CAJ | 학술논문

目的观察靶向小干扰RNA (siRNA)对小鼠肺缺血/再灌注损伤(PIRI)时细胞凋亡及CCAAT/增强子结合蛋白同源蛋白(CHOP)的影响.方法荧光标记法观察靶向siRNA在小鼠体内的分布并评估转染效率.C57BL/6J小鼠50只,随机分为5组(n=10):假手术组(Sham组)、缺血/再灌注组(I/R组)、I/R+载体组(VR+ Vehicle组)、I/R+阴性对照组(I/R+ Control-siRNA组)和I/R+ CHOP-siRNA组(I/R+ CHOP-siRNA组).取左肺,检测肺湿/干重比(W/D)、总肺水含量(TLW)和肺泡损伤定量评估(IQA),光镜和电镜观察肺组织结构改变,逆转录-聚合酶链反应(RT-PCR)、Western blot检测CHOP、葡萄糖调节蛋白78 (GRP78) mRNA和蛋白表达水平,原位末端转移酶标记(TUNEL)法检测细胞凋亡指数(AI).结果靶向siRNA通过滴鼻法可有效分布于肺脏中.与Sham组比较,I/R组中CHOP、GRP78 mRNA和蛋白表达水平(0.80±0.03、0.80±0.02;0.80±0.04、0.84±0.02)均升高(P ＜0.05),W/D(5.24±0.49)、TLW(4.24 ±0.49)、IQA[(42.80 ±4.21)％]和AI[(34.50±1.51)％]均明显增加(P＜0.01)；肺组织形态结构发生明显损伤；与I/R+ Vehicle组和I/R+ Control-siRNA组比较,I/R+ CHOP-siRNA组中GRP78 mRNA和蛋白表达水平(0.84±0.04、0.85±0.04)无明显变化(P＞0.05),而CHOP mRNA和蛋白表达水平(0.40±0.03、0.44±0.04)均降低(P＜0.05),W/D(2.54±0.24)、TLW(1.54±0.24)、IQA[(16.71±2.33)％]和AI[(11.50±1.58)％]亦均降低(P＜0.01)；肺组织形态结构损伤减轻.结论靶向siRNA对I/R损伤肺具有良好的保护作用,其机制可能与其对抗过度的未折叠蛋白反应(UPR)中CHOP介导的细胞凋亡有关.
목적 관찰파향소간우RNA (siRNA)대소서폐결혈/재관주손상(PIRI)시세포조망급CCAAT/증강자결합단백동원단백(CHOP)적영향.방법 형광표기법관찰파향siRNA재소서체내적분포병평고전염효솔.C57BL/6J소서50지,수궤분위5조(n=10):가수술조(Sham조)、결혈/재관주조(I/R조)、I/R+재체조(VR+ Vehicle조)、I/R+음성대조조(I/R+ Control-siRNA조)화I/R+ CHOP-siRNA조(I/R+ CHOP-siRNA조).취좌폐,검측폐습/간중비(W/D)、총폐수함량(TLW)화폐포손상정량평고(IQA),광경화전경관찰폐조직결구개변,역전록-취합매련반응(RT-PCR)、Western blot검측CHOP、포도당조절단백78 (GRP78) mRNA화단백표체수평,원위말단전이매표기(TUNEL)법검측세포조망지수(AI).결과 파향siRNA통과적비법가유효분포우폐장중.여Sham조비교,I/R조중CHOP、GRP78 mRNA화단백표체수평(0.80±0.03、0.80±0.02;0.80±0.04、0.84±0.02)균승고(P ＜0.05),W/D(5.24±0.49)、TLW(4.24 ±0.49)、IQA[(42.80 ±4.21)％]화AI[(34.50±1.51)％]균명현증가(P＜0.01)；폐조직형태결구발생명현손상；여I/R+ Vehicle조화I/R+ Control-siRNA조비교,I/R+ CHOP-siRNA조중GRP78 mRNA화단백표체수평(0.84±0.04、0.85±0.04)무명현변화(P＞0.05),이CHOP mRNA화단백표체수평(0.40±0.03、0.44±0.04)균강저(P＜0.05),W/D(2.54±0.24)、TLW(1.54±0.24)、IQA[(16.71±2.33)％]화AI[(11.50±1.58)％]역균강저(P＜0.01)；폐조직형태결구손상감경.결론 파향siRNA대I/R손상폐구유량호적보호작용,기궤제가능여기대항과도적미절첩단백반응(UPR)중CHOP개도적세포조망유관.
Objective To explore the effects of targeted small interfering RNA (siRNA) on pneumocyte apoptosis and CCAAT/enhancer-binding protein homologous protein (CHOP) in pulmonary ischemia/reperfusion injury (PIRI) in mice.Methods The location of targeted siRNA and transfection efficiency in mice were determined by using fluorescence labeling method.Fifty mice were randomly allocated into five groups (n =10):sham operation group (Sham group),ischemia/reperfusion group (I/R group),I/R + vehicle group (I/R + Vehicle group),L/R + negative control group (I/R + Control-siRNA group),and I/R + CHOP-siRNA group (I/R + CHOP-siRNA group).Left lung tissue was excised.Wet lung weight to dry lung weight (W/D),total lung water content (TLW) and index of quantitative evaluation for alveolar damage (IQA) were tested.Under light microscope and electron microscope,changes in lung tissues were observed.The expression levels of CHOP and glucose regulated protein (GRP78) was detected by using reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting.Apoptosis index (AI) of lung tissue was measured by using TdT-mediated dUTP nick end labeling (TUNEL)method.Results Targeted siRNA could be effectively distributed in the lung by intranasal delivery.Compared to Sham group,the expression levels of CHOP and GRP78 mRNA and protein (0.80 ±0.03,0.80 ±0.02; 0.80 ±0.04,0.84 ±0.02) were all significantly increased (P＜0.05) in I/R group,and W/D (5.24 ±0.49),TLW (4.24 ±0.49),IQA [(42.80 ±4.21)％] and AI [(34.50 ± 1.51) ％] were all very notably increased (P ＜ 0.01).The structural injury in lung tissue was notably observed in I/R group.Compared to I/R + Vehicle group and I/R + Control-siRNA group,there was no significant difference (P ＞0.05) in the expression levels of GRP78 mRNA and protein (0.84 ± 0.04 ; 0.85 ± 0.04) in I/R ± CHOPsiRNA group,but the expression levels of CHOP mRNA and protein (0.40 ±0.03,0.44 ±0.04) were reduced (P＜0.05),W/D (2.54±0.24),TLW (1.54 ±0.24),IQA [(16.71 ±2.33)％] and AI [(11.50 ± 1.58) ％] were all decreased (P ＜0.01).The structural injury in lung tissue was alleviated in I/R + CHOP-siRNA group.Conclusion Targeted siRNA has great effect on lung protection against I/R injury,which may be related to inhibition of pneumocyte apoptosis mediated by CHOP in excessive unfolded protein response (UPR).