马度米星铵致H9c2心肌细胞和C2C12骨骼肌细胞毒性作用机制研究
发布时间:2018-06-21 19:41
本文选题:马度米星铵 + 毒性 ; 参考:《南京农业大学》2015年博士论文
【摘要】:为探讨马度米星铵对心肌和骨骼肌细胞的毒性作用机制,本研究采用H9c2和C2C12细胞作为模型,考察马度米星铵的毒性,检测其对细胞周期分布、细胞凋亡和细胞自噬的影响,探讨细胞内相关信号通路在马度米星铵所致细胞毒性中的作用。具体分为以下几个部分:1.马度米星铵在心肌细胞和骨骼肌细胞中的毒性作用分别以H9c2细胞和C2C12细胞作为心肌细胞和骨骼肌细胞模型,不同浓度马度米星铵(H9c2细胞:0~5μg/mL,C2C12细胞:0~1μg/mL)处理5天,显微镜下观察细胞形态的变化并拍照,胰酶消化后计数;不同浓度马度米星铵(H9c2细胞:0~5μg/mL,C2C12细胞:0~1μg/mL)处理24、48或72h,One solution法检测细胞活性,台盼蓝染色法检测细胞存活率。结果显示,马度米星铵处理5天后,细胞生长被抑制,形态变圆,部分细胞脱落漂浮于培养液中,贴壁细胞中出现空泡,处理组细胞数量明显少于未处理组,呈浓度依赖性;马度米星铵处理24、48或72 h后,细胞活性显著降低,细胞存活率显著下降,呈浓度和时间依赖性。结果表明,马度米星铵抑制H9c2细胞和C2C12细胞生长,降低细胞活性,增加细胞死亡率,马度米星铵对心肌细胞和骨骼肌细胞具有毒性作用。2.马度米星铵对心肌细胞和骨骼肌细胞细胞周期分布的影响及其机制不同浓度马度米星铵(H9c2细胞:0~5μg/mL,C2C12细胞:0~1μg/mL)处理24h或0.5μg/mL马度米星铵处理不同时间(H9c2细胞:0、36、48和72h,C2C12细胞:0、24、48和72 h),PI染色后经流式细胞仪检测细胞周期的分布;不同浓度马度米星铵(H9c2细胞:0~5μg/mL,C2C12细胞:0~1μg/mL)处理24 h,Western blot检测周期相关蛋白的表达水平。结果显示,不同浓度马度米星铵处理24 h后,H9c2细胞G_0/G_1期比例先升高后降低,S期比例先降低后升高,G_2/M期比例逐渐降低;C2C12细胞G_0/G_1期比例逐渐升高,S期比例逐渐降低,G_2/M期比例逐渐降低;0.5μg/mL马度米星铵处理不同时间后,H9c2细胞和C2C12细胞G_0/G_1期比例逐渐升高,S期比例逐渐降低,G_2/M期比例逐渐降低;不同浓度马度米星铵处理24 h后,H9c2细胞周期素、CDK6、CDC25B表达增加;C2C12细胞Cyclin D1、CDK4、CDK6和CDC25A表达量减少,p21Cip1和p27Kip1表达增加,Rb条带出现位移、磷酸化水平降低。结果表明,马度米星铵对H9c2细胞和C2C12细胞有明显的细胞周期阻滞作用,抑制细胞增殖。3.马度米星铵对心肌细胞和骨骼肌细胞细胞凋亡的影响及其机制不同浓度马度米星铵(H9c2细胞:0~5μg/mL,C2C12细胞:0~1μg/mL)处理72h,Annexin V-PI双染经流式细胞仪检测细胞凋亡率的变化;不同浓度马度米星铵(H9c2细胞:0~5μg/mL,C2C12细胞:0~1μg/mL)处理24h,Western blot检测凋亡相关蛋白的表达量;z-VAD-fmk预处理1h后马度米星铵(0.5μg/mL和1μg/mL)处理24或48 h,Western blot检测Cleaved caspase 3和Cleaved PARP的表达情况,台盼蓝染色检测细胞死亡率的变化情况;不同浓度马度米星铵(H9c2细胞:0~5μg/mL,C2C12细胞:0~1μg/mL)处理24h,Western blot检测AIF蛋白的表达量,免疫荧光法定位细胞内AIF蛋白。结果显示,马度米星铵处理72 h后,细胞凋亡率显著升高,呈浓度依赖性;马度米星铵处理24h后,H9c2细胞中TRAIL、DR4、Cleaved caspase 8、Cleaved caspase 3和Cleaved PARP的表达水平升高,C2C12细胞中BAK、BAD、TRAIL、DR4、TRADD、Cleaved caspase 9、Cleaved caspase 8、Cleaved caspase 3和Cleaved PARP蛋白表达水平升高;z-VAD-fmk预处理可削弱马度米星铵导致的Caspase 3和PARP蛋白裂解及细胞死亡;马度米星铵处理24 h后,H9c2细胞中AIF表达增加且更多地转位到细胞核中,C2C12细胞中AIF表达量不变但转位到细胞核中的AIF增多。结果表明,马度米星铵能诱导H9c2细胞和C2C12细胞凋亡,从而导致其对心肌细胞和骨骼肌细胞的毒性作用。4.马度米星按对心肌细胞和骨骼肌细胞细胞自噬的影响不同浓度马度米星铵(H9c2细胞:0~5μg/mL,C2C12细胞:0~1μg/mL)处理24 h,Western blot检测自噬相关蛋白的表达量;腺病毒干扰24 h表达GFP标记的LC3蛋白,不同浓度马度米星按(H9c2细胞:0~5μg/mL,C2C12细胞:0~1μg/mL)处理24 h,荧光显微镜下观察定位细胞内LC3蛋白。结果显示,马度米星铵处理24 h后,细胞中LC3Ⅱ和p62蛋白水平升高;腺病毒干扰表达GFP标记的LC3蛋白,马度米星铵处理24h后,荧光显微镜下观察,药物处理组与对照组相比,细胞内有大量绿色荧光点,表明LC3蛋白聚集。结果表明,马度米星铵能诱导H9c2细胞和C2C12细胞自噬并阻断自噬流。5.马度米星铵对心肌细胞和骨骼肌细胞MAPK通路蛋白和蛋白磷酸酶的影响不同浓度马度米星按(0~1μg/mL)处理H9c2细胞和C2C12细胞24 h,Western blot检测MAPK通路相关蛋白和蛋白磷酸酶的表达量;腺病毒干扰表达MKK1-R4F和显性失活PP2A,0.5μg/mL和1μg/mL马度米星铵处理H9c2细胞24 h或48 h,Western blot检测p-ERK蛋白的表达量,显微镜下观察细胞形态的变化并拍照,One solution法检测细胞活性;PP2A抑制剂Okadaic acid预处理1 h,0.5μg/mL和1μg/mL马度米星铵处理H9c2细胞48 h,显微镜下观察细胞形态的变化并拍照,One solution法检测细胞活性;腺病毒干扰表达显性失活c-Jun,0.5μg/mL和1μg/mL马度米星铵处理C2C12细胞24h或48h,Western blot检测c-Jun蛋白的表达量,显微镜下观察细胞形态的变化并拍照,One solution法检测细胞活性;腺病毒干扰过表达PP5,0.5μg/mL和1μg/mL马度米星铵处理C2C12细胞24 h或48 h,Western blot检测p-JNK和p-c-Jun蛋白的表达量,显微镜下观察细胞形态的变化并拍照,One solution法检测细胞活性;JNK抑制剂SP600125预处理1 h,0.5μg/mL和1μg/mL马度米星铵处理C2C12细胞48 h,显微镜下观察细胞形态的变化并拍照,One solution法检测细胞活性。