【作者】 杨军；

【导师】 张连阳；

【作者基本信息】 第三军医大学 ， 外科学， 2008， 硕士

【摘要】 严重创伤(AIS>3)感染发生率较高,是导致创伤病人死亡的第二位死因。严重创伤患者存在免疫功能紊乱,发生感染后单纯抗感染疗效差。T淋巴细胞亚群是具有特殊标志的从胸腺中输出的细胞,执行细胞免疫效应及免疫调节功能,其中CD4+和CD8+是一对相互制约的T细胞亚群,细胞免疫的自我稳定依赖于这对亚群间的平衡。IL-2主要由T淋巴细胞产生,在免疫应答及调节中占有重要地位。肿瘤坏死因子TNF-α具有广泛的生物学活性,可参与炎症反应和免疫应答。创伤后TNF-α水平明显升高,如失控性释放,可引起炎症反应“级联效应”导致的全身炎症反应综合征(SIRS),是多器官功能障碍综合征(MODS)的发病基础。免疫球蛋白是具有抗体活性的球蛋白,在体液免疫抗感染方面具有极其重要的作用。随着对亚低温认识的加深以及其技术的进步,越来越多的实验及临床研究报告肯定了亚低温的脑保护效果。对创伤性失血动物和病人实施有目的的全身降温,可降低代谢率,减轻组织损伤,抑制炎症反应,延长创伤患者、尤其是大量失血患者急救的“黄金时间”,提高存活率。目前,亚低温对严重创伤后机体免疫功能影响的研究甚少。本实验采用大鼠创伤性失血性休克模型,初步探讨亚低温治疗对严重创伤后机体免疫功能的影响,为研究亚低温对严重创伤后机体的保护机制提供实验室依据。方法:60只SD大鼠(体重200～280 g,雌雄不限),随机分为3组,实验中因创伤、失血、低温等因素死亡10只,剩余50只。⑴对照组:大鼠不予创伤和失血(n=10);⑵正常体温组:制作创伤性失血性休克模型+正常体温,分别与6h、24h两个时相点检测指标(n=20);⑶亚低温组:制作创伤性失血性休克模型+亚低温,分别与6h、24h两个时相点检测指标(n=20)。除⑴组外,实验动物以3%戊巴比妥钠30 mg/kg腹腔麻醉,双股骨中段闭合性骨折后,股动脉插管放血至血压40 mmHg制成创伤性失血性休克模型。维持1 h后,回输全部失血及2倍量林格液。测定距肛门2cm直肠内温度。正常体温组用烤灯照射,维持大鼠直肠温度于38～39℃,持续3h;亚低温组用冰水降温,维持大鼠直肠温度于32.5～33.5℃,持续3h。于创伤性失血性休克模型完成后6h,24h于腹主动脉抽血,取1.6ml血浆,用于T细胞亚群的测定(FCM法)。取4ml血浆离心,取上清液于-20℃冰箱中保存,用于外周血免疫球蛋白IgG等的测定(ELISA法)。取脾并培养脾淋巴细胞,按照试剂盒的要求,用双抗体夹心ELISA法测定经ConA刺激脾淋巴细胞分泌的细胞因子IL-2、TNF-α。并观察各组实验动物的死亡情况。结果:1.在创伤性失血性休克后,外周血中CD4+明显降低(P<0.01),CD8+升高(P<0.05),CD4+/CD8+明显降低(P<0.01)。创伤后亚低温组与正常体温组相比,6h组无显著性差异(P>0.05); 24h组外周血中CD4+升高(P<0.05),CD8+下降(P<0.05),CD4+/CD8+升高(P<0.01)。2.创伤性失血性休克后,经ConA刺激脾淋巴细胞分泌的细胞因子IL-2降低(P<0.01),TNF-α升高(P<0.01)。创伤后亚低温组与正常体温组相比细胞因子IL-2升高(P<0.05),TNF-α降低(P<0.01)。3.与正常对照组比较,大鼠创伤性失血性休克后外周血免疫球蛋白IgG无显著性差异(P>0.05)。创伤后亚低温组与正常体温组相比,外周血免疫球蛋白IgG无显著性差异(P>0.05)。4.实验过程中对照组无动物死亡,亚低温组与正常体温组动物均有动物死亡,死亡率有所差别,但无统计学意义(P>0.05)。结论:1.创伤性失血性休克后早期(24h内)模型大鼠存在细胞免疫功能抑制,表现为:外周血T淋巴细胞亚群CD8+升高,CD4+、CD4+/CD8+明显降低;经ConA刺激脾淋巴细胞分泌的细胞因子IL-2明显降低,而TNF-α明显升高。应用亚低温治疗后,T淋巴细胞亚群CD8+降低,CD4+、CD4+/CD8+升高; IL-2增加,TNF-α明显下降,提示亚低温可能改善严重创伤性失血性休克后机体早期的细胞免疫功能抑制。2.在创伤性失血性休克后早期,外周血免疫球蛋白IgG变化不明显,应用亚低温治疗后也无显著变化。3.虽然应用亚低温治疗可能改善机体的细胞免疫功能抑制,但动物的早期存活率并没有显著提高。更多还原

