摘要
为了研究RNA干扰NF-κBp65 对鼠肺缺血再灌注损伤肺组织中NF-κB、TNF-α表达的影响,并探讨减轻鼠肺损伤的保护机制。本研究构建了体外靶向NF-κBp65 的短发夹RNA (short hairpin RNA, shRNA)重组表达载体,并采用QPCR检测NF-κBp65的沉默结果。供试的16只SD大鼠随机分为4组:假手术组4只、缺血再灌注+生理盐水组4 只,缺血再灌注+ NF-κBp65 shRNA 组4 只,缺血再灌注+空载组4 只,假手术组无缺血再灌注损伤,开胸游离左肺门180 min,缺血再灌注+生理盐水组左肺门阻断前术前24 h 给予生理盐水处理,开胸游离左肺门,左肺缺血60 min,再灌注120 min,缺血再灌注+NF-κBp65 shRNA 组左肺门阻断前术前24 h 滴鼻给予NF-κB shRNA 腺病毒(1×1010 pfu, 100 μL/只),开胸游离左肺门,左肺缺血60 min,再灌注120 min,缺血再灌注+空载组左肺门阻断前术前24 h 滴鼻给予空载shRNA 腺病毒(1×1010 pfu, 100 μL/只),开胸游离左肺门,左肺缺血60 min,再灌注120 min。实验结束后每组分别留取左肺组织,留取左肺上叶肺组织测定肺湿/干重比(W/D),部分肺组织光镜下观察病理变化,ELISA 法测量NF-κB 和TNF-α的表达含量。研究结果表明:成功构建重组NF-κBp65 载体,并获得稳定转染的NF-κBp65 shRNA 细胞,与转染空载体的阴性对照组(negative control, NC)比较,NF-κBp65 shRNA 能明显使NF-κBp65 基因沉默(p<0.05),肺组织NF-κB、TNF-α的表达量及W/D值与假手术组比较,缺血再灌注+生理盐水组和缺血再灌注+空载组有明显升高(p<0.05);而与缺血再和灌注+生理盐水组和缺血再灌注+空载组比较,缺血再灌注+NF-κBp65 shRNA组明显降低(p<0.05),病理学检查表明:假手术组肺组织无明显的炎症损伤;缺血再灌注+NF-κBp65 shRNA组肺炎性损伤较缺血再灌注+生理盐水组和缺血再灌注+空载组明显减轻。本研究结果初步说明,RNA 干扰NF-κBp65 能明显减轻早期肺移植损伤,其机制可能与抑制NF-κB 和TNF-α�
To investigate the effect of RNA interference of NF-κB (Nuclear factor-kappa B) p65 on the levels of NF-κB and TNF-α in reperfusion injured rat lung. The recombinant vector in which the shRNA (Short hairpin RNA) targeting NF-κBp65 was constructed. The tested Sixteen adult male Sprague Dawley rats were randomly divided into four different groups: sham-operation group (n=4), ischemia-reperfusion (I/R)+saline group (n=4), ischemiareperfusion (I/R)+NF-κBp65 shRNA group and ischemia-reperfusion (I/R)+empty vector group were 4 pairs in each group in which left lung were done with "60 min ischemia, 120 min reperfusion". In the ischemiareperfusion (I/R)+saline group, saline treatment before interrupting of pulmonary perfusion and ventilation 24 hours ago. In the ischemia-reperfusion (I/R)+NF-κBp65 shRNA and ischemia-reperfusion (I/R)+empty vector group, NF-κBp65 shRNA group and empty vector treatment before interrupting of pulmonary perfusion and ventilation 24 hours ago. The expression of NF-κB, TNF-α observed by ELISA, the wet to dry weight ratio (W/D) detected and pathological changes of the lung tissue with hematoxylin-eosin (HE) staining examined. The results showed that the recombinant NF-κBp65-shRNA was successfully constructed, and the stably transfected cells were established as well. As compared with NC (negative control) NF-κBp65 shRNA which were transfected with an empty vector, the abilities of proliferation of the NF-κBp65 cells were obviously decreased (p<0.05). The levels of NF-κB and TNF-α concentration, W/D were significantly lower in sham group than that in ischemia-reperfusion (I/R)+saline group and ischemia-reperfusion (I/R)+empty carrier group (p<0.05). The levels of NF-κB and TNF-α concentration, W/D were significantly lower in ischemia-reperfusion (I/R)+NF-κBp65 shRNA group than that in ischemia-reperfusion (I/R)+saline group and ischemia reperfusion (I/R)+empty carrier group (p<0.05). Histological examination showed that the lung injury in ischemia-reperfusion (I/R)+NF-κBp65 shR
作者
彭坚
施媛
杨镭镭
吴辉振
Peng Jian;Shi Yuan;Yang Leilei;Wu Huizhen(Third Hospital ofWuhan City,Wuhan,430072)
出处
《基因组学与应用生物学》
CAS
CSCD
北大核心
2019年第9期4230-4235,共6页
Genomics and Applied Biology
基金
2014年国家自然科学基金资助项目课题(资助批准号:81371251)
2016年市卫计委面上一般项目课题(项目编号:WX16C06)
2018年武汉市卫计委面上重点项目(项目编号:WX18B03)共同资助
