模拟失重所致人肺微血管内皮细胞凋亡的机制研究
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摘要
失重／模拟失重可导致肺组织形态学的改变,肺动脉高压,肺微血管的变化,同时可引起内皮细胞的凋亡。但是,失重／模拟失重所致的肺微血管内皮细胞(HPMVEC)凋亡情况已有报导,其时间效应和发生机制仍不清楚。因此,本课题的目的如下：
1.观察回转模拟失重对人肺微血管内皮细胞凋亡时间效应的影响；
2.研究线粒体信号转导通路在回转模拟失重所致的人肺微血管内皮细胞凋亡中的作用；
3.观察PI3K/Akt、NF-κB在回转模拟失重所致的人肺微血管内皮细胞凋亡中的变化情况,进一步探讨与细胞凋亡的关系；
4.观察回转模拟失重条件下,人肺微血管内皮细胞的细胞骨架的变化与细胞凋亡的关系。
方法
为达到以上研究目的,本课题采用以下实验方法进行研究：
1.将人肺微血管内皮细胞进行培养和传代,内皮细胞贴壁覆盖率达到80%时,收集细胞,并用于回转模拟失重实验。回转模拟失重时间设为4个时间点,12h、24h、48h和72h,同时每个时间点设立同步1g对照。采用流式细胞仪检测细胞凋亡和透射电镜观察模拟失重细胞超微结构的变化,观察模拟失重对人肺微血管内皮细胞凋亡时间效应的影响；
2.找出细胞凋亡最显著的时间点后，选择与线粒体信号传导通路相关的几个关键基因和蛋白,Bcl-2, Bax,细胞色素C, Caspase-9和Caspase-3;用real timePCR技术来检测模拟失重后人肺微血管内皮细胞中Caspase-3, Bcl-2和Bax基因的表达；用Western blot技术来检测模拟失重后人肺微血管内皮细胞中细胞色素C, Caspase-9和Caspase-3的表达；
3.用Westernblot技术来检测模拟失重后人肺微血管内皮细胞中PI3K、Akt及磷酸化水平的变化,以及NF-κB水平的变化；
4.采用免疫荧光标记技术,以phalloidin标记人肺微血管内皮细胞微丝肌动蛋白(F-actin),观察回转模拟失重所致人肺微血管内皮细胞的细胞骨架的变化与细胞凋亡的关系。
结果
采用以上的实验方法,本课题得出以下结果：
1.流式细胞仪结果显示,回转模拟失重12h和24h,细胞凋亡略有增加,但回转组和对照组比较,无明显差异,两组凋亡率为C12:2.29±0.78,Clinostat 12: 3.89±1.24,p=0.13; C24:2.35±1.48, Clinostat 24:4.08±0.72, p=0.14;回转模拟失重48h和72h,细胞凋亡显著,回转组和对照组比较,差异显著,以72h更为明显,两组凋亡率为C48:2.07±0.49, Clinostat 48:3.47±0.32, p=0.0386; C72:2.55±0.78, Clinostat72:8.18±0.48, p=0.0132,透射电镜观察模拟失重72h细胞超微结构,显示内皮细胞胞体缩小,变圆；细胞突起减少,细胞间连接消失；胞质基质密度增加,核膜皱缩、破损、消失,核内染色质浓缩凝聚趋边于核膜下；胞质内线粒体个数增加,但多肿胀变性,嵴变短、消失,甚至空泡化,粗面内质网扩张,肿胀成囊泡状；细胞溶酶体大量增殖,另还可见凋亡小体。
2. Real time PCR结果显示,回转模拟失重72h,人肺微血管内皮细胞中,Bcl-2表达下调,Bax和Caspase-3表达上调；Western blot结果显示：人肺微血管内皮细胞中,回转组胞浆中细胞色素C浓度高于线粒体中细胞色素C浓度,同时,Caspase-9和Caspase-3表达上调。
3. Western blot结果显示：模拟失重72h,人肺微血管内皮细胞中,PI3K的表达上调,Akt磷酸化水平降低,NF-κB水平增加。
4. phalloidin标记模拟失重48h时,人肺微血管内皮细胞微丝肌动蛋白,F-actin的表达明显减少,分布很不均匀,排列紊乱,72h时,F-actin的表达进一步减少。
结论
1.回转模拟失重可导致人肺血管内皮细胞发生早期凋亡,其中回转模拟失重72h,细胞凋亡最显著；
2.线粒体信号通路在回转模拟失重所致的人肺微血管内皮细胞凋亡中起主要作用；
3.回转模拟失重所致的人肺微血管内皮细胞凋亡中,PI3K-Akt参与细胞凋亡,回转模拟失重抑制PI3K,进一步抑制Akt磷酸化水平,促进细胞凋亡；同时,NF-κB表达水平升高,促进细胞凋亡。
4.回转模拟失重可导致人肺微血管内皮细胞的细胞骨架成分F-actin发生解聚,其可能参与细胞凋亡。
Objective
Microgravity or simulated microgravity can induce the change of pulmonary histomorphology, pulmonary hypertension and pulmonary microvascular change. Meanwhile, Microgravity or simulated microgravity can induce the apoptosis of endothelial cell. However, the apoptosis of human pulmonary microvascular endothelial cells (HPMVECs) which was caused by microgravity or simulated microgravity is not reported until now. The time effect and mechanism of the apoptosis are nor clear. So, the purpose of this study is following:
1. Find the optimal time of the apoptosis of human pulmonary microvascular endothelial cells which was caused by simulated microgravity by clinostat.
