血管活性肠肽对高氧损伤肺泡Ⅱ型上皮细胞再生修复的调控及其STAT3信号机制

发布时间：2018-06-28 17:14

本文选题：高氧 + AECⅡ　；参考：《重庆医科大学》2011年博士论文

【摘要】：第一部分高氧暴露对肺泡Ⅱ型上皮细胞存活的影响目的研究高氧诱导的AECⅡ损伤、死亡情况及高氧暴露后细胞增殖的变化,从而了解高氧对AECⅡ存活的影响。方法将MLE-12细胞暴露于90%体积分数的氧制备高氧损伤模型,在高氧暴露不同时点利用Annexin V/PI双标记流式细胞仪检测细胞早期凋亡、晚期凋亡及坏死,酶标仪检测凋亡相关蛋白caspase-3活性,DAPI染色荧光显微镜观察细胞核及核内染色质形态,透射电镜观察线粒体超微结构的改变、流式细胞仪检测线粒体膜电位变化以了解高氧对线粒体结构及功能的损害, MTT法及细胞周期检测反映高氧状态下的MLE-12细胞增殖情况。结果Annexin V/PI双标记流式细胞仪检测结果显示高氧暴露0.5 h,1 h,2 h,4 h细胞早期凋亡、晚期凋亡及坏死率与空气对照组相比无显著差异,高氧暴露后6 h细胞早期凋亡、晚期凋亡及坏死率明显增加(p 0.01),高氧暴露后12 h、24 h更多细胞发生早期凋亡、晚期凋亡及坏死(p 0.01),且发生死亡的细胞大部分集中于代表晚期凋亡及坏死的右上象限。酶标仪检测caspase-3活性结果显示与空气对照组比较,高氧暴露后0.5 h,1 h,2 h,4 h,6 h,12 h,24 h各组OD_(405nm)无显著差异。DAPI染色荧光显微镜观察,与空气对照组比较,高氧暴露0.5 h,1 h,2 h,4 h细胞核及染色质形态未见明显变化,胞核呈圆形,边缘清晰,染色均匀,高氧暴露6 h,12 h,24 h细胞核肿胀,体积变大,未见核固缩及染色质聚集现象。JC-1染色流式细胞仪分析及荧光显微镜观察,与空气对照组相比,高氧暴露0.5 h,1 h,2 h,4 h线粒体膜电位无明显变化,高氧暴露后6 h,12 h,24 h,随暴露时间延长,线粒体膜电位下降趋势愈发明显(p 0.01)。透射电镜观察发现,高氧暴露后线粒体发生肿胀,内膜及外膜均遭到破坏。MTT结果显示,与同时点空气对照组相比,高氧暴露0.5 h,1 h,2 h,4 h细胞OD_(490nm)值无显著变化,高氧暴露6 h,12 h,24 h随暴露时间延长OD_(490nm)值降低趋势越为明显(p0.01)。细胞周期分析显示高氧暴露0.5 h,1 h,2 h,4 h G)_0/G_1、S、G2/M各期细胞比例与空气对照组相比无显著差异,大部分细胞分布于S及G2/M期即DNA合成及分裂期,高氧暴露6 h,12 h,24 h G_0/G_1期细胞比例明显增加(p0.01),而S期、G_2/M比例显著降低(p0.01),大部分细胞分布于G_0/G_1期即静止期及DNA合成前期。结论高氧可以时间依赖性的方式诱导AECⅡ损伤及死亡,抑制AECⅡ增殖,从而使得AECⅡ存活减少。90%氧体积分数的高氧暴露后,AECⅡ表现为细胞膜的破坏、细胞核及线粒体的肿胀等胀亡的特征,而磷脂酰丝氨酸外翻、核固缩、染色质聚集、凋亡相关蛋白酶活化等凋亡特征不明显。第二部分血管活性肠肽对高氧损伤肺泡Ⅱ型上皮细胞再生修复的影响目的研究VIP干预后高氧暴露的AECⅡ损伤、死亡情况及细胞增殖的变化,从而了解VIP对高氧损伤后AECⅡ再生修复的影响。方法MLE-12细胞进行高氧暴露及VIP干预,MTT法及细胞周期检测细胞增殖,了解VIP对高氧暴露细胞增殖的影响,流式细胞仪检测线粒体膜电位的变化,Annexin V/PI双标记流式细胞学检测细胞早期凋亡、晚期凋亡及坏死,DAPI染色荧光显微镜观察细胞核及核内染色质形态,了解VIP对高氧诱导的细胞死亡的影响。