骨髓间充质干细胞对创伤弧菌脓毒症肺损伤的保护作用

The protective effect of bone marrow mesenchymal stem cell on lung injury induced by vibrio vulnificus sepsis

目的：探讨骨髓间充质干细胞（BMSC）对创伤弧菌脓毒症肺损伤的保护作用及机制。方法采用全骨髓贴壁培养法分离小鼠BMSC。按照随机数字表法将雄性ICR小鼠分为生理盐水对照组（NS组）、生理盐水＋BMSC对照组（NSB组）、创伤弧菌脓毒症组（VV组）、创伤弧菌脓毒症＋BMSC治疗组（VVB组），每组40只。于小鼠左侧腹腔注射1＆#215;107 cfu/mL创伤弧菌悬液5 mL/kg制备脓毒症模型；于制模后经尾静脉注射4＆#215;105 cfu/mL BMSC 5 mL/kg进行干预。各组分别于染菌后6、12、24、48 h取10只小鼠的肺组织，计算肺湿/干质量（W/D）比值；蛋白质免疫印迹试验（Western Blot）检测细胞核内核转录因子-κBp65（NF-κBp65）表达；酶联免疫吸附试验（ELISA）检测肺组织肿瘤坏死因子-α（TNF-α）、白细胞介素（IL-1β、IL-6）水平；苏木素-伊红（HE）染色及醋酸铀-枸橼酸铅双染后于镜下观察肺组织病理学改变。结果 VV组染毒后各时间点肺W/D比值、肺组织细胞核内NF-κBp65表达量及肺组织TNF-α、IL-1β、IL-6表达均较NS组明显升高，且于12 h达峰值；VVB组肺W/D比值、NF-κBp65表达量及TNF-α、IL-1β、IL-6水平均明显低于VV组同时间点〔12 h VV组比NS组：肺W/D比值7.22±0.03比5.21±0.02，NF-κBp65表达量（灰度值）1.86±0.74比0.75±0.07，TNF-α（ng/L）433.24±3.23比106.57±1.21，IL-1β（ng/L）35.64±0.15比10.64±0.48，IL-6（ng/L）58.84±0.55比17.69±1.35，均P＜0.05；12 h VVB组比VV组：肺W/D比值6.49±0.06比7.22±0.03， NF-κBp65表达量（A值）1.16±0.08比1.86±0.74，TNF-α（ng/L）357.22±3.25比433.24±3.23，IL-1β（ng/L）27.77±0.59比35.64±0.15，IL-6（ng/L）38.68±1.29比58.84±0.55，均P＜0.05〕。NS组和NSB组各指标比较差异均无统计学意义。光镜下显示NS组和NSB组肺组织病理改变不明显；VV组肺组织充血、水肿，肺泡腔内可见水肿液、红细胞，炎性细胞浸润；VVB组上述肺组织损害减轻。透射电镜下显示NS组和NSB组Ⅰ型、Ⅱ型肺泡上皮细胞结构完整，未见明显改变；VV组肺泡壁完整结构破坏，Ⅰ型上皮细胞胞质肿胀、鼓泡、破裂，Ⅱ型上皮细胞胞质减少呈空泡变、嗜锇性板层小体排空、溶酶体增生、微绒毛减少；VVB组Ⅰ型、Ⅱ型肺泡上皮细胞上述改变明显减轻。结论创伤弧菌脓毒症可引起急性肺损伤和肺水肿；BMSC能降低炎症因子水平，减轻创伤弧菌脓毒症导致的肺损伤。

Objective To discuss the protective effect of bone marrow mesenchymal stem cell（BMSC）on lung injury induced by vibrio vulnificus sepsis and its mechanism. Methods BMSCs were isolated by whole bone marrow adherent culture from mouse. Male ICR mice were randomly divided into normal saline control group（NS group）,normal saline＋BMSC control group（NSB group）,vibrio vulnificus sepsis group（VV group）,vibrio vulnificus sepsis ＋ BMSC group（VVB group）according to random number table,with 40 mice in each group. Sepsis mouse model was reproduced by injecting vibrio vulnificus（1＆#215;107 cfu/mL）5 mL/kg through the left side peritoneal cavity, and caudal intravenous injection of BMSC（4＆#215;105 cfu/mL）5 mL/kg for intervention after model reproduction. Ten mice in each group were sacrificed at 6,12,24 or 48 hours after injecting vibiro vulnificus,and their lung tissues were harvested. The lung wet/dry（W/D）ratio was calculated. The expression of nuclear factor-κBp65（NF-κBp65）in nucleus was measured by Western Blot. The levels of tumor necrosis factor-α（TNF-α）and interleukins（IL-1β, IL-6）in lung tissue were detected by enzyme-linked immunosorbent assay（ELISA）. The pathological changes in lung tissue were observed after hematoxylin-eosin（HE）staining and uranyl acetate-lead citrate staining. Results After vibrio vulnificus injection,lung W/D ratio,the expression of NF-κBp65 in nucleus,and the levels of TNF-α, IL-1β,IL-6 in the lung tissues were significantly increased in VV group compared with those in NS group at all the time points,and peaked at 12 hours. Compared with the VV group,the VVB group had significantly decreased levels of lung W/D ratio,NF-κBp65 expression,and the levels of TNF-α,IL-1β,IL-6,with significant differences at all the time points〔VV group vs. NS group at 12 hours：lung W/D ratio 7.22±0.03 vs. 5.21±0.02,NF-κBp65 expression （glay scale）1.86±0.74 vs. 0.75±0.07,TNF-α（ng/L）433.24±3.23 vs. 106.57±1.21,IL-1β（ng/L）35.64±0.15 vs. 10.64±0.48,IL-6（ng/L）58.84±0.55 vs. 17.69±1.35,all P〈0.05;VVB group vs. VV group at 12 hours：lung W/D ratio 6.49±0.06 vs. 7.22±0.03,NF-κBp65 expression（A value）1.16±0.08 vs. 1.86±0.74,TNF-α（ng/L）357.22±3.25 vs. 433.24±3.23,IL-1β（ng/L）27.77±0.59 vs. 35.64±0.15,IL-6（ng/L）38.68±1.29 vs. 58.84±0.55,all P〈0.05〕. There were no significant differences in above indexes between NS group and NSB group. In the NS and NSB groups pathological changes were not obvious under light microscopy,in the VV group lung tissue hyperemia and edema was significant,the edema fluid,red blood cells and inflammatory cells also could be seen, and in the VVB group lung damage that mentioned above could be alleviated. In the NS and NSB groups epithelial cell structure of type Ⅰ and type Ⅱ was completed,and the changes were not obvious under the transmission electron microscopy. In the VV group the alveolar walls were damaged significantly,with type Ⅰ epithelial cell cytoplasm swelling,bubbling and rupture,with type Ⅱ epithelial cells visible cytoplasm decrease,cavitation,addiction to osmium lamellar corpuscle emptying,lysosome hyperplasia,microvilli reduction,and in the VVB group the above damage was alleviated. Conclusion Vibrio vulnificus sepsis can cause acute lung damage and edema,and BMSC can down regulate inflammatory cytokines,reduce lung injury caused by vibrio vulnificus sepsis.