Intermedin对缺氧/复氧诱导的大鼠近端肾小管上皮细胞凋亡的影响
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摘要
目的：以大鼠近端肾小管上皮细胞(NRK-52E)缺氧复氧(H／R)模型模拟在体缺血再灌注(I／R)损伤,通过转染高表达IMD质粒,探讨中介素(IMD)对缺氧／复氧诱导大鼠近端肾小管上皮细胞(NRK-52E)凋亡的影响及机制。
方法：(1)利用三气培养箱调整氮气压力形成缺氧条件,缺氧4h,复氧12h后检测NRK-52E活细胞计数、细胞存活率和培养液上清乳酸脱氢酶(LDH)判断模型制备成功与否。(2)利用Fugene HD转染试剂,将pIRES2-EGFP/IMD表达质粒和pIRES2-EGFP空质粒转染至NRK-52E细胞内,于倒置荧光显微镜和流式细胞仪检测转染效率,选定最优转染条件；选转染效率高的细胞用含G418 300μg/ml的10%胎牛血清的DMEM/F12完全培养基进行筛选,2周后获得稳定表达IMD的阳性克隆NRK-52E细胞。(3)实验分对照组,模型组,空质粒组和IMD质粒组共四组,后三组行H／R实验,留取细胞上清液及细胞,分别以流式细胞仪测细胞凋亡率,酶联免疫法测丝氨酸-苏氨酸激酶(Akt)活性,比色法测Caspase-3活性。
结果：(1)经缺氧4h,复氧12h后,NRK-52E细胞计数量降低,细胞存活率下降,细胞上清液中LDH含量显著增加,造模成功。(2)于荧光显微镜和流式细胞仪观察转染率,结果显示质粒3μg:FuGENE HD 12μl的比例转染在48h的转染效率最高；G418筛选第3天起转染阳性的细胞逐渐出现克隆样增殖,第5天起未转染的细胞出现批量死亡,以后阳性细胞克隆逐渐增加,2周后可见融合成片状的阳性克隆细胞。(3)细胞上清液磷酸化丝氨酸-苏氨酸激酶(p-Akt)含量,与对照组相比,H／R后各组细胞Akt磷酸化水平增高,其中IMD质粒组分别与模型组和空质粒组相比,Akt磷酸化水平明显增高,模型组和空质粒组相比无明显差别。(4)细胞Caspase-3活性,与对照组相比,H／R后各组细胞Caspase-3活性增强,其中IMD质粒组分别与模型组和空质粒组相比,Caspase-3活性明显降低,模型组与空质粒组相比无明显差别。(5)细胞凋亡率,与对照组相比,H／R后各组细胞凋亡细胞显著增多,其中IMD质粒组分别与模型组和空质粒组相比,细胞凋亡率明显降低,模型组与空质粒组细胞凋亡率相比无明显差别。
结论：IMD可减轻H／R诱导的NRK-52E细胞凋亡,具体机制与IMD增强Akt活性,降低Caspase-3活性有关。
Objective The hypoxia/reoxygenation(H/R) injury models of rat renal tubular epithelial cells (NRK-52E), which were transfected the eukaryotic expression vector of rat IMD, were established to simulate I/R injury in vivo, to investigate the effect and mechanism of intermedin (IMD) on hypoxia/reoxygenation(H/R)-induced rat renal tubular epithelial cells (NRK-52E) apoptosis.
Methods (1) To establish the H/R injury model of NRK-52E by regulating the pressure of N2 in incubator to hypoxia condition, the cells were cultured with hypoxia for 4h and then with reoxygenation for 12h. The models were evaluated by detecting living cell count, cell viability and the activity of lactate dehydrogenase (LDH) in the culture medium. (2) NRK-52E cells were transfected by transfection complex comprising optimal proportion of pIRES2-EGFP/IMD plasmid and Fugene HD reagents. Transfection efficiency was tested by observation for EGFP made by fluorescent microscope and flow cytometery (FCM) after 48h. The cell groups with high transfection efficiency were screened for two weeks or so by incubating with medium containing G418(300μg/ml), and the positive cloning NRK-52E cells of stable expression IMD obtained. (3) NRK-52E cells were divided into 4 groups:control group, model group, primitive vector group and IMD vector group. The last three groups were exposed to H/R condition, and the apoptosis of NRK-52E cells were determined by flow cytometry, the serine-threonine kinase (Akt) activity were determined by enzyme linked immunosorbent assay, and the Caspase-3 activity were determined by colorimetry after H/R.