结果显示,马度米星铵处理24 h后,H9c2细胞中p-ERK、p-PP2A和demethylated PP2A(De-PP2A)蛋白水平降低,methylated PP2A(Me-PP2A)表达量升高;C2C12细胞中p-JNK和p-c-Jun蛋白水平升高,PP5蛋白水平降低。H9c2细胞中腺病毒干扰表达MKK1-R4F和显性失活PP2A可削弱马度米星铵对ERK蛋白磷酸化的抑制及细胞毒性;Okadaic acid预处理可削弱马度米星铵细胞毒性。C2C12细胞中腺病毒干扰表达显性失活c-Jun可削弱马度米星铵细胞毒性;过表达PP5可削弱马度米星铵对JNK和c-Jun蛋白磷酸化的激活及细胞毒性;SP600125预处理可削弱马度米星铵细胞毒性。结果表明,马度米星铵增强H9c2细胞PP2A活性,引起ERK蛋白磷酸化水平降低,导致其对心肌细胞的毒性作用;马度米星铵抑制C2C12细胞PP5蛋白表达,使JNK和c-Jun磷酸化水平升高,导致其对骨骼肌细胞的毒性作用。6.马度米星铵对心肌细胞和骨骼肌细胞Akt1-FoxO3a通路的影响不同浓度马度米星铵处理H9c2细胞和C2C12细胞24 h,Western blot检测Akt1、p-Akt1(S473)、p-Akt1(T308)、FoxO3a和p-FoxO3a(Thr132)蛋白的表达量,免疫荧光定位细胞内FoxO3a蛋白;腺病毒干扰表达持续激活Akt,0.5μg/mL和1μg/mL马度米星铵处理细胞24h或48h,Western blot检测p-FoxO3a(Thr132)蛋白的表达量,免疫荧光定位细胞内FoxO3a蛋白,显微镜下观察细胞形态的变化并拍照,One solution法检测细胞活性。结果显示,马度米星铵处理24h后,细胞中p-Akt1(S473)、p-Akt1(T308)和p-FoxO3a(Thr132)蛋白水平降低,细胞核中FoxO3a含量增加。腺病毒干扰表达持续激活Akt可削弱马度米星铵对FoxO3a蛋白磷酸化和胞浆转移的抑制及细胞毒性。结果表明,马度米星铵抑制H9c2细胞和C2C12细胞中Akt1活性,导致FoxO3a蛋白磷酸化水平降低,抑制其胞浆转移,从而导致其对心肌细胞和骨骼肌细胞的毒性作用。7.马度米星铵对心肌细胞和骨骼肌细胞AMPK蛋白的影响不同浓度马度米星铵(H9c2细胞:0~5μg/mL,C2C12细胞:0~1μg/mL)处理24 h,Western blot检测AMPK和p-AMPK(Thr172)蛋白的表达量;腺病毒干扰表达显性失活AMPK,0.5μg/mL和1μg/mL马度米星铵处理细胞24 h或48 h,Western blot检测AMPK蛋白的表达量,显微镜下观察细胞形态的变化并拍照,One solution法检测细胞活性;AMPK抑制剂Compound C预处理1 h,0.5μg/mL和1μg/mL马度米星铵处理细胞48 h,显微镜下观察细胞形态的变化并拍照,One solution法检测细胞活性。结果显示,马度米星铵处理24h后,细胞中p-AMPK(Thr172)蛋白水平升高;腺病毒干扰表达显性失活AMPK和Compound C预处理可削弱马度米星铵细胞毒性。结果表明,马度米星铵增加细胞AMPK蛋白磷酸化水平,从而导致其对心肌细胞和骨骼肌细胞的毒性作用。8.马度米星铵致心肌细胞和骨骼肌细胞氧化应激和内质网应激不同浓度马度米星铵(H9c2细胞:0~5μg/mL,C2C12细胞:0~1μg/mL)处理24或48 h,利用CM-H2DCFDA检测细胞内活性氧水平,Western blot检测PERK、p-PERK(Thr980)、eIF2α、p-eIF2α(Ser51)蛋白表达水平。结果显示,马度米星铵处理24或48 h后,细胞内活性氧水平升高;马度米星铵处理24 h后,细胞内PERK和eIF2α蛋白磷酸化水平升高,呈浓度依赖性。结果表明,马度米星铵能引起H9c2细胞和C2C12细胞氧化应激和内质网应激。
[Abstract]:In order to investigate the toxic mechanism of ammonium bromide on myocardium and skeletal muscle cells, H9c2 and C2C12 cells were used as a model to investigate the toxicity of ammonium bromide, and to detect the effect of its cell cycle distribution, cell apoptosis and autophagy, and to explore the role of intracellular signaling pathway in the cytotoxicity of makhm ammonium. It is divided into the following parts: 1. the toxic effects of 1. M. M. in cardiomyocytes and skeletal muscle cells are treated with H9c2 cells and C2C12 cells as cardiac myocytes and skeletal muscle cells, with different concentrations of H9c2 cells (0~5 mu g/mL, C2C12 cells: 0~1 mu g/mL) for 5 days, and the morphological changes of cells are observed under microscope. The cell viability was detected by the One solution method and 24,48 or 72h, and the cell viability was detected by trypan blue staining. The results showed that the cell growth was