2. Study the effect of mitochondrial signal transduction pathway on the apoptosis of human pulmonary microvascular endothelial cells which was caused by simulated microgravity.
3. Observe the change of PI3K/Akt and NF-κB in the human pulmonary microvascular endothelial cells of apoptosis caused by simulated microgravity.
4. Observe the change of cytoskeleton in human pulmonary microvascular endothelial cells by simulated microgravity, and find the relationship between cytoskeleton and cell apoptosis.
Methods
To obtain the aim of study above, the experimental methods were applied as following:
1. The human pulmonary microvascular endothelial cells were cultured and passaged. When the adherence of human pulmonary microvascular endothelial cells and fraction of coverage was 80%, they were gathered and were applied in the experiment of simulated microgravity by clinostat.4 time point selected were 12h,24h,48h and 72h, respectively, meanwhile,1g control was set at every time point. Cell apoptosis was detected by the flow cytometry, and transmission electron microscope was used to observe the ultrastrcture of human pulmonary microvascular endothelial cells after simulated microgravity. According to the results, the optimal time of the apoptosis of human pulmonary microvascular endothelial cells.
2. After the optimal time of the apoptosis of human pulmonary microvascular endothelial cells was found, some important genes and proteins, such as Bcl-2, Bax, Cytochrome C, Caspase-9 and Caspase-3, correlated with the mitochondrial signal transduction pathway were selected. Caspase-3, Bcl-2 and Bax were detected by real time PCR, and ctytochrome C, Caspase-9 and Caspase-3 were measured by Western blot.
3. PI3K, Akt, P-Akt and NF-κB in the human pulmonary microvascular endothelial cells after simulated microgravity were measured by Western blot.
4. By inmunfluorescence technique, phalloidin was used to incubate with human pulmonary microvascular endothelial cells to detect the change of microfilament cytoskeleton at the different time of cell apoptosis.
Results
1. By the flow cytometry, when 12h and 24h of simulated microgravity by clinostat, cell apoptosis was not obvious; Compared 12h with 24h, there was no significantly difference (C12:2.29±0.78, Clinostat 12:3.89±1.24, p=0.13; C24: 2.35±1.48, Clinostat 24:4.08±0.72, p=0.14). When 48h and 72h of simulated microgravity by clinostat, cell apoptosis can by observed; however, Compared 48h with 72h, the apoptosis of human pulmonary microvascular endothelial cells was most obvious at 72h (C48:2.07±0.49, Clinostat 48:3.47±0.32, p=0.0386; C72:2.55±0.78, Clinostat72:8.18±0.48, p=0.0132). The results from transmission electron microscopy showed that the cell body of endothelial cells decreased and became round, the cell process decreased, the density of cytoplasmic matrix increased, nuclear membrane shrinkaged, intranuclear chromatin concentrated and aggregated below the nuclear membrane and mitochondrion became hyperplasia and swelling. Meanwhile, the apoptotic body can be observed when 72h of simulated microgravity.
2. The results by real time PCR showed that the expression of Bcl-2 was decreased, and the expression of Bax and Caspase-3 were increased in the human pulmonary microvascular endothelial cells when 72h of simulated microgravity. The results by Western blot showed the concentration of Cytochrome C in the cytoplasm was higher than that in the mitochondrion and the expression of Caspase-9 and Caspase-3 were increased when 72h of simulated microgravity.
