脓毒症大鼠脾脏淋巴细胞凋亡的实验研究

Study on apoptosis of splenic lymphocytes in rats with sepsis

目的观察脓毒症大鼠脾脏T、B淋巴细胞的数量变化及细胞凋亡情况，探讨脓毒症时免疫失衡的机制。方法健康雄性Wistar大鼠80只，按随机数字表法分为假手术组（30只）、模型组（50只）。采用盲肠结扎穿孔术（CLP）制备脓毒症大鼠模型，于制模后6、12、24、48、96h活杀动物取脾脏，行苏木素一伊红（HE）染色，光镜下观察脾脏组织病理变化；采用免疫组化法检测脾脏CD4＋、CD8＋T淋巴细胞和B淋巴细胞以及Bax、Bcl一2蛋白表达；采用原位末端缺刻标记法（TUNEL）检测脾脏细胞凋亡指数。结果光镜下观察模型大鼠脾脏白髓逐渐萎缩，淋巴小结结构破坏。制模6、12、24、48、96h，模型组大鼠脾脏CD4＋T淋巴细胞数、B淋巴细胞数、Bcl一2蛋白表达均较假手术组明显降低（P均〈0．01），CD8＋T淋巴细胞数与假手术组比较差异无统计学意义（P均〉0．05），细胞凋亡指数及Bax蛋白表达均较假手术组显著增加（P均〈0．01）。相关分析表明：Bcl一2蛋白表达与细胞凋亡指数呈负相关（r=-0．659，P〈0．01），而Bax蛋白表达与细胞凋亡指数呈正相关p=0．522，P〈0．01）。结论脓毒症早期免疫功能处于紊乱状态，表现为脾脏CD4＋T淋巴细胞数、B淋巴细胞数减少，细胞凋亡显著增加；Bax、Bcl一2在脓毒症脾细胞凋亡中发挥了关键作用。

Objective To observe the change in number of spleen T lymphocytes and B lymphocytes and apoptosis of splenocytes in rats with sepsis, and explore the mechanism of immune imba｜ance in sepsis. Methods Eighty healthy male Wistar rats were randomly divided into two groups： sham operation （n=30） and sepsis model （n= 50）. Sepsis mode[ was reproduced by cecal [igation and puncture （CLP）. The rats were sacrificed and their spleens were obtained at 6, 12, 24, 48, 96 hours after the model was reproduced. The pathological changes in spleen were observed by optical microscopy. Immunohistochemistry method was used to examine the positive expression of CD4＋/CD8＋T lymphocyte and B lymphocyte, Bax and Bcl-2. Apoptotic cells of spleen were detected by means of terminal deoxynu-cleotidyl transferase mediated dUTP nick end labeling （TUNEL）. Results By optical microscopy, white pulps were atrophied and splenic nodules were destroyed after CLP. At 6, 12 , 24, 48, 96 hours after CLP, the number of CD4＋ T lymphocyte, B lymphocyte and Bcl-2 positive expressed cells in sepsis model group were obviously reduced as compared with sham operation group （all P〈0. 01）. CD8＋ positive lymphocytes were not significant changed （all P〈0.05）. Apoptotic splenocytes and Bax positive cells were obviously increased compared with those of sham operation group （all P〈0.01）. Correlation analysis showed that Bcl-2 expression and cell apoptosis was negatively correlated （r =- 0. 659, P〈 0.01）, but Bax positive cells and apotosis showed positive correlation （r=0. 522, P〈0. 01）. Conclusion Disturbance of immunological function exists in the rats with sepsis at 24 hours after CLP. The number of CD4＋ T lymphocyte, B lymphocyte was obviously reduced, apoptotic splenocytes were obviously increased. Bax and Bcl-2 play an important role in apoptosis of splenocytes in sepsis.