【作者】 杨艳琴；

【导师】 古妙宁；

【作者基本信息】 南方医科大学 ， 麻醉学， 2010， 硕士

【摘要】 体外循环(cardiopulmonary bypass,CPB)为手术或其他治疗方法提供了有利条件,但也引起一系列术后并发症,包括心、肺、肾功能的损伤,以及由炎症因子失衡和免疫功能失调介导的复杂炎症反应。研究表明,CPB术后创伤反应及并发症是机体免疫功能失调所致。CPB结合其他因素诱导了机体复杂的体液、细胞炎性反应。一方面,CPB引起补体激活、内毒素释放、粒细胞活化、黏附分子表达、致炎介质释放,表现为全身性、非特异性炎性反应；另一方面,通过产生抗炎介质,促使免疫细胞功能下降、凋亡,导致机体免疫系统的短暂失能,抑制炎性反应。二者均是机体对CPB产生的正常保护性反应,对于减轻创伤、抵抗感染是至关重要的。大多数CPB手术病例,致炎、抗炎反应系统相互制衡,保证机体平安度过围手术期。如果失衡,大量炎性介质入血,致炎性介质级联释放,抗炎介质不足以拮抗,即发生过度炎性反应；相反,若抗炎介质释放过多,则产生免疫功能抑制,易继发感染引起败血症等。炎性介质或抗炎介质由对机体的防御作用变为破坏作用,最终致内环境自稳态失衡,器官功能障碍。因此,对CPB手术中机体致炎／抗炎反应调节机制的研究尤为重要。研究表明,体外循环后外周血单个核细胞(peripheral blood mononuclear cell, PBMC)分泌一系列的细胞因子参与炎症反应,但外周血单个核细胞参与这一过程的分子机制仍未完全明确。为了深入探讨外周血单个核细胞参与炎症的具体机制,我们采用了蛋白质组学的思路和方法来进行进一步的研究。为临床控制体外循环后全身炎症反应的发生发展提供重要的理论基础。材料和方法1.临床资料：选择在我院行心脏瓣膜置换术的患者11例,男女不限,年龄20-60岁,ASAⅡ-Ⅲ级。在体外循环心脏停跳下手术。术前均无风湿活动和其它免疫系统疾病。术前半年内均未用免疫抑制剂。2.麻醉实施：麻醉前：入室后鼻导管吸氧2-5L/min,监测5导联ECG, SpO2, NIBP和BIS。局麻下建立前臂静脉通道、桡动脉置管监测直接动脉压、右颈内静脉穿刺置入3-lumen中心静脉导管,建立CVP测压并连接血管活性药泵组。全麻诱导：芬太尼4ug/kg,异丙酚2.0mg/kg,顺式阿曲库铵0.15mg/kg,气管插管成功后吸入纯氧+七氟烷2%-2.5%,调节七氟烷ET浓度在0.8-1.1MAC之间以维持BIS值在40-60区间,ETCO2在30-40mmHg。静注吗啡5-10mg。麻醉维持：切皮后增加七氟烷吸入浓度以加深麻醉,劈胸骨前3-5min补注芬太尼3-4ug/kg。胸腔操作及心脏、主动脉插管期间根据血流动力学的变化调整麻醉深度。体外循环开始后关闭七氟烷并间断补注吗啡和顺式阿曲库铵。给予异丙酚(1.0mg/kg)一次静注后继以异丙酚(100ug/kg*min)持续输注,直至术毕。手术全程连续监测心电图、脉氧饱和度、桡动脉压、CVP、鼻咽温、尿量,定时查动脉血气。体外循环期间根据血气分析的结果和尿量,视情况给与碳酸氢钠、甘露醇、速尿、氯化钾、氯化钙等。根据公式及患者的体重计算预充液的用量,使病人的Hct维持在20%-28%之间。麻醉后：术毕以异丙酚(50ug/kg min)持续输注镇静,送入ICU行机械通气。3.手术方法：在体外循环下阻断升主动脉,心脏停跳下手术,鼻咽温度约32℃-35℃。4.标本的采集与处理患者分别于麻醉后手术前(TO)、转机后1h(T1)和手术结束时(T2)三个时点抽取动脉血3-5ml,肝素钠抗凝,常温下送实验室,取3ml血样用单个核细胞分离液按密度梯度离心法分离外周血单个核细胞(PBMC)。随后将分离的外周血单个核细胞进行裂解,获取蛋白,并将蛋白进行2-DE分离。利用UMAX PowerLook 1100透射扫描仪获取图像,用PDQuest 7.1.0软件包进行图像分析,选取差异蛋白质点。将差异点挖出,处理后送入质谱,按照4000Series Explorer Software软件操作,打击样品获得MS和MS/MS图谱,搜素SwissProt数据库,解析蛋白序列,分析可能参与炎症反应的单个核细胞分泌的蛋白。对与有意义的差异蛋白,采用Western blot进行进一步验证。5.统计学方法：计量资料以均数±标准差(x±s)表示,统计学处理采用ANOVO Test,所有统计分析均采用SPSS13.0软件进行处理。P<0.05为差异有统计学意义。结果根据差异蛋白质点的选取标准：麻醉后手术前(T0)、体外循环开始后1h(T1)和手术结束时(T2)三时点蛋白表达存在显著不同。分析2-DE分离得到的差异点,获得有效鉴定的蛋白12种,其中4个蛋白表达量在T1组达到高峰,后开始下降。5个蛋白在T0组表达量最高,T1组表达量最低；1个蛋白随着手术的进行表达量逐渐降低；2个蛋白随着手术的进行表达量逐渐升高。获得的12个差异蛋白质谱(MALDI-TOF MS/MS质谱分析)鉴定全部成功。其中S100A9是由外周血单个核细胞分泌的,在炎症反应中发挥重要作用。S100A9的表达量在T1组达到高峰,T2组开始下降,但仍高于T0组(One-Way ANOVO, F=371.174, P=0.000)结论1)体外循环可诱导外周血单个核细胞产生S100A9,其表达量在体外循环开始后1h达到高峰,手术结束时开始下降,但未达到术前水平。2)S100A9可能参与体外循环术后全身炎症反应综合症的发生发展。更多还原

【Abstract】 Cardiopulmonary bypass (CPB) is widely used in cardiovascular surgery. Unfortunately, CPB causes postoperative complications, including damage to cardiac, pulmonary, and renal tissues, complications associated with inflammation mediated by changes in cytokine balance, and immunomodulatory effects. Studies have shown that, trauma reactions and complications after CPB are due to the immune dysfunction. CPB combined with other factors induced the body’s complex inflammatory response, including humoral and cellular immunity. On the one hand, CPB caused complement activation、endotoxin release、granulocyte activation、adhesion molecule expression pro-inflammatory cytokines