【摘要】目的通过猪脓毒症模型研究体外循环细胞干预性治疗(extracorporeal immune cells intervention therapy, EICIT)对临床及免疫学指标的影响。方法采用孵育猪自体粪便腹腔注射建立猪脓毒症模型，24h后接受一般性液体治疗(对照组)及在此基础上的体外循环细胞采集治疗(实验组)，体外循环细胞采集使用COBE Spectra血细胞分离机选用白细胞采集程序进行，右侧颈内静脉导管作为血管通路。于造模前(T1)、造模后24h干预前0h(T2)、治疗后即刻(T3)和治疗后24h(T4)4个时间点留取血液标本，进行血气分析、细胞因子检测和免疫细胞的流式细胞检测，并于T4时点在麻醉状态下取材送病理并处死动物，观察组织形态学变化。结果在T2时点，成功建立猪脓毒症模型，表现为平均动脉压降低、心率增快、低氧血症及血乳酸水平升高。血清促炎因子如降钙素原(procalcitonin, PCT)、C 反应蛋白(C reactive protein, CRP)、肿瘤坏死因子α(tumor necrosis factor-α, TNF-α)、干扰素γ(interferon-γ, IFN-γ) 呈上升趋势，白细胞介素(interleukin,IL)-6 降低(t=-3.067，P=0.025)、IL-10 呈下降趋势。T4时点，与对照组相比，实验组猪血流动力学指标、血氧分压显著改善，血乳酸水平显著降低，组织炎细胞浸润减轻；实验组循环中CD8+T 细胞显著升高(t=4.373,P=0.006)，粒细胞髓过氧化物酶(myeloperoxidase, MPO)活性显著升高(t=3.637,P =0.005)，但CD4+T 细胞、B 细胞、树突细胞(dentric cell,DC)、单核细胞和中性粒细胞计数无明显变化。结论通过猪自体粪便注射建立脓毒症模型，基础治疗联合EICIT 治疗能够改善脓毒症猪的血流动力学、氧合状态及血乳酸水平，同时还可改善脏器炎性细胞浸润。
【Abstract】Objective To observe the effect of extracorporeal immune cell intervention therapy(EICIT)on clinical and immunological indexes in a septic pig model. Methods The sepsis model was established by intraperitoneal injection of autologous incubated feces. After 24 hours, the pigs were treated with general fluid therapy (control group) or with EICIT in addition to the general fluid therapy (experimental group). Extracorporeal blood cell separation was conducted via right internal jugular vein catheter using the white blood cell acquisition program of the COBE Spectra Blood Cell Separator. Arterial blood gas, cytokine measurement, and immune cells by flow cytometry were performed at the points before building the model (T1), after building up the model for 24 hours but before treatment (T2), immediately after treatment (T3), and after treatment for 24 hours (T4). At the T4 time point, pathological examinations were performed under anesthesia and the animals were sacrificed. Results At T2 time point, we successfully established the sepsis model and the pigs showed lower mean arterial pressure, tachycardia, hypoxemia and higher blood lactic acid. Pro-inflammatory factors including procalcitonin (PCT), C-reactive protein (CRP), tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) increased, and interleukin (IL)-6 decreased significantly (t=-3.067, P=0.025), accompanied by a
reduced tendency of IL- 10. At T4 time point in the experimental group, hemodynamics parameters, partial pressure of blood oxygen, lactic acid level and infiltration of inflammatory cells improved significantly, as compared with those in the control group. In experimental group, the number of circulated CD8+ T-cells increased (t=4.373, P=0.006), granulocytic myeloperoxidase (MPO) activity became higher (t=3.637,P=0.005), but the numbers of CD4+T cells, B cells, dentric cells, monocytes and neutrophils had no significant changes. Conclusion We proved that EICIT combined with basic fluid treatment could improve the clinical symptoms of sepsis, including the improvement of hemodynamics, oxygenation status, blood lactic acid level, and the infiltration of inflammatory cells in organs in a septic pig model.
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