肌腱.术后3天时、10天时分别从A组、B组、C组、D组随机抽取3只,近端自肌肉肌腱移行处,远端自跟腱附着处切断(包括近、远端吻合口),进行大体观察,组织学观察;术后4W、8W时分别从A组、B组、C组、D组随机抽取6只,进行大体观察,组织学观察,最大载荷测定.结果:保存10天以上的三个时间段的肌腱移植后和保存1天的比较,保存10天以上的肌腱术后粘连较轻,肌腱抗拉力强度降低.保存10天以上三个时间段的肌腱移植后大体观察、组织学观察无明显差异;最大载荷测定,结果:分析无统计学意义(P＞0.05).结论:肌腱在深低温(-80℃)环境中长时间保存后,其用于功能修复,愈合情况无明显差别.     

胰岛素对大鼠肠缺血再灌注损伤的影响

目的：观察胰岛素对大鼠肠缺血再灌注后小肠组织损伤的影响。方法：雄性SD大鼠40只随机分为4组,每组10只,手术对照组、单纯缺血组、再灌注组、胰岛素干预组。于30min缺血和120min再灌注后,进行组织病理学和生化检测。结果：（1）单纯缺血组肠粘膜损害较手术对照组明显升高（P〈0.01）,超氧化物歧化酶（SOD）活性无明显变化;（2）再灌注组SOD活性明显降低,与手术对照组和单纯缺血组相比较差异均有显著性（P〈0.01）;（3）胰岛素组SOD活性与再灌注组相比有明显改善（P〈0.01）。结论：肠缺血可以引起肠粘膜损伤,再灌注则可加重这种损伤,胰岛素可以减轻再灌注损伤。     

胰岛素对大鼠肠缺血再灌注损伤的影响

目的:观察胰岛素对大鼠肠缺血再灌注后小肠组织损伤的影响。方法:雄性SD大鼠40只随机分为4组,每组10只,手术对照组、单纯缺血组、再灌注组、胰岛素干预组。于30min缺血和120min再灌注后,进行组织病理学和生化检测。结果:(1)单纯缺血组肠粘膜损害较手术对照组明显升高(P<0.01),超氧化物歧化酶(SOD)活性无明显变化;(2)再灌注组SOD活性明显降低,与手术对照组和单纯缺血组相比较差异均有显著性(P<0.01);(3)胰岛素组SOD活性与再灌注组相比有明显改善(P<0.01)。结论:肠缺血可以引起肠粘膜损伤,再灌注则可加重这种损伤,胰岛素可以减轻再灌注损伤。     

运用HPLC-MS对鸦胆子油自微乳给药系统肠吸收的研究

目的:建立高效液相色谱-串联质谱法检测油酸和亚油酸含量的方法,从而对鸦胆子油自微乳给药系统中鸦胆子油的肠吸收进行研究.方法:以甲醇-水(95∶5 v/v)为流动相,流速为0.4 mL/min,柱温为35℃作为高效液相色谱的检测条件.利用大鼠小肠膜建立体外药物扩散体系研究鸦胆子油的肠吸收特性.结果:油酸和亚油酸的保留时间分别为10.46± 0.02和8.55±0.01 min,线性范围分别为0.50～50.0 ng/mL和5.06～101.2 ng/mL,平均绝对回收率分别为97.49±3.11％和105.76± 3.13％.日间和日内精密度都小于5％.在肠吸收实验中,鸦胆子油自微乳体系中测得油酸和亚油酸含量是单独给予鸦胆子油测得量的2.8和4.1倍.结论:该方法高效、灵敏、选择性高,可以用于鸦胆子油及鸦胆子油自微乳肠吸收中油酸和亚油酸的质量测定.     

Pulsatile flow during cardiopulmonary bypass preserves postoperative microcirculatory perfusion irrespective of systemic hemodynamics

The onset of nonpulsatile cardiopulmonary bypass is known to deteriorate microcirculatory perfusion, but it has never been investigated whether this may be prevented by restoration of pulsatility during extracorporeal circulation. We therefore investigated the distinct effects of nonpulsatile and pulsatile flow on microcirculatory perfusion during on-pump cardiac surgery. Patients undergoing coronary artery bypass graft surgery were randomized into a nonpulsatile (n = 17) or pulsatile (n = 16) cardiopulmonary bypass group. Sublingual mucosal microvascular perfusion was measured at distinct perioperative time intervals using sidestream dark field imaging, and quantified as the level of perfused small vessel density and microvascular flow index (vessel diameter < 20 μm). Microcirculation measurements were paralleled by hemodynamic and free hemoglobin analyses. The pulse wave during pulsatile bypass estimated 58 ± 17% of the baseline blood pressure waveform. The observed reduction in perfused vessel density during aorta cross-clamping was only restored in the pulsatile flow group and increased from 15.5 ± 2.4 to 20.3 ± 3.7 mm/mm(2) upon intensive care admission (P < 0.01). The median postoperative microvascular flow index was higher in the pulsatile group [2.6 (2.5-2.9)] than in the nonpulsatile group [2.1 (1.7-2.5); P = 0.001]. Pulsatile flow was not associated with augmentation of free hemoglobin production and was paralleled by improved oxygen consumption from 70 ± 14 to 82 ± 16 ml·min(-1)·m(-2) (P = 0.01) at the end of aortic cross-clamping. In conclusion, pulsatile cardiopulmonary bypass preserves microcirculatory perfusion throughout the early postoperative period, irrespective of systemic hemodynamics. This observation is paralleled by an increase in oxygen consumption during pulsatile flow, which may hint toward decreased microcirculatory heterogeneity during extracorporeal circulation and preservation of microcirculatory perfusion throughout the perioperative period.     