结果MTT结果显示,与空气对照组相比,高氧组氧暴露后24 h,MLE-12细胞OD_(490nm)值显著降低(p0.05),高氧VIP各浓度组与高氧组比较,随加入的VIP终浓度由10~(-9)至10~(-6) M升高,OD_(490nm)值亦渐升高,高氧VIP10~(-7)M组与高氧VIP10~(-10)M组、高氧VIP10~(-9)M组、高氧VIP10~(-8)M组比较,OD_(490nm)值差异有显著性(p0.01)。Annexin-V/PI双标记流式细胞仪检测结果显示,高氧VIP10~(-7)M组细胞早期凋亡率、晚期凋亡及坏死率与单纯高氧暴露组相比明显降低( p 0.01),但仍高于空气对照组,VIP干预后死亡的细胞仍大部分分布于代表晚期凋亡及坏死的右上象限。JC-1染色流式细胞仪分析显示,与单纯高氧组相比,高氧VIP10~(-7)M组线粒体膜电位下降幅度减少(p 0.01)。DAPI染色荧光显微镜下观察,VIP干预后高氧暴露的MLE-12细胞仍可见增大的胞核,而无核固缩及染色质聚集。流式细胞学细胞周期检测结果发现,高氧VIP10~(-7)M组与单纯高氧组比较,G_0/G_1期细胞比例明显减少(p0.01),S期、G2/M期比例显著增高(p0.01),但两者均未达空气对照组水平。结论VIP可以剂量依赖性的方式促进高氧暴露的AECⅡ的增殖,减轻高氧暴露对细胞增殖的抑制作用,同时VIP通过稳定线粒体功能,减少细胞高氧性损伤及死亡,从而改善高氧暴露后AECⅡ的存活。VIP干预后高氧暴露细胞仍表现出胞膜破坏、胞核肿胀等胀亡的特征,提示VIP可减少高氧诱导的AECⅡ的胀亡,胀亡可能与凋亡一样也是可调控的过程。第三部分血管活性肠肽促进高氧损伤肺泡Ⅱ型上皮细胞再生修复的STAT3信号机制目的研究高氧暴露及VIP干预后JAK2/STAT3的活化以及STAT3表达敲低后VIP对高氧暴露AECⅡ增殖、死亡的影响,探讨VIP调控高氧损伤AECⅡ再生修复的可能信号机制。方法Western Blot及凝胶迁移实验(Electrophoretic Mobility Shift Assay, EMSA)检测VIP干预前后高氧暴露的MLE-12细胞JAK2、磷酸化JAK2(p-JAK2),STAT3、磷酸化STAT3(p-STAT3)的表达以及STAT3与DNA结合活性,了解高氧损伤过程中JAK2/STAT3活化情况和VIP对JAK2/STAT3活化影响,RNAi技术敲低MLE-12细胞STAT3表达后采用Annexin V/PI双标记流式细胞学检测细胞死亡、MTT法检测细胞增殖情况,了解STAT3是否介导了VIP对高氧损伤AECⅡ再生修复的调控。结果Western Blot结果显示,在高氧暴露后2 h,p-JAK2蛋白表达增加,4 h达峰值,高氧暴露后6 h表达有所下降,高氧暴露后12 h p-JAK2蛋白表达回复至未干预时水平,与此同时,高氧暴露24 h内的各时点JAK2总蛋白水平并无显著变化,高氧暴露VIP 10~(-7)M组细胞与单纯高氧暴露组细胞相比,24 h内的各检测时间点JAK2总蛋白水平、JAK2发生磷酸化的时点及p-JAK2表达无显著变化。p-STAT3蛋白表达水平在高氧暴露后2 h增加,4 h最强,6 h后有所下降,高氧暴露后12 h p-STAT3蛋白表达与未干预时水平无差异,在p-STAT3表达增强的同时,STAT3总蛋白表达在高氧暴露24 h内各时点无明显差异,10~(-7)M VIP干预后,高氧暴露细胞STAT3总蛋白水平和STAT3磷酸化的时效性均无变化,但p-STAT3表达较单纯高氧暴露组明显增加(p0.05)。EMSA结果显示,高氧暴露后2 h即可见STAT3 DNA结合活性增加,4 h达峰值,6 h减弱,12 h降至对照组水平,10~(-7)M VIP干预后,高氧暴露细胞中STAT3与DNA结合活性进一步增强(p0.01)。