【作者】 张旭；

【导师】 戴朝六；

【作者基本信息】 中国医科大学 ， 外科学， 2003， 硕士

【摘要】 前言 在肝脏外科中，肝脏缺血再灌注损伤(Hepatic ischemia reperfusion injury,HIRI)可导致肝细胞发生一系列结构与功能的损害，进而引起肝脏功能衰竭，甚至全身多脏器功能衰竭(MODS)，是影响疾病预后、手术成功率以及病人生存率的主要因素之一。HIRI的确切机制目前尚不完全清楚，多数学者认为与氧自由基及钙离子超载等因素有关。 在以往的研究中，已有多种方法应用于HIRI的防护，如缺血预处理、门腔转流或部分人肝血流阻断等，但由于其本身所具有的机体损害性、应用的局限性或操作的复杂性等原因，临床应用受到限制；同时亦有多种药物应用于HIRI的保护，如钙离子拮抗剂、蛋白酶抑制剂等，但效果不确定或并不令人满意。因此，有必要对HIRI的防治行进一步的研究。 还原型谷胱甘肽(Glutathione,GSH)是体内一种重要的抗氧化剂。研究证实，应激时提高与维持组织中GSH的含量，可有效地减轻组织的损伤；热休克蛋白70(Heat shock proteins 70,HSP70)是生物细胞在应激过程中产生的一类生物进化上高度保守的蛋白质，其可以提高组织细胞对应激的耐受能力，抵御各种损害因素的影响，是一种内源性的保护机制。有关GSH、HSP70在HIRI中所起的作用及相关因素对之的影响，一直是实验与临床研究的热点问题。 作为条件必需氨基酸，谷氨酰胺(Glutamine,Gln)对务器官和组织细胞具有重要而独特的代谢功能。研究发现，在多种疾病及应激状态下，组织中Gln的水平与疾病的预后及转归密切相关，而补充GIn则可以缩短患者住院时间、改善疾病的预后及病人的生存率。目前在临床中，多采用丙氨酸一谷氨酸胺（Alanyl－Gluta－mine，Ua－Gi）二肽作为Gi的供体。 以前的研究证实，Gin可维持组织中GSH的含量或增强脏器HSP70的表达，从而保护心肌、骨骼肌及肠道等脏器的缺血再灌注损伤。然而，有关Gin在HIRI中是否具有保护作用，及其对肝脏GSH含量、HSpoo表达影响，目前应用研究不多。 我们利用大鼠HIRI模型，观察Ala－Gi（GIn）对肝脏生化酶类以及还原型GSH、HSpoo表达的影响，初步探讨la－Gin（Gi）对HIRI的保护作用及其可能机制。 材料与方法 雄性Wistar大鼠，参照第三军医大学的静脉营养模型，行右侧颈外静脉插管，微量泵输注 0．9％Nacl液问讪hL观察 3天。然后，大鼠随机分为Gi组＊组）与对照组＊组人人组经静脉导管给予 3％Ala－GIn溶液（速度 Zrnl／h八 C组以相同速度给予同等量0．9％生理盐水，均输注3天K8价d人第7天开腹，离断肝周韧带，消除肝脏侧支循环，夹闭肝门（Pingle ％法）使肝脏缺血，持续30分钟后松开血管夹使肝血供恢复。分别于再灌注后1／J’时、24小时上8小时自腹主动脉取血，用全自动生化分析仪测定血清中的谷丙转氨酶（ALTX乳酸脱氢酶（LDH）水平；取肝组织按谷济甘肽（GSH入超氧化物歧化酶门OD）试剂盒说明测定其GSH、SOD的水平；用Western Blotting法测定驱注后l、24、48／J’时肝组织中HSpoo的表达；并取肝组织置于10％福尔马林溶液中固定以用于作病理学检查；再取肝组织置于2．5％戊二醛溶液中固定以用于作电镜检查；计算两组大鼠的术后24小时生存率。 血清生化酶 ALT、LDH，肝组织中 GSHSOD的数值以 i。S表 ·2·示，用 t检验进行统计学处理；术后生存率用 X‘检验。所有数据资料均采用SPSS软件进行处理。 结 果 l．血清生化酶水平HIRI后1／J’时，G组血清ALT、LDH的水平分别为499．25 t 120．8什【／L6956．00。ZM 3．931U／L；C组的水平为823．56土328．71 IU／L、11715．31土2993．50IU／L人组显著低于C组（P＜o．05；P＜O．01）。再灌注后24小时人组血清＊u。LDH的水平分别为176．69 t 151．84IU／L、415．38 L 213．68IU／L＊组的水平为548．25。257．25 o：／o：／LJ958．so。687．321U／L，G组显著低于C组（P＜o．0二）。 2．缺血再灌注后肝脏组织中GSHJOD的水平＊ 后1／J’时人组肝组织中 GSH水平为 1216．09。152．78mgGSH／mgprot，明显高于 C组的 856．68土互 17．64mgGSH／mgProt（P＜ 0．01）；HIRI后24 ／J’时，G组肝组织中GSH 7k平为899．73 t 57．75mgGSH／mg－Prot，明显高于 C组的 800．50 t 94．79mgGSH／mgProt（P＜ 0．05）。HIRI后1小时及24小时人组肝组织中SOD活性与C组无显著性差异（145．09 t 14·66NU／mgProt vs 135．30 i 3．93NU／mg’prot，136．90。6．46 mgprot vs 136．35 t 14．25 mgl）rot少＞0．05）。 3．缺血融注后肝脏组织中HSP70的表达C组于再灌注后1小时仅有微量的HSpoo表达，其表达程度与G组无显著性差异，至再灌注后24小时A8小时其HSP70的表达明显增强；而G组在再灌注后24小时产8小时仅有轻度的增强，其表达程度明显低于C组。 4．术后生存率术后 24小时生存率 G组为 78．57％O八人明显高于C组的45．45％（10／22）（X‘二3．86，p＜0．05）。更多还原