Transport and metabolism of carnitine precursors in various organs of the rat

Isolated, vascularly perfused small intestine, liver, and kidney were used to investigate their interdependence in the absorption and metabolism of carnitine precursors in the rat. During 30 min of recirculating perfusion, the small intestine absorbed trimethyllysine, hydroxytrimethyllysine, and trimethylaminobutyrate fairly well when they were administered via the lumen or the perfusate. Trimethylaminobutyrate was synthesized from either trimethyllysine or hydroxytrimethyllysine by the small intestine, but further hydroxylation of trimethylaminobutyrate to carnitine did not occur. Trimethyllysine and hydroxytrimethyllysine were not readily absorbed by the liver. In contrast, trimethylaminobutyrate and trimethylaminobutyraldehyde were rapidly absorbed from the perfusate and readily incorporated into carnitine by the liver. Trimethyllysine and hydroxytrimethyllysine were taken up slowly by the kiodney and partially converted to trimethylaminobutyrate during 3409 min of perfusion. Trimethylaminobutyrate was neither absorbed readily by the kidney nor was it hydroxylated to carnitine. These results were compared to whole animal studies performed over an equivalent time period. The data suggest that the isolted small intestine absorbs trimethyllysine well, but it probably plays a minor role in metabolizing physiological quantities of this compound in the whole animal where other organs are competing for the same substrate. In both the isolated organ and in the whole animal, the kidney absorbs and metabolizes trimethyllysine more readily than the liver; whereas the liver absorbs trimethylaminobutyrate more rapidly than either the kidney or the small intestine and, unlike these organs, converts it to carnitine.     

单人建立大鼠原位肝移植模型的手术体会

目的:探讨单人建立稳定大鼠原位肝移植模型手术中的难点及对策。方法:单人及双人裸视下采用改良二袖套法制备大鼠原位肝移植模型各50例。结果:单人及双人组供体手术时间、无肝期、受体手术手术时间分别为:(39.16±2.89)min和(38.36±3.04)min、(19.92±1.36)min和(19.70±1.40)min、(61.98±3.46)min和(58.65±3.94)min;单人及双人组手术成功率、1周存活率、一月存活率分别为:94.0%(47/50)和92.0%(46/50)、86.0%(43/50)和88.0%(44/50)、86%(43/50)和84%(42/50)。结论:通过改进手术方法,简化操作,单人即可建立稳定的大鼠原位肝移植模型。     

Difructose anhydrides III and IV equally promote calcium absorption from the luminally perfused rat small intestine

We examined the effects of di-D-fructose anhydride (DFA) III and IV on Ca absorption in luminally perfused segments of the small intestine in anesthetized rats. The calcium absorption rate with perfusion of 10 mmol/l CaCl2 was similarly increased by addition of 100 mmol/l DFAIII or IV, and these promotive effects of both DFAs were pronounced at perfusion rate of 0.15 ml/min than at 0.3 ml/min. The promotive effects were higher in the duodenojejunum than in the ileum.     

Liver glycogen metabolism during intestinal perfusion in the rat.

The effects of metabolic constituents on glycogen metabolism in the liver can be studied by perfusing the small intestine with those constituents capable of being absorbed across the gut mucosa. Perfusion of the gut with glucose to give concentrations of approx. 20 mM in the portal vein produced a 5-fold increase in liver glycogen after 60 min. The rate of synthesis over the 15–30 min period of perfusion of 1.24 μmol/min/g liver fell to 0.34 in the 30–60 min period. This stimulation was accompanied by a transient activation of glycogen synthase which occurred in the first 30 min period of perfusion but which had disappeared by 60 min. Glucose perfusion had no effect on total and active phosphorylase at the time intervals studied. While the pattern of insulin secretion during perfusion could not be ascertained, the activation of glycogen synthase and increase in glycogen synthesis correlated with the production of gastric inhibitory polypeptide (GIP), a potent stimulator of insulin secretion. Perfusion of the gut with fructose did not stimulate glycogen synthesis or activate glycogen synthase or stimulate GIP secretion, but instead it produced a marked activation of phosphorylase after 30 min of perfusion.     