Annexin-V/PI双标记流式细胞仪检测结果显示,与单纯高氧暴露的未转染组细胞比较,高氧STAT3 siRNA组早期凋亡细胞、晚期凋亡及坏死细胞显著增多(p0.01),而高氧VIP未转染组早期凋亡细胞及晚期凋亡和坏死细胞显著降低(p0.01),与高氧VIP未转染组相比,高氧VIP STAT3 siRNA组早期凋亡细胞、晚期凋亡及坏死细胞数明显增加(p0.05)。MTT结果显示,与高氧未转染组相比,高氧STAT3 siRNA组OD_(490nm)值明显降低(p0.01),高氧VIP未转染组OD_(490nm)值显著增高(p0.01),高氧VIP STAT3 siRNA组较之高氧VIP未转染组,OD_(490nm)值明显降低(p0.01)。结论VIP可通过促进高氧诱导的STAT3的活化,减轻细胞损伤死亡,促进细胞增殖,改善细胞存活,但VIP活化STAT3的过程与经典的JAK2/STAT3途径激活不同,并不影响JAK2的活性。
[Abstract]:Part one the effect of hyperoxia exposure on the survival of alveolar type II epithelial cells
Objective to study the effects of hyperoxia induced AEC II injury, death and cell proliferation after hyperoxia exposure, so as to understand the effect of hyperoxia on AEC II survival.
Methods the MLE-12 cells were exposed to 90% volume fraction of oxygen to prepare the hyperoxia damage model. The early apoptosis, late apoptosis and necrosis were detected by Annexin V/PI double standard flow cytometry at different levels of hyperoxia exposure. The activity of apoptosis related protein caspase-3 was detected by the enzyme labeling instrument, and the nucleus and chromatin in nucleus were observed by DAPI staining fluorescence microscope. Morphology and transmission electron microscopy were used to observe the ultrastructural changes of mitochondria. The changes of mitochondrial membrane potential were detected by flow cytometry to understand the damage of hyperoxia to mitochondria structure and function. The MTT method and cell cycle detection reflect the proliferation of MLE-12 cells in hyperoxia.