suppressed, the morphology became round and some cells fell off after 5 days after the principle of trypan blue staining, with different concentrations of H9c2 cells (0~5 Mu g/mL, C2C12 cells: 0~1 g/mL). In the culture fluid, vacuoles appeared in the adherent cells, and the number of cells treated in the treatment group was significantly less than that in the untreated group. The cell viability was significantly reduced after 24,48 or 72 h was treated, and the cell survival rate decreased significantly. The results showed that the growth of H9c2 and C2C12 cells was inhibited by mnammonium. Reducing cell activity, increasing cell mortality, the toxic effect of mad ammonium on cardiomyocytes and skeletal muscle cells.2. the effect of mad ammonium on the cell cycle distribution of cardiomyocytes and skeletal muscle cells and its mechanism with different concentrations of mad M ammonium (H9c2 cells: 0~5 mu g/mL, C2C12 cells: 0~1 mu g/mL) treatment 24h or 0.5 mu g/mL horse degree At different time (H9c2 cells: 0,36,48 and 72h, C2C12 cells: 0,24,48 and 72 h), the distribution of cell cycle was detected by flow cytometry after PI staining, and 24 h was treated with different concentrations of mautoman (H9c2 cells: 0~5 micron, C2C12 cells: 0~1 mu g/mL). The results showed that the expression level of the cycle related proteins was different. After the treatment of 24 h, the G_0/G_1 phase ratio of H9c2 cells increased first and then decreased, the proportion of S phase decreased first, the proportion of G_2/M stage decreased gradually, the proportion of G_0/G_1 phase in C2C12 cells gradually increased, the proportion of S phase gradually decreased, and the proportion of G_2/M phase gradually decreased; after the treatment of different time, H9c2 cells and C2C12 cells were treated for different time. The proportion of the 1 phase gradually increased, the proportion of S phase gradually decreased and the proportion of G_2/M phase decreased gradually; the expression of H9c2 Cell Cyclin, CDK6 and CDC25B increased after the treatment of 24 h with different concentrations of MADM ammonium. C2C12 cells Cyclin D1, CDK4, CDK6 and CDC25A expressions increased, the bands appeared displacements and phosphorylation levels decreased. The results showed that H9c2 cells and C2C12 cells have obvious cell cycle blocking effect on H9c2 and C2C12 cells, which inhibit the effect of.3. Ma on cardiomyocyte and skeletal muscle cell apoptosis and its mechanism with different concentrations of mad M ammonium (H9c2 cells: 0~5 mu g/mL, C2C12 cells: 0~1 mu g/mL) to treat 72h, Annexin V-PI double dyed meridian cells The changes in the apoptosis rate were detected by the instrument. The expression of apoptosis related proteins was detected by different concentrations of H9c2 cells (H9c2 cells: 0~5 