A simple and rapid ion-pair HPLC method for simultaneous quantitation of 4-nitrophenol and its glucuronide and sulfate conjugates

Because of its simple and well characterized metabolic profile, 4-nitrophenol is widely used as a model substrate to investigate the influence of drug therapy, disease, nutrient deficiencies and other physiologically altered conditions on conjugative drug metabolism in animal studies. For simultaneous determination of 4-nitrophenol (PNP), 4-nitrophenyl-beta-D-glucuronide (PNP-G) and 4-nitrophenyl-sulfate (PNP-S) in samples generated in rat small intestine luminal perfusion experiments, an ion-pair HPLC assay coupled with UV detection was set up. The RP-HPLC separation was achieved with a methanol-water mixture (50:50, v/v) containing 0.01 M tetrabutyl-ammonium-bromide with UV detection of the analytes at 290 nm. The isocratic system was operated at ambient temperature and required less than 7 min of chromatographic time. The method provided good enough within-day precision, between-day precision and linearity in the target concentration ranges of 6-1200 microM (PNP) and 2.5-100 microM (PNP-G and PNP-S). The instrumental limit of quantification for PNP-G and PNP-S was found to be 2.7 microM and 2.1 microM, respectively. The assay was applied for determination of PNP, PNP-G and PNP-S in rat small intestine perfusates.     

心内直视手术中七十岁以上患者的体外循环管理

目的总结分析心内直视手术中七十岁以上患者的体外循环特点和管理方法。方法 2005年3月至2008年7月间7000余例手术中70岁以上患者共有194例,分类进行心内直视手术。体外循环时全部应用进口膜肺,勃脉力A和胶体预充,常规加入白蛋白、激素和乌司他丁;心肌保护采用间断灌注4：1冷含血停搏液,顺灌逆灌和桥灌相结合;常规监测混合静脉氧饱和度和血细胞压积,积极应用超滤技术。结果术中转流平稳,血流动力学稳定,监测指标均在正常范围,平均体外循环时间和主动脉阻断时间分别为111.5±40.4m in和63.6±21.0m in,自动复跳率52.6%,平均搭桥数目3.4±0.8支,术后平均气管拔管时间24.0±12.7h,平均ICU时间4.7±3.5d。结论 70岁以上患者以瓣膜和冠状动脉病变为主、病变复杂,体外循环时间和主动脉阻断时间较长,各脏器保护要求高,针对高龄患者的特点,制定相应的体外循环管理方法,是保证手术成功的重要因素。     

Small bowel transplantation in mice

Since 1990, the development of tacrolimus-based immunosuppression and improved surgical techniques, the increased array of potent immunosuppressive medications, infection prophylaxis, and suitable patient selection helped improve actuarial graft and patient survival rates for all types of intestine transplantation. Patients with irreversible intestinal failure and complications of parenteral nutrition should now be routinely considered for small intestine transplantation. However, Survival rates for small intestinal transplantation have been slow to improve compares increasingly favorably with renal, liver, heart and lung. The small bowel transplantation is still unsatisfactory compared with other organs. Further progress may depend on better understanding of immunology and physiology of the graft and can be greatly facilitated by animal models. A wider use of mouse small bowel transplantation model is needed in the study of immunology and physiology of the transplantation gut as well as efficient methods in diagnosing early rejection. However, this model is limited to use because the techniques involved is an extremely technically challenging. We have developed a modified technique. When making anastomosis of portal vein and inferior vena cava, two stay sutures are made at the proximal apex and distal apex of the recipient s inferior vena cava with the donor s portal vein. The left wall of the inferior vena cava and donor s portal vein is closed with continuing sutures in the inside of the inferior vena cava after, after one knot with the proximal apex stay suture the right wall of the inferior vena cava and the donor s portal vein are closed with continuing sutures outside the inferior vena cave with 10-0 sutures. This method is easier to perform because anastomosis is made just on the one side of the inferior vena cava and 10-0 sutures is the right size to avoid bleeding and thrombosis. In this article, we provide details of the technique to supplement the video.     

小鼠腹部心脏移植模型的制作与改进

目的建立并改进小鼠腹部异位心脏移植模型,为器官移植研究提供技术支持。方法SPF级近交系小鼠112只进行心脏移植手术,在传统方法的基础上进行改进,观察改进后手术效果并分析其优势。结果手术成功率为89.28％。总手术时间（103.9±16.5）min,受体手术时间（73.0±7.9）min。改进的方法简化了操作步骤,降低了手术难度。结论改进的小鼠腹部异位心脏移植技术是一种简便、有效、成功率高的模型制作方法。