Results (1) After hypoxia 4h and reoxygenation 12h, cell count and cell viability decreased significantly and the activity of LDH increased significantly; the model was established successfully. (2) NRK-52E cells was transfected with the optimal transfection proportion of pIRES2-EGFP/IMD to Fugene HD reagent as 3μg:12μl, transfection efficiency evaluated by fluorescence microscope and FCM showed that the transfection efficiency increased with the prolongation of time. The positive transfected cells after G418 screening were found clone-like proliferation on the third day, and the untransfected cells died in large bulk on the fifth day. Then the positive transfected cells increased gradually; and the positive cloning cells blending into slices were found on the fourteenth day. (3) Phosphorylated Akt:Compared with the control group, the level of Akt phosphorylation in last three groups significantly increased after H/R. Compared with model group and the empty vector group, Akt phosphorylation levels significantly increased in IMD group. There is no significant diffenrence between model group and the empty vector group. (4) Caspase-3 activity:Compared with the control group, the activity of Caspase-3 in last three groups significantly increased after H/R. Compared with model group and the empty vector group, the activity of Caspase-3 significantly reduced in IMD group. There is no significant diffenrence between model group and the empty vector group. (5) Apoptosis:Compared with the control group, the apoptosis of the cells in last three groups significantly increased after H/R. The apoptosis of the cells asignificantly reduced in IMD group, contrasted with model group and primitive vector group, respectively. There is no significant diffenrence between model group and the empty vector group.
Conclusion IMD may against hypoxia/reoxygenation-induced NRK-52E apoptosis by activating Akt to decrease Caspase-3 activity.
方法：(1)利用三气培养箱调整氮气压力形成缺氧条件,缺氧4h,复氧12h后检测NRK-52E活细胞计数、细胞存活率和培养液上清乳酸脱氢酶(LDH)判断模型制备成功与否。(2)利用Fugene HD转染试剂,将pIRES2-EGFP/IMD表达质粒和pIRES2-EGFP空质粒转染至NRK-52E细胞内,于倒置荧光显微镜和流式细胞仪检测转染效率,选定最优转染条件；选转染效率高的细胞用含G418 300μg/ml的10%胎牛血清的DMEM/F12完全培养基进行筛选,2周后获得稳定表达IMD的阳性克隆NRK-52E细胞。(3)实验分对照组,模型组,空质粒组和IMD质粒组共四组,后三组行H／R实验,留取细胞上清液及细胞,分别以流式细胞仪测细胞凋亡率,酶联免疫法测丝氨酸-苏氨酸激酶(Akt)活性,比色法测Caspase-3活性。
结果：(1)经缺氧4h,复氧12h后,NRK-52E细胞计数量降低,细胞存活率下降,细胞上清液中LDH含量显著增加,造模成功。(2)于荧光显微镜和流式细胞仪观察转染率,结果显示质粒3μg:FuGENE HD 12μl的比例转染在48h的转染效率最高；G418筛选第3天起转染阳性的细胞逐渐出现克隆样增殖,第5天起未转染的细胞出现批量死亡,以后阳性细胞克隆逐渐增加,2周后可见融合成片状的阳性克隆细胞。(3)细胞上清液磷酸化丝氨酸-苏氨酸激酶(p-Akt)含量,与对照组相比,H／R后各组细胞Akt磷酸化水平增高,其中IMD质粒组分别与模型组和空质粒组相比,Akt磷酸化水平明显增高,模型组和空质粒组相比无明显差别。(4)细胞Caspase-3活性,与对照组相比,H／R后各组细胞Caspase-3活性增强,其中IMD质粒组分别与模型组和空质粒组相比,Caspase-3活性明显降低,模型组与空质粒组相比无明显差别。(5)细胞凋亡率,与对照组相比,H／R后各组细胞凋亡细胞显著增多,其中IMD质粒组分别与模型组和空质粒组相比,细胞凋亡率明显降低,模型组与空质粒组细胞凋亡率相比无明显差别。
结论：IMD可减轻H／R诱导的NRK-52E细胞凋亡,具体机制与IMD增强Akt活性,降低Caspase-3活性有关。
Objective The hypoxia/reoxygenation(H/R) injury models of rat renal tubular epithelial cells (NRK-52E), which were transfected the eukaryotic expression vector of rat IMD, were established to simulate I/R injury in vivo, to investigate the effect and mechanism of intermedin (IMD) on hypoxia/reoxygenation(H/R)-induced rat renal tubular epithelial cells (NRK-52E) apoptosis.