mu g/mL, C2C12 cells: 0~1 g/mL), and Western blot to detect the expression of apoptosis related proteins; z-VAD-fmk pretreatment of 1H (0.5 mu g/mL and 1 mu g/mL) treated 24 or 48 h. At the same time, trypan blue staining was used to detect the change of cell death rate; different concentrations of H9c2 cells (0~5 mu g/mL, C2C12 cells: 0~1 g/mL) were treated with 24h, Western blot was used to detect the expression of AIF protein, and the intracellular AIF protein was detected by immunofluorescence. The results showed that the apoptosis rate of the cells was significantly increased after the treatment of 72 h. The expression level of TRAIL, DR4, Cleaved caspase 8, Cleaved caspase 3 and Cleaved PARP increased in H9c2 cells after the treatment of 24h, and the expression level of Cleaved caspase 3 and Cleaved PARP increased. Caspase 3 and PARP protein lysis and cell death caused by NH4 were induced. After the treatment of 24 h, the expression of AIF in H9c2 cells increased and more transposition into the nucleus. The expression of AIF in C2C12 cells was unchanged but the AIF in the nucleus increased. The results showed that the apoptosis of H9c2 and C2C12 cells was induced. Toxic effects on cardiac myocytes and skeletal muscle cells.4. mad m star was treated with different concentrations of autophagy to cardiomyocytes and skeletal muscle cells in different concentrations (H9c2 cells: 0~5 mu g/mL, C2C12 cells: 0~1 mu g/mL) to treat 24 h and Western blot to detect the expression of autophagy protein; adenovirus interference 24 h expression GFP The labeled LC3 protein was treated with 24 h by (H9c2 cells: 0~5 mu g/mL, C2C12 cells: 0~1 mu g/mL). The intracellular LC3 protein was observed under the fluorescence microscope. The results showed that the level of LC3 II and p62 protein in the cells increased after 24 h treatment. After 24h, it was observed under the fluorescence microscope that there were a large number of green fluorescence points in the cells compared with the control group. The results showed that the LC3 protein could induce autophagy in H9c2 cells and C2C12 cells and blocked the autophagy,.5. mad, and the MAPK pathway protein and protein phosphatase of cardiomyocytes and skeletal muscle cells. The expression of MAPK pathway related protein and protein phosphatase in H9c2 cells and C2C12 cells was measured by (0~1 mu g/mL), and the expression of MAPK pathway related protein and protein phosphatase was detected by Western blot. The adenovirus interfered with the expression of MKK1-R4F and dominant inactivation PP2A, 0.5 mu g/mL and 1 mu g/mL Malayan ammonium treatment H9c2 cells 24 or 48 The cell morphologic changes were observed under the microscope and the cell activity was taken by the One solution method. The PP2A inhibitor Okadaic acid was pretreated