Results the results of Annexin V/PI double standard flow cytometry showed that hyperoxia was exposed to 0.5 h, 1 h, 2 h, 4 h cells early apoptosis, and there was no significant difference in the late apoptosis and necrosis rate compared with the air control group. After hyperoxia exposure, 6 h cells were apoptotic, late apoptosis and necrosis were significantly increased (P 0.01), 12 h after hyperoxia exposure, and 24 h more cells. Early apoptosis, late apoptosis and necrosis (P 0.01), and most of the dead cells were concentrated on the right upper quadrant of late apoptosis and necrosis. The results of caspase-3 activity detected by the enzyme labelled instrument showed that compared with the air control group, 0.5 h, 1 h, 2 h, 4 h, 6 h, 12 h, 24 h OD_ (405nm) were not significantly different from the.DAPI staining fluorescence microscope Compared with the air control group, high oxygen exposure was 0.5 h, 1 h, 2 h, and 4 h had no significant changes in nucleus and chromatin form. The nucleus was round, the edge was clear, the staining was uniform, the hyperoxia exposure 6 h, 12 h, 24 h were swelling and the volume became larger, no nuclear condensation and chromatin aggregation were observed by.JC-1 staining flow cytometer analysis and fluorescence microscope observation. Compared with the air control group, the mitochondrial membrane potential of hyperoxia exposure 0.5 h, 1 h, 2 h and 4 h had no obvious change, 6 h, 12 h, 24 h after hyperoxia exposure. With the prolonged exposure time, the decrease trend of mitochondrial membrane potential became more obvious (P 0.01). Transmission electron microscope observed that the mitochondria swelled after hyperoxia exposure and the endintima and outer membrane were destroyed by.MTT results. Compared with the same point air control group, high oxygen exposure was 0.5 h, 1 h, 2 h, and 4 h cells OD_ (490nm) values did not change significantly. Hyperoxia exposure 6 h, 12 h, 24 h decreased as OD_ (490nm) decreased with exposure time (P0.01). Cell cycle analysis showed that the ratio of hyperoxia 0.5, 2, 2, 4 Compared with no significant difference, most of the cells were distributed at S and G2/M phase DNA synthesis and mitosis stage. Hyperoxia exposure was 6 h, 12 h, and 24 h G_0/G_1 cells increased significantly (P0.01), while S phase was significantly decreased (P0.01). Most of the cells were distributed in the G_0/G_1 stage at rest and in the prophase of synthesis.
Conclusion hyperoxia can induce AEC II injury and death in a time dependent manner, and inhibit the proliferation of AEC II. Thus, after AEC II survives the hyperoxia exposure of.90% oxygen volume fraction, AEC II is characterized by the destruction of cell membrane, swelling of nuclei and mitochondria, and phosphatidylserine ectropion, nuclear condensation, chromatin aggregation, and withering. The apoptotic characteristics, such as the activation of the perish protease, were not obvious.
The second part is the effect of vasoactive intestinal peptide on regeneration and repair of alveolar type II epithelial cells injured by hyperoxia.
Objective to study the effects of VIP on AEC II injury, death and cell proliferation in hyperoxia exposed rats, so as to understand the effect of VIP on AEC II regeneration and repair after hyperoxia.
Methods MLE-12 cells were exposed to hyperoxia and VIP intervention, MTT method and cell cycle were used to detect cell proliferation. The effect of VIP on the proliferation of hyperoxia exposed cells was investigated. Flow cytometry was used to detect the changes of mitochondrial membrane potential. Annexin V/PI double labeled flow cytology was used to detect early apoptosis and late apoptosis and necrosis. DAPI staining fluorescence microscopy was used to observe the cell proliferation. The morphology of chromatin in nucleus and nucleus was studied to understand the effect of VIP on hyperoxia induced cell death.
Results the results of MTT showed that compared with the air control group, the OD_ (490nm) value of MLE-12 cells decreased significantly (P0.05) after oxygen exposure in the hyperoxic group, and the OD_ (490nm) of MLE-12 cells was significantly higher than that in the hyperoxic group. The final concentration of VIP was increased from 10~ (-9) to 10~ (-6). Compared with the group of hyperoxic VIP10~ (-8) M, the difference of OD_ (490nm) value was significant (P0.01).Annexin-V/PI double standard flow cytometry results showed that the early apoptosis rate, the late apoptosis and necrosis rate of the hyperoxic VIP10~ (-7) M group decreased significantly compared with the hyperoxia group (P 0.01), but it was still higher than that in the air control group. Most of the right upper quadrant.JC-1 staining flow cytometry, which represented late apoptosis and necrosis, showed that the decrease of mitochondrial membrane potential decreased in the hyperoxic VIP10~ (-7) M group compared with those in the hyperoxia group (P 0.01).DAPI staining fluorescence microscopy, and the enlarged nucleus was still visible in MLE-12 cells exposed to hyperoxia after VIP. Nuclear condensation and chromatin aggregation. Flow cytometry cell cycle detection results showed that the proportion of G_0/G_1 phase cells decreased significantly (P0.01), S phase and G2/M phase (P0.01) in hyperoxic VIP10~ (-7) M group compared with pure hyperoxia group, but both did not reach the level of air control group. Conclusion VIP can be used in a dose dependent manner to promote hyperoxia exposure. The proliferation of AEC II reduces the inhibitory effect of hyperoxia exposure on cell proliferation, while VIP reduces hyperoxic damage and death by stabilizing mitochondrial function, thus improving the survival of AEC II after hyperoxia exposure to the survival.VIP, the hyperoxic exposed cells still exhibit the characteristics of cell membrane destruction, swelling of the nucleus and so on, suggesting that VIP can reduce hyperoxia induction. The swelling and death of AEC II may be a regulatory process as well as apoptosis.