Methods (1) To establish the H/R injury model of NRK-52E by regulating the pressure of N2 in incubator to hypoxia condition, the cells were cultured with hypoxia for 4h and then with reoxygenation for 12h. The models were evaluated by detecting living cell count, cell viability and the activity of lactate dehydrogenase (LDH) in the culture medium. (2) NRK-52E cells were transfected by transfection complex comprising optimal proportion of pIRES2-EGFP/IMD plasmid and Fugene HD reagents. Transfection efficiency was tested by observation for EGFP made by fluorescent microscope and flow cytometery (FCM) after 48h. The cell groups with high transfection efficiency were screened for two weeks or so by incubating with medium containing G418(300μg/ml), and the positive cloning NRK-52E cells of stable expression IMD obtained. (3) NRK-52E cells were divided into 4 groups:control group, model group, primitive vector group and IMD vector group. The last three groups were exposed to H/R condition, and the apoptosis of NRK-52E cells were determined by flow cytometry, the serine-threonine kinase (Akt) activity were determined by enzyme linked immunosorbent assay, and the Caspase-3 activity were determined by colorimetry after H/R.
Results (1) After hypoxia 4h and reoxygenation 12h, cell count and cell viability decreased significantly and the activity of LDH increased significantly; the model was established successfully. (2) NRK-52E cells was transfected with the optimal transfection proportion of pIRES2-EGFP/IMD to Fugene HD reagent as 3μg:12μl, transfection efficiency evaluated by fluorescence microscope and FCM showed that the transfection efficiency increased with the prolongation of time. The positive transfected cells after G418 screening were found clone-like proliferation on the third day, and the untransfected cells died in large bulk on the fifth day. Then the positive transfected cells increased gradually; and the positive cloning cells blending into slices were found on the fourteenth day. (3) Phosphorylated Akt:Compared with the control group, the level of Akt phosphorylation in last three groups significantly increased after H/R. Compared with model group and the empty vector group, Akt phosphorylation levels significantly increased in IMD group. There is no significant diffenrence between model group and the empty vector group. (4) Caspase-3 activity:Compared with the control group, the activity of Caspase-3 in last three groups significantly increased after H/R. Compared with model group and the empty vector group, the activity of Caspase-3 significantly reduced in IMD group. There is no significant diffenrence between model group and the empty vector group. (5) Apoptosis:Compared with the control group, the apoptosis of the cells in last three groups significantly increased after H/R. The apoptosis of the cells asignificantly reduced in IMD group, contrasted with model group and primitive vector group, respectively. There is no significant diffenrence between model group and the empty vector group.
Conclusion IMD may against hypoxia/reoxygenation-induced NRK-52E apoptosis by activating Akt to decrease Caspase-3 activity.
引文
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[16]Fujio Y, Nguyen T, Wencker D, et al. Akt promotessurvival of cardiomyocytes in vitro and protects against ischemia-reperfusion injury in mouse heart. Circulation.2000; 101(6): 660-667.
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[23]Hiroyuki O, Noritoshi N, Takefumi I, et al. Adrenomedullin infusion attenuates myocardial ischemia/reperfusion injury through the phosphatidylinositol3-kinase/Akt-dependent-pathway[J]. Circulation.2004; 109(2):242-248
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[30]Parameswaran N, Spielman WS, Brooks DP, et al. SB203580 reverses adrenomedullin's effect on proliferation and apoptosis in cultured mesangial cells[J]. Eur JPharma.1999; 371(1):75-82.
[31]Parameswaran N, Hall CS, McCabe LR, et al. Adrenomedullin increases AP-1 expression in rat mesangial cells via activation of protein kinase-A and p38 MAPK[J]. Cell Physiol Biochem.2003;13(6):367-374.
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[33]Boudouresque F, Berthois Y, Martin PM, et al. Role of adrenomedullin in glioblastomas growth[J]. Bull Cancer.2005;92(4):317-326.
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[41]Josthna Penchalaneni, Sunil J. Wimalawansa, Chandrasekhar Yallampalli. Adrenomedullin antagonist treatment during early gestation in rats causes fetoplacental growth restriction through apoptosis[J]. Biology of reproduction.2004;71(5):1475-1483.