Xia Pei-Yuan, Zheng Jiang, Zhou Hong, Pan Wen-Dong, Qin Xiao-Jian, Xiao Guan-Xia
Department of Pharmacy and Clinical Pharmacology, Southwestern Hospital, Third Military Medical University, Chongqing 400038, China.
World J Gastroenterol. 2002 Jun;8(3):546-50. doi: 10.3748/wjg.v8.i3.546.
To investigate the relationship between lymphocyte apoptosis in peripheral blood, spleen and mesenteric lymph nodes(MLN) and endotoxin translocation after thermal injury in rats.
In a Wistar rat model inflicted with 30% TBSA III degree scalding, serum LPS levels in portal vein and vena cava were quantified by tachypleus amebocyte lysate (TAL) technique. The analysis of peripheral blood lymphocyte was employed in in situ Cell Death Detection Kit and evaluated by flow cytometry. Apoptotic lymphocytes in paraffin-embedded spleen and MLN sections were examined by histologic analysis, in situ deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) and peroxidase (POD) staining. The images were taken by Cooldccd camera system, and the count and optical density value (transmission light) of apoptotic lymphocytes were analyzed with software Spot and Imagine proplus 4.10a(IPP4.10a).
In the period of 3 to 48 postburn hours (PBHs) serum LPS level (x10(3) EU.L(-1)) in portal vein (2.11+/-0.02, 5.66+/-0.20, 3.70+/-0.22, 2.56+/-0.28, 0.90+/-0.11) was higher than that in vena cava (0.63+/-0.01, 1.53+/-0.18, 0.83+/-0.32, 0.52+/-0.12, 0.23+/-0.02, P<0.01), but both increased sharply in postburn rats (P<0.01) and reached a peak at 6 PBH. Analysis of apoptotic lymphocytes showed that the proportion (%) of postburn apoptotic cells was much higher than that in healthy rats (8.34+/-1.53, 8.13+/-1.81, 20.77+/-3.94, 23.90+/-3.92, 11.23+/-1.35 and 13.26+/-2.09 at 3, 6, 12, 24, 48 and 72 PBH, respectively, vs 3.99+/-1.72, P<0.01), especially after 6 PBH. The concentrations of lymphocytic apoptosis at 12 and 24 PBH were markedly higher than that at other time points. Meantime, few apoptotic lymphocytes were found in normal MLN, but increased postburn obviously (3+/-1 vs 546+/-83, 285+/-39, 149+/-30, 58+/-10, 36+/-11 and 33+/-9 in turn, P<0.01), especially at 3 PBH, whereas apoptotic lymphocytes were concentrated in splenic cortex before the burn and decreased obviously during 72 PBHs (499+/-186 vs 12+/-8, 19+/-15, 12+/-7, 100+/-15, 123+/-25 and 226+/-26 in turn, P<0.01) though a slight rise was found in the medulla after 24 PBH. Optical density of apoptotic lymphocytes was significantly reduced in spleen in the 24 PBHs and raised in MLN during 48 PBHs than that prior to the burn, respectively.
Gut-origin LPS is a major cause of endotoxemia taken place early in rats following severe thermal injury and could induce extensive lymphocyte apoptosis in blood and MLN, which suggests an immunosuppression state could follow the initial injury and favores a septic state based on apoptotic mechanism.
探讨大鼠热损伤后外周血、脾脏和肠系膜淋巴结（MLN）中淋巴细胞凋亡与内毒素移位之间的关系。
在Wistar大鼠30%体表面积Ⅲ度烫伤模型中，采用鲎试剂（TAL）技术定量检测门静脉和腔静脉血清LPS水平。采用原位细胞死亡检测试剂盒对外周血淋巴细胞进行分析，并通过流式细胞术进行评估。通过组织学分析、原位脱氧核苷酸末端转移酶dUTP缺口末端标记（TUNEL）和过氧化物酶（POD）染色检测石蜡包埋的脾脏和MLN切片中的凋亡淋巴细胞。用Cooldccd摄像系统拍摄图像，并用软件Spot和Imagine proplus 4.10a（IPP4.10a）分析凋亡淋巴细胞的计数和光密度值（透射光）。
烧伤后3至48小时（PBH）期间，门静脉血清LPS水平（x10(3) EU.L(-1)）（2.11±0.02、5.66±0.20、3.70±0.22、2.56±0.28、0.90±0.11）高于腔静脉（0.63±0.01、1.53±0.18、0.83±0.32、0.52±0.12、0.23±0.02，P<0.01），但烧伤大鼠两者均急剧升高（P<0.01），并在烧伤后6小时达到峰值。凋亡淋巴细胞分析显示，烧伤后凋亡细胞比例（%）远高于健康大鼠（烧伤后3、6、12、24、48和72小时分别为8.34±1.53、8.13±1.81、20.77±3.94、23.90±3.92、11.23±1.35和13.26±2.09，而健康大鼠为3.99±1.72，P<0.01），尤其是在烧伤后6小时后。烧伤后12和24小时淋巴细胞凋亡浓度明显高于其他时间点。同时，正常MLN中凋亡淋巴细胞很少，但烧伤后明显增加（依次为3±1 vs 546±83、285±39、149±30、58±10、36±11和33±9，P<0.01），尤其是在烧伤后3小时，而烧伤前凋亡淋巴细胞集中在脾皮质，在72小时内明显减少（依次为499±186 vs 12±8、19±15、12±7、100±15、123±25和226±26，P<0.01），尽管在烧伤后24小时髓质有轻微升高。烧伤后24小时脾脏中凋亡淋巴细胞的光密度明显降低，烧伤后48小时MLN中的光密度比烧伤前升高。
肠道源性LPS是大鼠严重热损伤后早期发生内毒素血症的主要原因，可诱导血液和MLN中广泛的淋巴细胞凋亡，这表明初始损伤后可能出现免疫抑制状态，并基于凋亡机制有利于脓毒症状态的发生。