Third part of vasoactive intestinal peptide promotes STAT3 signaling mechanism of alveolar type II epithelial cell regeneration and repair after hyperoxia injury.
Objective to study the activation of JAK2/STAT3 after hyperoxia exposure and VIP intervention, and the effect of VIP on AEC II proliferation and death of hyperoxia exposed AEC II, and to explore the possible signal mechanism of VIP regulation of hyperoxia induced AEC II regeneration.
Methods Western Blot and gel migration tests (Electrophoretic Mobility Shift Assay, EMSA) were used to detect the expression of MLE-12 cell JAK2, JAK2 phosphorylation, phosphorylation, phosphorylation and binding activity before and after the intervention of VIP. After RNAi technology knocked down the expression of MLE-12 cell STAT3, Annexin V/PI double standard flow cytology was used to detect cell death, MTT method was used to detect cell proliferation, and whether STAT3 mediated the regulation of VIP on hyperoxic injury AEC II regeneration and repair.
Results Western Blot results showed that 2 h after hyperoxia exposure, the expression of p-JAK2 protein increased, the peak value of 4 h was reached, the expression of 6 h decreased after hyperoxia exposure, and the expression of 12 h p-JAK2 protein after hyperoxia exposure to the level of non dry preconditioning, while the total protein level of JAK2 was not significantly changed at all time points in the high oxygen exposure 24 h, and the VIP 10~ (VIP 10~) was exposed to high oxygen exposure. In group M, the total protein level of JAK2 in 24 h, the time point of JAK2 phosphorylation and the expression of p-JAK2 were not significantly changed, and the expression level of.P-STAT3 protein was increased at 2 h after hyperoxia exposure, 4 h was the strongest, and then decreased after 6 h. The expression of 12 h p-STAT3 protein after hyperoxia exposure was no longer than that of Undry. The difference, while the expression of p-STAT3 was enhanced, there was no significant difference in the total protein expression of STAT3 in the 24 h of hyperoxia exposure. The prognosis of 10~ (-7) M VIP and the aging of STAT3 total protein level and STAT3 phosphorylation in hyperoxic exposed cells were not changed, but the expression of p-STAT3 was significantly higher than that in the simple hyperoxic exposure group (P0.05).EMSA results showed, hyperoxia exposure After 2 h, STAT3 DNA binding activity increased, 4 h reached its peak value, 6 h weakened, and 12 h decreased to the control group. 10~ (-7) M VIP was the prognosis, and the STAT3 and DNA binding activity in hyperoxic exposed cells was further enhanced. The early apoptosis and necrotic cells in the RNA group were significantly increased (P0.01), while the early apoptotic cells and late apoptotic and necrotic cells in the hyperoxic VIP untransfected group were significantly decreased (P0.01). Compared with the hyperoxic VIP untransfected group, the early apoptotic cells of the hyperoxic VIP STAT3 siRNA group, the late apoptosis and the number of necrotic cells increased significantly (P0.05).MTT results. The values of OD_ (490nm) in the hyperoxic STAT3 siRNA group were significantly lower than those in the hyperoxia group (P0.01), and the value of OD_ (490nm) in the hyperoxic VIP untransfected group was significantly higher (P0.01), and the value of the hyperoxic VIP STAT3 siRNA group was significantly lower than that of the hyperoxic VIP group.
Conclusion VIP can promote the activation of STAT3 induced by hyperoxia, reduce cell death, promote cell proliferation and improve cell survival, but the activation of STAT3 by VIP is different from that of the classical JAK2/STAT3 pathway, and does not affect the activity of JAK2.
【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R363

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