【作者】 张匀；

【导师】 梁廷波；

【作者基本信息】 浙江大学 ， 外科学， 2006， 硕士

【摘要】 研究背景及目的严重创伤性休克为青壮年患者的最大死亡原因之一，创伤性休克复苏后常出现各种并发症，导致住院时间延长，医疗费用增加。许多学者在损伤控制、液体复苏方法以及复苏后机体免疫紊乱等方面大量的研究表明，在损伤已控制的基础上有效的容量复苏不仅能增加早期复苏的成功率，并且能减轻全身的炎症反应，改善机体免疫功能紊乱，降低全身严重感染及多脏器功能衰竭的发生以提高严重创伤性休克的治愈率。然而在选择早期复苏液体的种类上，一直存在着晶体／胶体、自然胶体／人工胶体孰优孰劣的争论，同时临床上也缺乏一些评价机体免疫功能紊乱的可靠指标。机体在严重创伤休克时会出现毛细血管内皮损伤及渗漏导致白蛋白及约75％输入的晶体渗至间质，引起组织水肿并进一步加重毛细血管对液体的扣押和组织的缺氧，同时机体细胞免疫功能特别是T细胞功能明显受抑，表现为T细胞IL-2mRNA表达及产生和分泌减少，IL-2受体表达降低以及T细胞亚群及其细胞因子表达降低。羟乙基淀粉(Hydroxyethyl starch，HES)具有“堵塞”毛细血管渗漏的作用，能有效改善微循环的血液流变学异常，增加组织氧供。HES可明显减轻创伤休克后全身炎症反应及血管内皮的损伤，同时可能通过破坏以血清IL-2，IL-4，IL-10，γ-IFN等水平为标志的促炎反应及抗炎反应的平衡而诱导淋巴细胞的增殖与分化的改变。HES(130／0.4)作为第三代中分子量(130kd)低取代级(C2／C6∶0.4)羟乙基淀粉产品更具有扩容效果佳，体内蓄积少，微循环及组织氧张力改善佳，凝血功能及肾功能障碍等并发症少的优点。本研究的目的为在建立一个稳定可靠的创伤性休克模型的基础上探讨HES(130／0.4)是否能改善创伤性休克大鼠淋巴细胞的增殖与分化功能紊乱。对象与方法1．采用“断股骨+动脉放血+液体复苏”大鼠创伤性休克的模型。钝性离断右侧股骨作为主要创伤因素。动脉快速放血，以平均动脉压降至30mmHg为目标。2．分别以不同的复苏液体(林格氏液，6％HES(130／0.4)，5％白蛋白)及不同的取材时间(复苏后24，48小时)多组进行对照研究。复苏液体量按照大鼠体重计量。3．监测创伤后0，1，3，24小时血清乳酸，pH值，动脉血氧分压，碱剩余，血清钾及肌酐浓度等生化指标以及肾肺脏器病理改变评价模型的稳定性。4．分别用AnnexinV／PI染色法(流式细胞仪Coulter XL)及末端脱氧核苷酸转移酶介导的原位末端标记法(TUNEL法)检测骨髓单个核细胞凋亡比例。5．用流式细胞仪法检测创伤前后外周血淋巴细胞CD4+、CD8+T细胞数量及CD4+／CD8+比值。6．用细胞内因子检测法(流式细胞仪Coulter XL)检测创伤前后外周血CD4+细胞向TH1／TH2的分化能力。7．用CD4+／Annexin V法(流式细胞仪Coulter XL)检测脾脏CD4+细胞凋亡。8．统计学方法数据用spss12.O统计软件分析。组间采用t检验，P＜0.05具有统计学意义。结果1．大鼠创伤性休克模型建立成功，模型鼠能达到平均动脉压30mmHg的休克要求，具有一定的复苏成功率(90％)。同时创伤休克后大鼠血氧合能力，乳酸水平，肾脏功能，电解质改变明显并且稳定。肺肾等脏器的病理学改变亦较为显著，模型建立成功。2．创伤休克后CD4+细胞数量明显下降，而HES复苏后第48小时，CD4+细胞数量即能恢复到正常水平。CD8+细胞数量未发现明显改变。3．创伤性休克后外周血CD4+淋巴细胞分化出现“TH1向TH2漂移”的改变，且HES能改变此趋势。4．与对照组比较，HES组复苏后CD4+T细胞凋亡比例未呈现先升后降的改变。5．与对照组比较，流式细胞仪法和TUNNEL法均发现HES组复苏后骨髓单个核细胞凋亡比例未见明显升高。结论1．该创伤性休克动物模型可造成机体明显的病生理损伤，且操作简便，易控制。2．创伤性休克后大鼠外周血CD4+T淋巴细胞数量明显减少，CD4+T细胞的进一步分化能力表现为“TH1向TH2漂移”，脾CD4+T细胞及骨髓单个核细胞凋亡明显增加等。机体细胞免疫功能受到明显抑制。3．羟乙基淀粉(130／0.4)可抑制创伤休克后CD4+T淋巴细胞数量下降及“TH1向TH2漂移”的趋势，同时缓解脾CD4+T细胞及骨髓单个核细胞凋亡急剧增加的趋势，从而改善机体细胞免疫功能。更多还原