with 1 h, 0.5 mu g/mL and 1 micron ammonium bromide treated H9c2 cell 48 h. The morphological changes of the cells were observed under the microscope and the cell activity was photographed by the One solution method; the adenovirus interfered with the expression of dominant inactivation. C-Jun, 0.5 mu g/mL and 1 g/mL M. M. NH4 treated C2C12 cells 24h or 48h, Western blot detected the expression of c-Jun protein, observed cell morphological changes under microscope and photographed, One solution method was used to detect cell activity; adenovirus interfered over expression PP5,0.5 muon and 1 mu ammonium bromide treated 24 or 48 cells The expression of p-JNK and p-c-Jun protein was measured, the morphological changes of cells were observed under microscope and the cell activity was detected by One solution method. The JNK inhibitor SP600125 pretreated 1 h, 0.5 mu g/mL and 1 micron ammonium bromide treated C2C12 cells 48 h. The cell morphology was observed under microscope and photographed. The cell activity was detected by One assay. After the treatment of 24 h, the level of p-ERK, p-PP2A and demethylated PP2A (De-PP2A) protein in H9c2 cells decreased, and the expression of methylated PP2A (Me-PP2A) increased, and the level of p-JNK and protein in C2C12 cells increased. The inhibition and cytotoxicity of ERK protein phosphorylation by stellar ammonium, Okadaic acid pretreatment can weaken the interference expression of adenovirus in.C2C12 cells of madeimatin cytotoxic c-Jun to weaken the toxicity of the cytotoxicity of the cytotoxicity of madeimatin; overexpression of PP5 can weaken the activation and cytotoxicity of JNK and c-Jun protein phosphorylation by the overexpression of PP5; SP600125 The pretreatment could weaken the cytotoxicity of the H9c2 cells. The results showed that the PP2A activity of H9c2 cells was enhanced and the level of phosphorylation of ERK protein decreased, which resulted in its toxic effect on cardiac myocytes; the expression of PP5 protein in C2C12 cells was inhibited by M. marem, and the level of JNK and c-Jun phosphorylation was increased, resulting in its toxicity to skeletal muscle cells. Effects of.6. on Akt1-FoxO3a pathway in cardiac myocytes and skeletal muscle cells with different concentrations of H9c2 cells and C2C12 cells in H9c2 cells and C2C12 cells, Western blot to detect Akt1, p-Akt1 (S473), p-Akt1 (T308), protein expression, immunofluorescent localization of intracellular proteins; adenovirus interference expression Continuous activation of Akt, 0.5 mu g/mL and 1 g/mL Ma ammonium ammonium treated cells 24h or 48h, Western blot to detect the expression of p-FoxO3a (Thr132) protein, immunofluorescence localization of intracellular FoxO3a protein, microscopic observation of cell morphology changes and photographing, One