摘要
目的通过体外构建携带抗氧化基因核因子E2相关因子2（Nrf2）的骨髓间充质干细胞（BMSC），观察Nrf2对百草枯所致肺损伤模型小鼠的保护作用。方法构建携带有Nrf2基因的过表达慢病毒并转染BMSCs，建立携带有抗氧化基因Nrf2的BMSCs体系。将120只清洁级Balb／c小鼠按随机数字表法分为正常对照组、P0染毒模型组、BMSCs治疗组、携带空载基因的BMSCs治疗组（BMSC—Cherry治疗组）、携带Nrf2基因的BMSCs治疗组（BMSC—Nrf2治疗组）5组，每组24只。腹腔注射PQ溶液25mg／kg复制小鼠PQ中毒肺损伤模型，正常对照组给予等体积生理盐水；BMSC治疗组、BMSC—Cherry治疗组、BMSC—Nrf2治疗组于制模后6h经球后注射相应的BMSCs0．3mL，每只1×106个。制模后处死小鼠取肺组织，用蛋白质免疫印迹试验（WesternBlot）检测肺组织Nrf2蛋白表达；留取血液标本，用化学比色法检测超氧化物歧化酶（SOD）、丙二醛（MDA）、谷胱甘肽（GSH）等氧化应激指标，用酶联免疫吸附试验（ELISA）检测白细胞介素-1β（IL-1β）、肿瘤坏死因子-α（TNF-α）等炎症指标，用原位末端缺刻标记试验（TUNEL）检测肺组织细胞凋亡情况。结果与正常对照组比较，制模后3d和21d，PQ染毒模型组、BMSC治疗组、BMSC—Cherry治疗组、BMSC—Nrf2治疗组的Nrf2蛋白表达均显著升高，且以BMSC—Nff2组21d升高最明显（灰度值：3．52±0．26比1．00±0．12，P〈0．05）。PQ染毒模型组血清MDA、IL-1β、TNF-α水平均较正常对照组明显升高，SOD、GSH均较正常对照组明显降低；用BMSC、BMSC—Cherry、BMSC—Nrf2干预后，随着时间延长，各组MDA、IL-1β和TNF-α水平均较PQ染毒模型组逐渐降低，SOD、GSH均较P0染毒模型组逐渐升高，以BMSC—Nrf2组制模后21d变化最显著（MDA（umol／L）：2．09±0．28比3．11±0．11，SOD（kU／L）：23．88±0．68比16．12±0．87，GSH（ixm01]L）：2．92±0．21比2．33±0．15，IL-1β（ng／L）：55．58±4．54比87．23±9．28，TNF-α（ng／L）：179．25±9．40比228．38±13．30，均P〈0．05J。与正常对照组比较，PQ染毒模型组TUNEL染色阳性细胞明显增多[（45．06±6．78）％比（3．11±0．99）％]；与PQ染毒模型组比较，BMSC治疗组、BMSC-Cherry治疗组、BMSC—Nrf2治疗组阳性细胞明显减少【（30．34±1．79）％、（26．25±4．97）％、（18．00±3．15）％比（45．06±6．78）％]，且以BMSC—Nrf2治疗组下降最明显（P〈0．05）。结论BMSCs及携带有抗氧化基因Nrf2的BMSCs均能有效减轻PQ所致肺损伤，且以BMSCs—Nrf2的作用更显著；其保护作用可能与拮抗体内氧化应激和炎症因子等有关。
Objective We constructed bone marrow mesenchymal stem cells （BMSCs） bringing anti- oxidant gene nuclear factor erythroid 2-related factor 2 （Nrf2）, in vitro to explore the protective effect of Nrf2 on lung injury induced by paraquat （PQ） in mice. Methods Lentiviral vector carrying Nrf2 gene was built and transfected into BMSCs, creating the cell line of BMSCs with antioxidant gene Nrf2. A total of clean 120 Balb/c mice were randomly divided into five groups： normal control group, PQ poisoning model group, BMSCs therapy group, BMSCs- Cherry （carrying no load of gene） therapy group, BMSCs-Nrf2 therapy group, 24 mice in each group. 20% PQ solution （25 mg/kg） was injected intraperitoneally to establish PQ poisoning model and the same volume of normal saline （NS） was given to normal control group. After modeling for 6 hours in BMSC, BMSC-Cherry and BMSC-Nrf2 groups, 0.3 mL corresponding BMSCs was injected into a retro-bulbar vein, respectively, each mouse 1 -106 unit. The mice were killed after cell treatment and the lung tissues were taken from each group and the protein expression of Nrf2 in lung tissue was detected by Western Blot. After blood samples were taken, the activity of superoxide dismutase （SOD） and the levels of malondialdehyde （MDA）, glutathione （GSH）, etc. oxygenation stress indexes were detected by chemical colorimetry, the interleukin-1β（IL-1β ）, tumor necrosis factor--α （TNF-α ） etc. inflammatory indexes were detected by enzyme linked immunosorbent assay （ELISA）, and the apoptosis of lung tissues was observed by terminal-deoxynucleoitidyl transferase mediated nick end labeling （TUNEL）. Results Compared with normal control group, the Nrf2 protein expressions on 3 days and 21 days after modeling in PQ poisoning model, BMSC, BMSC-Cherry and BMSC-Nrf2 groups were significantly increased, and in the BMSC-Nrf2 therapy group after molding for 21 days, the elevation of Nrf2 protein expression was mostly obvious （gray value： 3.52 ±0.26 vs. 1.00 ± 0.12, P 〈 0.05）. The levels of serum MDA, IL-113 and TNF-α of PQ poisoning model group showed significantly elevated, while the levels of SOD and GSH were markedly reduced. After BMSC, BMSC-Cherry and BMSC-Nrf2 treatment, with the time prolongation, compared with the PQ poisoning model group, the levels of MDA, IL-1β and TNF-αof each group were gradually decreased, while the levels of GSH and SOD were gradually increased, and after modeling for 21 days the above changes in BMSC-Nrf2 group showed mostly obvious [MDA （umol/L）： 2.09± 0.28 vs. 3.11 ± 0.11, SOD （kU/L）： 23.88± 0.68 vs. 16.12 ± 0.87, GSH （umol/L）： 2.92 ±0.21 vs. 2.33 ±0.15, IL-1 13 （rig/L）： 55.58 ± 4.54 vs. 87.23 ±9.28, TNF-α （ng/L）： 179.25± 9.40 vs. 228.38 ± 13.30, all P 〈 0.05]. Compared with normal control group, the positive TUNEL staining cells were visibly increased in PQ poisoning model group [（45.06 ± 6.78）% vs. （3.11 ± 0.99）%, P 〈 0.05]; compared with PQ poisoning model group, the positive cells were remarkably decreased in BMSC, BMSC-Cherry and BMSC-Nrf2 treatment groups [（30.34± 1.79）%, （26.25 ± 4.97）%, （18.00± 3.15）% vs. 45.06 ± 6.78）%, all P 〈 0.05], and in the BMSC-Nrf2 group, the percentage of positive cells was the lowest among the 3 treatment groups（all P 〈 0.05）. Conclusions BMSCs and BMSCs carrying antioxidant gene Nrf2 can effectively attenuate the lung injury caused by PQ, and the latter is more effective than the former. The protective effect may be related to body anti-oxidative stress, inflammation, anti- inflammatory factors and so on.
出处
《中国中西医结合急救杂志》
CAS
北大核心
2016年第1期36-41,共6页
Chinese Journal of Integrated Traditional and Western Medicine in Intensive and Critical Care
基金
浙江省自然科学基金资助项目（LZ12H26001）
浙江省医药卫生平台研究计划重点项目（2012ZDA034）
浙江省医学创新学科建设计划（11-CX26）
浙江省中医药重点学科计划（2012-XK-A28）
浙江省“十二五”重点学科建设项目（2012-207）
作者简介
通讯作者：卢中秋，Email：lzq640815@163．com
