创伤性休克大鼠模型的制作及羟乙基淀粉对其淋巴细胞功能影响的研究

发布时间：2018-03-03 01:14

本文选题：创伤　切入点：休克　出处：《浙江大学》2006年硕士论文　论文类型：学位论文

【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。
[Abstract]:Background and purpose of research
Severe traumatic shock is one of the largest cause of death in young patients, the complications often occur after resuscitation from traumatic shock, resulting in prolonged hospitalization, medical expenses increased. Many scholars in the study of a lot of damage control, fluid resuscitation method and after the recovery of immune disorders and other aspects that effectively based on the damage control capacity early recovery can not only increase the success rate of recovery, and can reduce the systemic inflammatory reaction, improve the immune function disorder, reduce serious systemic infection and multiple organ failure occurred in order to improve the cure rate of severe traumatic shock. However, in the choice of early resuscitation fluid, there has been a natural colloidal crystal / colloid. The artificial colloid / the merits of arguments, clinical and lack of reliable indicators of evaluation of the disorder of the immune function.
The body will appear capillary endothelial damage and lead to leakage of albumin and about 75% input crystals into the interstitial infiltration in severe traumatic shock, causing edema and the increase of capillary liquid seizure and tissue hypoxia, while the cellular immune function especially the function of T cells was significantly inhibited, the expression of IL-2mRNA and T were produced and reduce the secretion of IL-2 receptor, and decreased expression of T cell subsets and cytokines. The decreased expression of hydroxyethyl starch (Hydroxyethyl starch, HES) has a "jam" capillary leakage effect, can effectively improve blood rheology microcirculation abnormal, increase oxygen supply.HES can significantly reduce the systemic inflammatory response after traumatic shock and vascular endothelial at the same time through injury, damage to the serum IL-2, IL-4, IL-10, -IFN etc. as a symbol of the gamma level of proinflammatory and anti-inflammatory responses and balance Induce the proliferation and differentiation of lymphocyte.
HES (130 / 0.4) as the third generation of molecular weight (130kd) low substitution (C2 / C6: 0.4) hydroxyethyl starch products have more expansion effect, less accumulation, improve microcirculation and tissue oxygen tension is good, the advantages of blood coagulation and renal dysfunction and other complications. The purpose of this study is in establish a stable and reliable model of traumatic shock on the HES (130 / 0.4) the proliferation and differentiation of lymphocytes can improve dysfunction in rats with traumatic shock.
Object and method
1., a rat model of traumatic shock was established by breaking the femoral artery + blood letting + fluid resuscitation. The right femur was the main trauma factor, and the artery was fast bleeding. The mean arterial pressure drop to 30mmHg was the target.
2., different groups of resuscitate fluid (Ringer's solution, 6%HES (130 / 0.4), 5% albumin) and different time of extraction (24,48 hours after recovery) were compared in different groups.
3. monitoring serum lactic acid, pH value, arterial oxygen partial pressure, alkali residual, serum potassium and creatinine concentration and other biochemical indicators of 0,1,3,24 hours after trauma, as well as the stability of renal and lung organ pathological changes evaluation model.
4., the apoptosis ratio of bone marrow mononuclear cells was detected by AnnexinV / PI staining (flow cytometry Coulter XL) and terminal deoxynucleotidyl transferase mediated in situ end labeling (TUNEL).
5. flow cytometry was used to detect the number of CD4+, CD8+T cells and the ratio of CD4+ / CD8+ in peripheral blood lymphocytes before and after trauma.
6. the differentiation ability of CD4+ cells from peripheral blood to TH1 / TH2 was detected by intracellular factor detection (flow cytometry Coulter XL).
7. the apoptosis of spleen CD4+ cells was detected by CD4+ / Annexin V (flow cytometry Coulter XL).
8. statistics method data were analyzed by spss12.O statistical software. T test was used among groups, and P < 0.05 had statistical significance.
Result
1. rat model of traumatic shock rats can be successfully established, shock mean arterial pressure 30mmHg, has a certain success rate of recovery (90%). At the same time after traumatic shock in rats with blood oxygenation capacity, lactate levels, renal function, electrolyte changes and stability. The pathological lung kidney pathology is more significant that model was established successfully.
2., the number of CD4+ cells decreased significantly after traumatic shock, and the number of CD4+ cells returned to normal level after HES recovery for forty-eighth hours. The number of.CD8+ cells did not change significantly.
3. after traumatic shock, the differentiation of CD4+ lymphocyte in peripheral blood appeared "TH1 to TH2 drift", and HES could change this trend.
4. compared with the control group, the apoptosis ratio of CD4+T cells in HES group did not rise first and then descend after resuscitation.
5. compared with the control group, both flow cytometer method and TUNNEL method found no significant increase in the percentage of apoptosis in bone marrow mononuclear cells in group HES after resuscitation.
conclusion
1. the animal model of traumatic shock can cause obvious physiological damage to the body, and it is easy to operate and easy to control.
2., after traumatic shock, the number of CD4+T lymphocytes in peripheral blood of rats was significantly reduced, and the further differentiation ability of CD4+T cells was "TH1 drift to TH2", and the apoptosis of splenic CD4+T cells and bone marrow mononuclear cells increased significantly.
3. hydroxyethyl starch (130 / 0.4) can inhibit the decline of CD4+T lymphocyte and the trend of "TH1 drift to TH2" after trauma shock, and alleviate the trend of sharp increase in splenic CD4+T cells and bone marrow mononuclear cells apoptosis, so as to improve cellular immune function.

【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R-332

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