release, manifested as systemic, non-specific inflammatory response; the other hand, the activation of anti-inflammatory cytokines inhabited immune cell function promoted apoptosis, resulted in short-term loss of the immune system. Both are normal response, and are important to prevent trauma, and resist to infection. In most cases of CPB surgery, pro- and anti-inflammatory keep balance, and ensure the body safely during the perioperative period. If the balance were broken, a large number of pro-inflammatory cytokines were secreted into the blood, caused a series of inflammatory cytokines releasing, and lead to excessive inflammatory response; on the contrary, if too many anti-inflammatory cytokines were secreted, it will inhibit the immunological reaction and cause infections, even initiate septicemia. The function of pro- or anti-inflammatory cytokines will be changed from defensive to destructive, and ultimately it will break the internal environment and due to the organs dysfunction. Therefore, it is important to study the mechanism of pro-/anti-inflammatory response during preoperative period of CPB.Studies have shown that mononuclear cells secreted a series of cytokines during the period of cardiopulmonary bypass, but the molecular mechanisms involved in this process is not completely clear. To explore the specific mechanism of mononuclear cells during the inflammation responses, we adopted proteomics technology to think and research. The study will provide important theoretical basis for clinical.Materials and methods1. Patients11 patients aged 20—60 years, ASAⅡ-Ⅲ, undergoing valve replacement surgery using CPB in our institution. No rheumatism activities and other immune system diseases preoperative, and not use immunosuppressants 6 months before surgery.2. AnesthesiaAnesthesia:when the patients in the operating room, they are monitored by 5-lead ECG, SpO2, NIBP, and BIS. Under local anesthesia, we establish forearm vein channels and radial artery catheter to monitor direct arterial pressure; right internal jugular vein punctured by 3-lumen central venous catheter also under local anesthesia, to establish CVP manometry and connect with vasoactive drug pumps.Anesthesia induction:Fentanyl 4ug/kg, propofol 2.0mg/kg, cis-atracurium 0.15mg/kg, after the success of tracheal intubation, the patients inhalate pure oxygen+sevoflurane 2%-2.5%, sevoflurane ET concentration was regulated in the 0.8-1.1MAC, the BIS value was between 40-60, ETCO2 in the 30-40mmHg.5-10mg morphine was injected into intravenous.Anesthesia maintenance:After skin incision, we increased the inhalation concentration of sevoflurane in order to deepen the anesthesia,3-5min before sternotomy sidebar, we injected 3-4ug/kg fentanyl. During operation, we changed the depth of anesthesia according to hemodynamic. After pump shut, we turn off the sevoflurane and intermittent injected morphine and cis-atracurium. After given the first dose of propofol (1.0mg/kg) into vein, we infused propofol (100ug/kg