CAJ | 학술논문

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1×107 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4×105 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。目的：探討骨髓間充質榦細胞（BMSC）對創傷弧菌膿毒癥肺損傷的保護作用及機製。方法採用全骨髓貼壁培養法分離小鼠BMSC。按照隨機數字錶法將雄性ICR小鼠分為生理鹽水對照組（NS組）、生理鹽水+BMSC對照組（NSB組）、創傷弧菌膿毒癥組（VV組）、創傷弧菌膿毒癥+BMSC治療組（VVB組），每組40隻。于小鼠左側腹腔註射1×107 cfu/mL創傷弧菌懸液5 mL/kg製備膿毒癥模型；于製模後經尾靜脈註射4×105 cfu/mL BMSC 5 mL/kg進行榦預。各組分彆于染菌後6、12、24、48 h取10隻小鼠的肺組織，計算肺濕/榦質量（W/D）比值；蛋白質免疫印跡試驗（Western Blot）檢測細胞覈內覈轉錄因子-κBp65（NF-κBp65）錶達；酶聯免疫吸附試驗（ELISA）檢測肺組織腫瘤壞死因子-α（TNF-α）、白細胞介素（IL-1β、IL-6）水平；囌木素-伊紅（HE）染色及醋痠鈾-枸櫞痠鉛雙染後于鏡下觀察肺組織病理學改變。結果 VV組染毒後各時間點肺W/D比值、肺組織細胞覈內NF-κBp65錶達量及肺組織TNF-α、IL-1β、IL-6錶達均較NS組明顯升高，且于12 h達峰值；VVB組肺W/D比值、NF-κBp65錶達量及TNF-α、IL-1β、IL-6水平均明顯低于VV組同時間點〔12 h VV組比NS組：肺W/D比值7.22±0.03比5.21±0.02，NF-κBp65錶達量（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組比VV組：肺W/D比值6.49±0.06比7.22±0.03， NF-κBp65錶達量（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組和NSB組各指標比較差異均無統計學意義。光鏡下顯示NS組和NSB組肺組織病理改變不明顯；VV組肺組織充血、水腫，肺泡腔內可見水腫液、紅細胞，炎性細胞浸潤；VVB組上述肺組織損害減輕。透射電鏡下顯示NS組和NSB組Ⅰ型、Ⅱ型肺泡上皮細胞結構完整，未見明顯改變；VV組肺泡壁完整結構破壞，Ⅰ型上皮細胞胞質腫脹、鼓泡、破裂，Ⅱ型上皮細胞胞質減少呈空泡變、嗜鋨性闆層小體排空、溶酶體增生、微絨毛減少；VVB組Ⅰ型、Ⅱ型肺泡上皮細胞上述改變明顯減輕。結論創傷弧菌膿毒癥可引起急性肺損傷和肺水腫；BMSC能降低炎癥因子水平，減輕創傷弧菌膿毒癥導緻的肺損傷。
목적：탐토골수간충질간세포（BMSC）대창상호균농독증폐손상적보호작용급궤제。방법채용전골수첩벽배양법분리소서BMSC。안조수궤수자표법장웅성ICR소서분위생리염수대조조（NS조）、생리염수+BMSC대조조（NSB조）、창상호균농독증조（VV조）、창상호균농독증+BMSC치료조（VVB조），매조40지。우소서좌측복강주사1×107 cfu/mL창상호균현액5 mL/kg제비농독증모형；우제모후경미정맥주사4×105 cfu/mL BMSC 5 mL/kg진행간예。각조분별우염균후6、12、24、48 h취10지소서적폐조직，계산폐습/간질량（W/D）비치；단백질면역인적시험（Western Blot）검측세포핵내핵전록인자-κBp65（NF-κBp65）표체；매련면역흡부시험（ELISA）검측폐조직종류배사인자-α（TNF-α）、백세포개소（IL-1β、IL-6）수평；소목소-이홍（HE）염색급작산유-구연산연쌍염후우경하관찰폐조직병이학개변。결과 VV조염독후각시간점폐W/D비치、폐조직세포핵내NF-κBp65표체량급폐조직TNF-α、IL-1β、IL-6표체균교NS조명현승고，차우12 h체봉치；VVB조폐W/D비치、NF-κBp65표체량급TNF-α、IL-1β、IL-6수평균명현저우VV조동시간점〔12 h VV조비NS조：폐W/D비치7.22±0.03비5.21±0.02，NF-κBp65표체량（회도치）1.86±0.74비0.75±0.07，TNF-α（ng/L）433.24±3.23비106.57±1.21，IL-1β（ng/L）35.64±0.15비10.64±0.48，IL-6（ng/L）58.84±0.55비17.69±1.35，균P＜0.05；12 h VVB조비VV조：폐W/D비치6.49±0.06비7.22±0.03， NF-κBp65표체량（A치）1.16±0.08비1.86±0.74，TNF-α（ng/L）357.22±3.25비433.24±3.23，IL-1β（ng/L）27.77±0.59비35.64±0.15，IL-6（ng/L）38.68±1.29비58.84±0.55，균P＜0.05〕。NS조화NSB조각지표비교차이균무통계학의의。광경하현시NS조화NSB조폐조직병리개변불명현；VV조폐조직충혈、수종，폐포강내가견수종액、홍세포，염성세포침윤；VVB조상술폐조직손해감경。투사전경하현시NS조화NSB조Ⅰ형、Ⅱ형폐포상피세포결구완정，미견명현개변；VV조폐포벽완정결구파배，Ⅰ형상피세포포질종창、고포、파렬，Ⅱ형상피세포포질감소정공포변、기철성판층소체배공、용매체증생、미융모감소；VVB조Ⅰ형、Ⅱ형폐포상피세포상술개변명현감경。결론창상호균농독증가인기급성폐손상화폐수종；BMSC능강저염증인자수평，감경창상호균농독증도치적폐손상。
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(1×107 cfu/mL)5 mL/kg through the left side peritoneal cavity, and caudal intravenous injection of BMSC(4×105 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.