【Abstract】 BackgroundTrauma has been one of the most important factors of the increasing mortality of young people. There are three major causes of death of trauma: (1)failure of resuscitation of severe traumatic shock. (2)severe organ injury. (3)Development of immunosuppression, sepsis and MODS after trauma. Many severe traumatic/hemorragic shock and insufficient fluid resuscitation initiate systemic inflammatory process and alter the physiologic immune balance. Adequate volume replacement is paramount in the treatment of traumatic/hemorragic shock patients. However, controversy is still remaining on the volume therapy.Crystalloids are freely permeable to the vascular membrane and are therefore distributed mainly in the interstititial and/or intercellular compartment. Only 25% of the infused crystalloid solution remains in the intravascular space and subsequently lead to tissue edema. That’s why even a massive crystalloid resuscitation is less likely to achieve adequate restoration of microcirculation blood flow compared to a colloid-based volume replacement strategy. After world war II, a large variety of nature and synthetic colloid preparations are used world-wide. An infusion of albumin can not raise colloid osmotic and the pressure gradient of colloid osmotic-pulmonary arterial wedge pressure(PAWP). It was shown that the infusion of albumin resulted respiratory failure, especially in patients with sever shock compared to patients who did not receive albumin. This appears to be due to increased leakage into the interstitial pace. Whereas in inflammatory-related capillary leaks, hydroxyethyl starch (HES) has been reported to have "occlusive" effect on damaged capillaries, and subsequently limit the extravasation of fluid. In a recent clinical study, intravascular volume replacement with HES130/0.4 versus crystalloid solution improved tissue oxygenation in patients undergoing abdominal surgery. Meantime, the use of HES may result in less endothelial cell damage and improve microcirculation, andconsequently reduced the inflammatory response.Depressed T-cell activity has been a consistent finding during the development of immunosuppression after severe traumatic/hemorragic shock . Many reports show a shift to Th2 subset with resultant overproduction of IL-10 and IL-4. It has been suggested to be a pivotal contributor to post injury immunosuppression. On the otherwise exaggerate lymphocyte apoptosis is present in peripheral blood of patients with traumatic/hemorragic shock, and leads rapidly to a profound and persistent lymphocyte loss. Since HES may reduce inflammatory response in traumatic/hemorragic shock, can it prevent the development of immunosuppression versus other resuscitation fluid. This is the main goal of our study.Objects and methods1. T/HS animal model: SD rats underwent internal jugular vessel cannulation. The T/HS model involved: (1)femoral bone mutilation , (2) a controlled retrieval of blood, maintaining mean blood pressure at 30±5mmHg during 30 minutes. (3)resuscitation with lactated Ringer’s (control 1), HES130/0.4 (experimental) and 5%human albumin (control 2)(twice the blood volume retrieved)plus their shed blood.2. Evaluate Serum lactate , potassium, and creatinine level; PH, PO2 and sBE in arterial blood; and pathological change in tissue of lung and kidney in control group to evaluate the stability of the T/HS animal model.3. Determination of CD4+ and CD8+ lymphocyte subsets by dual parameter flow cytometry at time of 24, 48 hours after resuscitation (control group versus experimental group).4. Determination of TH1 and TH2 lymphocyte subset by detection of intracytoplasmic cytokine (IL-4, γ-IFN) using flow cytometry at time of 24, 48 hours after resuscitation (control group versus experimental group).5. Determination of apoptosis of CD4+ lymphocyte in spleen by detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V with Flow cytometric at time of 24, 48 hours after resuscitation (control group versus experimental group).6. Determination of apoptosis of PBMC of marrow by detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V with Flow cytometric at time of 24,48 hours after resuscitation (control group versus experimental group).7. Apoptosis of PBMC of marrow was assessed through terminal deoxynucleotide transferase-mediated dUTP nick-end labeling assay at time of 24, 48 hours after resuscitation (control group versus experimental group).Result1. T/HS animal model was established successfully. Through a controlled retrieving of blood from jugular vessel the mean arterial pressure was reduced to 30 mmHg and kept this level for 30 mins. Biochemical indicator such as serum lactate , potassium, and creatinine level; PH, PO2 and sBE in arterial blood; and pathological change in tissue of lung and kidney at time of 24,48 hours after resuscitation indicate the stability of the T/HS animal model in control group.2. The percentage area of CD3+,CD4+ T cell decreased obviously at time of 24 hours after resuscitation in each group, and risen quickly at time of 48 hours only in experimental group (HES130/0.4).3. The ratio of TH1/TH2 decreased obviously in control group 1 (Ringer’s) at time of 48 hours after resuscitation. This difference did not occur in experimental group at any time.4. Control group 1(Ringer’s) showed a striking increased number of apoptosis cells in CD4+T cell at 24 hours after resuscitation versus HES and albumin groups.5. A significantly increased number of apoptosis cells in PBMC of bone marrow was observed in control group1,2(Ringer’s and albumin) and not in experimental group(HES130/0.4).ConclusionsMany significant changes such as the decrease of percentage area of CD3+,CD4+T cell and ratio of TH1/TH2 and the increase number of apoptosis immunologic cell was observed after traumatic/hemorragic shock. HES130/0.4 as a resuscitation fluid may prevent those changes occurred in immunological function.更多还原