solution method detection of cell activity. The level of -Akt1 (S473), p-Akt1 (T308) and p-FoxO3a (Thr132) protein decreased, and the content of FoxO3a in the nucleus increased. The interference of adenovirus to continuously activate Akt could weaken the inhibition and cytotoxicity of marem ammonium on the phosphorylation and cytoplasmic transfer of FoxO3a protein. The results showed that the activity of Akt1 activity in H9c2 and C2C12 cells was inhibited. The level of phosphorylation of 3a protein decreased and the cytoplasmic transfer inhibited its cytoplasmic transfer, resulting in its toxic effect on cardiac myocytes and skeletal muscle cells.7.. The effect of mad m on AMPK protein of myocardial cells and skeletal muscle cells was different at different concentrations (H9c2 cells: 0~5 mu g/mL, C2C12 fine cell: 0~1 Mu g/mL) treatment 24 h, Western blot detection AMPK and P. The expression of -AMPK (Thr172) protein; adenovirus interfered with the expression of dominant inactivation AMPK, 0.5 g/mL and 1 mu g/mL treated cells 24 h or 48 h, Western blot detected the expression of AMPK protein, observed cell morphological changes under microscope and photographed, One solution method was used to detect cell activity. 1 The cell morphology was observed under microscope and the cell activity was photographed under the microscope and the cell activity was photographed under the microscope. The results showed that the level of p-AMPK (Thr172) protein in the cells increased after the treatment of 24h by One solution, and the preconditioning of adenovirus to express the dominant inactivation AMPK and Compound C could weaken the madeimonammonium cell. The results showed that the preprocessing of One and Compound C could weaken the madeimonammonium cell. Toxicity. The results showed that marem ammonium increased the level of phosphorylation of AMPK protein, resulting in its toxic effect on cardiomyocytes and skeletal muscle cells.8. mad M ammonium induced oxidative stress in cardiomyocytes and skeletal muscle cells and endoplasmic reticulum stress at different concentrations of H9c2 cells (H9c2 cells: 0~5 mu g/mL, C2C12 cells: 0~1 Mu g/mL) treatment 2 4 or 48 h, using CM-H2DCFDA to detect the intracellular reactive oxygen level, and Western blot to detect the expression level of PERK, p-PERK (Thr980), eIF2 a, and p-eIF2 alpha (Ser51) protein. The results showed that the intracellular active oxygen level was increased after the treatment of 24 or 48 h, and the level of phosphorylation of intracellular and alpha protein was increased after 24 h. The results showed that the treatment of H9c2 and C2C12 cells induced oxidative stress and endoplasmic reticulum stress.
【学位授予单位】:南京农业大学
【学位级别】:博士
【学位授予年份】:2015
【分类号】:S859.795
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