摘要
【摘要】目的分析髓源性抑制细胞（MDSC）亚群及其比值在脓毒症小鼠体内的动态变化，初步探讨其在脓毒症发展中的作用。方法将180只雄性C57BL／6小鼠按随机数字表法分为脓毒症模型组和假手术（Sham）组，采用盲肠结扎穿孔术（CLP）建立小鼠脓毒症模型；Sham组只开腹、关腹，不进行结扎和穿刺。两组先各取30只小鼠，观察术后存活情况，并计算20d存活率。两组再各另取60只小鼠，分别于术后0、3、7、12、20d取12只小鼠，采用流式细胞仪检测外周血、脾脏和骨髓中MDSC亚群的比例；于术后7d取小鼠脾脏，测定脾脏质量，并制备成单细胞悬液，进行脾细胞计数；取脾脏和肝脏组织进行苏木素一伊红（HE）染色，镜下观察病理形态学改变。结果①Sham组小鼠术后20d内无死亡，模型组小鼠术后20d死亡10只，两组小鼠20d存活率比较差异有统计学意义（100．0％比66．7％，x。=11．861，P=0．001）。②模型组小鼠术后7d脾脏明显增大，脾脏质量明显大于Sham组（mg：413．33±41．63比111．67±17．56，￡=11．564，P=0．000），且脾细胞计数明显高于Sham组（×109／L：21．20±2．43比1．87±0．06，f=13．578，P=0．005）。⑧HE染色显示：Sham组小鼠肝脏和脾脏组织基本正常。而模型组术后7d小鼠肝组织结构紊乱，毛细血管充血，中性粒细胞渗出，肝细胞水肿明显，部分肝细胞发生灶性坏死，呈急性炎性改变；脾组织红髓和白髓结构紊乱，脾窦显著扩张充血，被膜增厚，被膜下及血管周围组织可见局灶性呈环状核的非成熟髓系细胞增殖，脾索及脾窦内见大量造血细胞灶状聚集。④Sham组术后各时间点外周血、脾脏和骨髓中巨噬细胞型MDSC（M—MDSC）和粒细胞型MDSC（G—MDSC）的比例及其比值均无明显变化。模型组随时间延长，外周血、脾脏和骨髓中M—MDSC逐渐升高，于术后20d稍有下降；G—MDSC持续升高；而M—MDSC／G—MDSC比值则呈先升后降的趋势。术后3d起外周血M—MDSC／G—MDSC比值均〉1，且于12d达峰值（与0d比较：4．16±0．53比0．79±0．11，P〈0．05）；术后各时间点脾脏和骨髓M—MDSC／G—MDSC比值均〈1，且于7d达峰值（与0d比较：脾脏为0．70±0．06比0．25±0．02，骨髓为0．39±0．06比0．11±0．01，均P〈0．05），之后均逐渐下降。结论随着脓毒症病程的延长，MDSC两个亚群在外周血、脾脏和骨髓中均不断聚集，而M—MDSC／G—MDSC比值则呈先升后降的趋势，可能反映脓毒症不同阶段的免疫状态。
Objective To investigate the dynamic variation of subsets of myeloid derived suppressor cells （MDSC） and their ratio in septic mice, and to discuss their role in the development of sepsis. Methods Male C57BL/6 mice were randomly divided into sepsis model group and sham group according to random nμmber table. Polymicrobial sepsis was induced by using cecal ligation and puncture （CLP）, while mice in sham group only underwent laparotomy and laparorrhaphy without CLP. Thirty mice in each group were used to observe living condition, and the 20-day survival rate was compared between the two groups. In addition, subsets of MDSC in peripheral blood, spleen and bone marrow were analyzed with flow cytometry for other 60 mice （12 mice at each time point, as 0, 3, 7, 12 and 20 days）. Spleens were harvested at 7 days for weighing, and single cell suspension of spleen tissue was prepared for splenocyte counting. Histopathologic changes in spleen tissue and liver tissue were observed under light microscope after hematoxylin and eosin （HE） stain. Results （1） No mice died in sham group within 20 days after the operation. On the other hand, 10 mice in model group died within 20 days, and the difference in survival rate between the two groups was statistically significant （100.0% vs. 66.7%, χ2 = 11.861, P = 0.001）. （2） The spleens in model group showed obvious enlargement and significantly outweighed as compared with those in sham group （mg： 413.33 ± 41.63 vs. 111.67 ± 17.56, t = 11.564, P = 0.000）, and the total count of splenocytes was significantly higher than that in sham group （X 109/L： 21.20±2.43 vs. 1.87±0.06, t = 13.578, P = 0.005）. （3） Pathological sections with HE staining showed that the liver tissue and spleen tissue remained normal in sham group. In model group, the hepatic tissue showed acute inflammatory reaction, including tissue disruption, capillary congestion, infiltration of neutrophils, marked edema of hepatocytes and focal hepatocellular necrosis. Abnormalities were also found in the spleen tissue： the red pulp and white pulp were disordered, splenic sinus was congested with nμmerous red cells, the splenic capsule thickened, immature myeloid cells with circular nuclei proliferated in the subcapsular region and perivascular region, splenic cord and splenic sinus were infiltrated with a large nμmber of hematopoietic cells. （~） No significant changes in the monocytic MDSC （M-MDSC） and granulocytic MDSC （G-MDSC）, and their ratio were found in peripheral blood, spleen and bone marrow at every time point in Sham group. On the other hand, in model group, the ratio of M-MDSC and G-MDSC was continuously increased in peripheral blood, spleen and bone marrow, and M-MDSC only slightly decreased at 20 days. On the other hand, the ratio of M-MDSC/G-MDSC rose at first followed by a decrease. The ratio of M-MDSC/G-MDSC in peripheral blood was higher than 1 from 3 days after the operation, reaching the peak at 12 days （compared with 0 day： 4.16±0.53 vs. 0.79±0.11, P 〈 0.05）, while the ratio of M-MDSC/G-MDSC＇ in spleen and bone marrow after CLP were lower than 1 at all time points, reaching the peak on 7 days after the operation （compared with 0 day： 0.70 ± 0.06 vs. 0.25± 0.02 in spleen, 0.39 ± 0.06 vs. 0.11 ± 0.01 in bone marrow, both P 〈 0.05）, and then gradually decreased afterwards. Conclusion Subgroups of MDSCs were continuously aggregated in the peripheral blood, spleen and bone marrow, and their ratio rose first and decreased afterwards along with the development of sepsis, and the changes may reflect the change of immune status at different stages of sepsis.
出处
《中华危重病急救医学》
CAS
CSCD
北大核心
2016年第2期147-152,共6页
Chinese Critical Care Medicine
基金
卫生部医药卫生科技发展研究项目(WH2011-01-02)@@@@ Medical and Health Technology Development Research Program of the Ministry of Health
关键词
脓毒症
髓源性抑制细胞
巨噬细胞亚型
粒细胞亚型
Sepsis
Myeloid-derived suppressor cell
Monocytic subsets
Granulocytic subsets