【Abstract】 In the hepatic surgery, Hepatic ischemia reperfusion injury ( HI-RI) can lead to a series of structural and functional disorders of liver tissue and cause hepatic failure or multiple organ system failure. HIRI is one of the main causes affecting the prognosis of the diseases, success rate of the operations and survival rate of the patients. Although the pathogenesis of HIRI has not been completely understood, most of scholars believe that HIRI is related to free oxygen radicals and calcium overload, et al.In previously studies, lots of methods have been used to pertect liver against HIRI, including ischemia pretreatment, portal blood bypass, partial or transient intermittently inflow occlusion and the others. But these methods are Limited at practice for self - deleterious-ness , localization at clinical practice, increasing complexity of the operations or easy emergence of the complications. At same time some drugs, including calcium antagonists, protease inhibitors and oxygen radical eliminators have been used to pertect liver against injuries, but the effects are not definitive or satisfactory, So it is essential to study furtherly the protection methods against HIRI.Reduced glutathione (GSH) is a important antioxidant-that per-tects tissues from free radical injuries. Studies have demonstrated thatincreasing or maintaining the GSH stores in tissues can attenuate free radical injuries. Heat shock protein 70 (HSP70) is highly conserved and produced by bio - cell subjected to a variety of stress. In cells and tissues, HSP70 is an intrinsic perfection mechanism and can increase the tolerance to against stimuli and preelection effects on injuries. The proctection effects of GSH and HSP70 on HIRI and the related influences are always the focus of clinical and experimental studies.GLN, a conditional essential amino acid , occupies a particular and important role in the metabolism in cells and tissues. Studies indicated that Gin concentration in the blood and tissues was related to the prognosis of the diseases and survival rate of the patients in many severe diseases and stress. Supplementation of Gin cound preserve the structures and functions of tissues, decrease length of hospital stay and improve the prognosis and survival rate. For the low solubility and unstable character, it is usually replaced by alanyl - glutamine dipeptide (Ala-Gin) in clinic.It has beeb reported previously that Gin can pertect tissues from the ischemia reperfusion injuries by preserving tissue glutathione stores, enhancing the expression of HSP70 in skeletal muscle, cardiac muscle and intestines. In liver, However, the related reports were rare about its pertective effects on HIRI or effects on GSH level and HSP70 expression in tissues.In our study, we primarily investigated whether Ala - Gin ( Gin) has a perfection effects on HIRI through observing the effect of Ala -Gin (Gin) on the concentrations of the hepatic biochemical enzymes, and furtherly discussed the possible mechanisms through observing the effect on tissue GSH level and the expression of HSP70 in the HIRI model of rat.Materials and MethodsMale Wistar rats were placed central vena catheter through the right external jugular vein, according to the TPN model of the Third Military Medical University. After 2 hours, rats received 0.9% saline solution by minim pump (2ml/h). After observed for 2 days, rats were randomly divided into Gin (G) group and control (C) group. Rats in G group were received the 3% Ala - Gin solution (2ml/h) through the venous catheter and Rats in C group received 0. 9% saline solution at the same velocity and quantity. All solution was infused for 3 days (48ml/d) . At the seventh day, the rats were anesthetized before all operative procedures, and the abdomen was opened through a median laparotomy and the liver was detached from there ligaments. According to Pringle, s method, total hepatic ischemia with portal congestion was produced by clamping the hepatoduodenal ligament for 30 minutes with a microvascular clamp, and dec更多还原