min) continuously until the completion of surgery. The patients were monitored by electrocardiogram, pulse oxygen saturation, radial arterial pressure, CVP, nasopharyngeal temperature, urine output, regular check arterial blood gases continuously during the period of surgery. In the period of CPB, we injected different drugs basing on the result of blood gas analysis and urine. According to the calculation formula and the patient’s weight, we infused the fluid, maintained the patient’s Hct between 20%～28%.After anesthesia:we escorted the patients who were injected by propofol (50ug/kg min) continuously to ICU.3. Surgical methods:The aorta was blocked during the period of cardiopulmonary bypass, and the surgery continues when heart was stopped jumping. We keep the patients’ nasopharyngeal temperature about 32℃-35℃.4. Blood sampling3-5ml arterial blood was drawn in tubes with heparin sodium. Samples were drawn preoperatively after anesthesia (TO);1h after CPB(T1); and at the end of operation (T2). We separated peripheral blood mononuclear cells (PBMC) by using density gradient separation, and lyses PBMC to obtain protein. Two-dimensional protein electrophoresis was used to separate the proteins which were obtained from PBMC. Using UMAX PowerLook 1100 to get images, and using PDQuest 7.1.0 software to analysis the result. The difference points were dug up, and sent to MS. According to 4000Series Explorer Software, we obtain MS and MS/MS map. Then, we searched SwissProt database, and analyzed protein sequence which may be involved in the inflammatory response. Western blot were used to further verification.5. Statistics analysisAll date are reported as the mean±standard deviation (x±s). ANOVO Test was used to statistical analysis. SPSS 13.0 were used to analyze the date. P values of <0.05 were accepted as significant.ResultAccording to the selection criteria of protein spots:The expression of proteins are significantly different among preoperatively after anesthesia (T0)、1 h after CPB(T1) and at the end of operation (T2). Among differential points separated by 2-DE and effectively identified,12 protein up-regulated after CPB.4 proteins expressed at least in T0, but at peak in T1; 5 proteins expressed highest in T0, but least in T1; 1 protein expressed highest in T0, and least in T2; 2 proteins expressed least in T0, highest in T2. All of these proteins were identificated successful by spectrum identification (MALDI-TOF MS/MSMS). S100A9 is secreted by PBMC, and play an important role in the inflammatory response. Compared with T0, the levels of S100A9 significantly increased in T1 and T2. But Compared with T1, the levels of S100A9 decreased in T2. (One-Way ANOVO, F=371.174, P=0.000). Conclusion1) PBMC can secret S100A9 in acute inflammatory reaction after CPB, and S100A9 up-regulated in PBMC by CPB stimulation 1h, and show a contrary trend in T2 group.2) S100A9 may participate in the occurrence and development of systemic inflammatory response syndrome induced by CPB.更多还原