【Abstract】 The incidence of infection after severe injury (AIS>3)is extremely high. Many scholars find that severe injury bring about restrain of immune function. There are multiple changes in body immune function in acute stage of severe injury, such as the quantitative and functional descent of CD4, CD4 / CD8 ,while the increase of CD8. Interleukin 2 (IL-2) mostly comes from CD4 and CD8 T lymphocytes and has an important role in immune response and immune regulation. Tumor necrosis factorα(TNF-α) has extensive biologic activity and is often involved in inflammatory reaction and immune response. Immunoglobulin has antibody activity and has a function in anti-infection of humoral immunity. It exists with form of IgG in body. So the level of IgG generally can reflect the humoral immunity state.It is acceptable that the use of mild hypothermia therapeutics(32℃～35℃) in patients with trauma, especially severe craniocerebral trauma. Many reports confirmed brain safety effect of mild hypothermia without serious adverse effect in the past two decades. The mechanism of mild hypothermia therapeutics applicated in severe injury and body protection is not clearly explained. Some reports suggest that total body hypothermia after trauma and blood loss can decrease metabolic rate, soften tissue damage, repress inflammatory reaction, extend“golden time”of emergency treatment and elevate survival. The effect of mild hypothermia therapeutics on immune system after severe injury has not been elucidated. To investigate the influence of therapic mild hypothermia on immune function, rats with hemorrhagic shock induced by severe injury were used as model.MethodsIn this study, sixty SD rats ( weight: 200~300g, female and male) were randomly divided into three groups, ten died in experiment. Control group(group1); normal body temperature group (group2), examined at 6h and 24h; mild hypothermia group (group 3), examined at 6h and 24h. Except group 1, other two groups were the trauma and hemorrhagic shock model. Hemorrhagic shock was induced in forty adult rats to a mean arterial pressure of 40 mmHg. Hypotension was maintained for one hour, resuscitation was accomplished with the shed blood and lactated Ringer’s solution (2×blood volume). Intrarectal temperatures were measured replacing the central temperature (CT). The normal contro1 group used broiling lamp to maintain intrarectal temperature at 38～39℃; while The mild hypothermia therapeutics group used ice water to reduce the temperature and the intrarectal temperature was maintained at 32.5～33.5℃. The rats were executed at 6h and 24h through bloodletting in the abdominal aorta. Add 4ml blood plasma into heparinized centrifuge tube and centrifugalize. Keep the supernatant fluid at -20℃for measuring immunoglobulin IgG.1.6ml blood plasma was used for determining the subsets of T lymphocyte. Cell factors IL-2 and TNF-αwere examined using ELISA.Results1. The expression of CD4 and CD4/CD8 ratio obviously decreased, the expression of CD8 heightened in peripheral blood of rats with trauma and hemorrhagic shock. Compared with normal body temperature group, the expression of CD4 and CD4/CD8 ratio increased (P<0.01)while the expression of CD8 decreased significantly(P<0.05) in mild hypothermia therapeutics group in both 6h and 24h treatment condition.2. The expression of cell factor IL-2 lowered and TNF-αheightened in trauma and hemorrhagic shock models. Compared with normal body temperature group, the expression of IL-2 increased (P<0.01)while TNF-αdecreased significant (P<0.05)in mild hypothermia therapeutics group in both 6h and 24h treatment condition.3. The expression of immunoglobulin IgG of peripheral blood had no significant change(P>0.05)between normal contro1 group and mild hypothermia therapeutics group.4. The rats in normal contro1 group were all survival, while few of the rats in each experiment group died and the rate of death had difference but had not statistically significant(P>0.05).Conclusion1. In the early stage of trauma and hemorrhagic shock, the cellular immune function is repressed, CD4, CD4/CD8 and IL-2 all decrease obviously and inflammatory mediators such as TNF-αelevate .The application of mild hypothermia therapeutics can improve and stabilize cell immune function. 2. In the early stage of trauma and hemorrhagic shock, humoral immunity changes little even in the state of using mild hypothermia therapeutics.3. Although the use of mild hypothermia therapeutics can improve cell immune function, the early survival has no notable elevation. The effect of mild hypothermia on trauma is in many ways and has multifactor, which needs further study.更多还原