3. The results by Western blot showed that simulated microgravity significantly decreased the expression of PI3K and the phosphorylation level of Akt, meanwhile, increased the expression of NF-κB.
4. The results by phallodidin showed that the expression of F-actin was decreased and appeared depolymerization at 48h, and the change of that was more obvious at 72h.
Conclusion
1. Simulated microgravity by clinostat can induce human pulmonary microvascular endothelial cells prophase apoptosis and the optimal time was 72h.
2. The mitochondial signal transduction pathway played an important role in the apoptosis of human pulmonary microvascular endothelial cells by simulated microgravity.
3. PI3K-Akt and NF-κB took part in the apoptosis of human pulmonary microvascular endothelial cells by simulated microgravity.
4. The express of F-actin was decreased and appeared depolymerization in the human pulmonary microvascular endothelial cells by simulated microgravity, and this suggested that the change of F-actin can take part in the cell apoptosis.
1.观察回转模拟失重对人肺微血管内皮细胞凋亡时间效应的影响；
2.研究线粒体信号转导通路在回转模拟失重所致的人肺微血管内皮细胞凋亡中的作用；
3.观察PI3K/Akt、NF-κB在回转模拟失重所致的人肺微血管内皮细胞凋亡中的变化情况,进一步探讨与细胞凋亡的关系；
4.观察回转模拟失重条件下,人肺微血管内皮细胞的细胞骨架的变化与细胞凋亡的关系。
方法
为达到以上研究目的,本课题采用以下实验方法进行研究：
1.将人肺微血管内皮细胞进行培养和传代,内皮细胞贴壁覆盖率达到80%时,收集细胞,并用于回转模拟失重实验。回转模拟失重时间设为4个时间点,12h、24h、48h和72h,同时每个时间点设立同步1g对照。采用流式细胞仪检测细胞凋亡和透射电镜观察模拟失重细胞超微结构的变化,观察模拟失重对人肺微血管内皮细胞凋亡时间效应的影响；
2.找出细胞凋亡最显著的时间点后，选择与线粒体信号传导通路相关的几个关键基因和蛋白,Bcl-2, Bax,细胞色素C, Caspase-9和Caspase-3;用real timePCR技术来检测模拟失重后人肺微血管内皮细胞中Caspase-3, Bcl-2和Bax基因的表达；用Western blot技术来检测模拟失重后人肺微血管内皮细胞中细胞色素C, Caspase-9和Caspase-3的表达；
3.用Westernblot技术来检测模拟失重后人肺微血管内皮细胞中PI3K、Akt及磷酸化水平的变化,以及NF-κB水平的变化；
4.采用免疫荧光标记技术,以phalloidin标记人肺微血管内皮细胞微丝肌动蛋白(F-actin),观察回转模拟失重所致人肺微血管内皮细胞的细胞骨架的变化与细胞凋亡的关系。
结果
采用以上的实验方法,本课题得出以下结果：
1.流式细胞仪结果显示,回转模拟失重12h和24h,细胞凋亡略有增加,但回转组和对照组比较,无明显差异,两组凋亡率为C12:2.29±0.78,Clinostat 12: 3.89±1.24,p=0.13; C24:2.35±1.48, Clinostat 24:4.08±0.72, p=0.14;回转模拟失重48h和72h,细胞凋亡显著,回转组和对照组比较,差异显著,以72h更为明显,两组凋亡率为C48:2.07±0.49, Clinostat 48:3.47±0.32, p=0.0386; C72:2.55±0.78, Clinostat72:8.18±0.48, p=0.0132,透射电镜观察模拟失重72h细胞超微结构,显示内皮细胞胞体缩小,变圆；细胞突起减少,细胞间连接消失；胞质基质密度增加,核膜皱缩、破损、消失,核内染色质浓缩凝聚趋边于核膜下；胞质内线粒体个数增加,但多肿胀变性,嵴变短、消失,甚至空泡化,粗面内质网扩张,肿胀成囊泡状；细胞溶酶体大量增殖,另还可见凋亡小体。
2. Real time PCR结果显示,回转模拟失重72h,人肺微血管内皮细胞中,Bcl-2表达下调,Bax和Caspase-3表达上调；Western blot结果显示：人肺微血管内皮细胞中,回转组胞浆中细胞色素C浓度高于线粒体中细胞色素C浓度,同时,Caspase-9和Caspase-3表达上调。
3. Western blot结果显示：模拟失重72h,人肺微血管内皮细胞中,PI3K的表达上调,Akt磷酸化水平降低,NF-κB水平增加。
4. phalloidin标记模拟失重48h时,人肺微血管内皮细胞微丝肌动蛋白,F-actin的表达明显减少,分布很不均匀,排列紊乱,72h时,F-actin的表达进一步减少。
结论
1.回转模拟失重可导致人肺血管内皮细胞发生早期凋亡,其中回转模拟失重72h,细胞凋亡最显著；
2.线粒体信号通路在回转模拟失重所致的人肺微血管内皮细胞凋亡中起主要作用；
3.回转模拟失重所致的人肺微血管内皮细胞凋亡中,PI3K-Akt参与细胞凋亡,回转模拟失重抑制PI3K,进一步抑制Akt磷酸化水平,促进细胞凋亡；同时,NF-κB表达水平升高,促进细胞凋亡。
4.回转模拟失重可导致人肺微血管内皮细胞的细胞骨架成分F-actin发生解聚,其可能参与细胞凋亡。
Objective
Microgravity or simulated microgravity can induce the change of pulmonary histomorphology, pulmonary hypertension and pulmonary microvascular change. Meanwhile, Microgravity or simulated microgravity can induce the apoptosis of endothelial cell. However, the apoptosis of human pulmonary microvascular endothelial cells (HPMVECs) which was caused by microgravity or simulated microgravity is not reported until now. The time effect and mechanism of the apoptosis are nor clear. So, the purpose of this study is following:
1. Find the optimal time of the apoptosis of human pulmonary microvascular endothelial cells which was caused by simulated microgravity by clinostat.
2. Study the effect of mitochondrial signal transduction pathway on the apoptosis of human pulmonary microvascular endothelial cells which was caused by simulated microgravity.
3. Observe the change of PI3K/Akt and NF-κB in the human pulmonary microvascular endothelial cells of apoptosis caused by simulated microgravity.
4. Observe the change of cytoskeleton in human pulmonary microvascular endothelial cells by simulated microgravity, and find the relationship between cytoskeleton and cell apoptosis.
Methods
To obtain the aim of study above, the experimental methods were applied as following:
1. The human pulmonary microvascular endothelial cells were cultured and passaged. When the adherence of human pulmonary microvascular endothelial cells and fraction of coverage was 80%, they were gathered and were applied in the experiment of simulated microgravity by clinostat.4 time point selected were 12h,24h,48h and 72h, respectively, meanwhile,1g control was set at every time point. Cell apoptosis was detected by the flow cytometry, and transmission electron microscope was used to observe the ultrastrcture of human pulmonary microvascular endothelial cells after simulated microgravity. According to the results, the optimal time of the apoptosis of human pulmonary microvascular endothelial cells.
2. After the optimal time of the apoptosis of human pulmonary microvascular endothelial cells was found, some important genes and proteins, such as Bcl-2, Bax, Cytochrome C, Caspase-9 and Caspase-3, correlated with the mitochondrial signal transduction pathway were selected. Caspase-3, Bcl-2 and Bax were detected by real time PCR, and ctytochrome C, Caspase-9 and Caspase-3 were measured by Western blot.
3. PI3K, Akt, P-Akt and NF-κB in the human pulmonary microvascular endothelial cells after simulated microgravity were measured by Western blot.
4. By inmunfluorescence technique, phalloidin was used to incubate with human pulmonary microvascular endothelial cells to detect the change of microfilament cytoskeleton at the different time of cell apoptosis.
Results
1. By the flow cytometry, when 12h and 24h of simulated microgravity by clinostat, cell apoptosis was not obvious; Compared 12h with 24h, there was no significantly difference (C12:2.29±0.78, Clinostat 12:3.89±1.24, p=0.13; C24: 2.35±1.48, Clinostat 24:4.08±0.72, p=0.14). When 48h and 72h of simulated microgravity by clinostat, cell apoptosis can by observed; however, Compared 48h with 72h, the apoptosis of human pulmonary microvascular endothelial cells was most obvious at 72h (C48:2.07±0.49, Clinostat 48:3.47±0.32, p=0.0386; C72:2.55±0.78, Clinostat72:8.18±0.48, p=0.0132). The results from transmission electron microscopy showed that the cell body of endothelial cells decreased and became round, the cell process decreased, the density of cytoplasmic matrix increased, nuclear membrane shrinkaged, intranuclear chromatin concentrated and aggregated below the nuclear membrane and mitochondrion became hyperplasia and swelling. Meanwhile, the apoptotic body can be observed when 72h of simulated microgravity.
2. The results by real time PCR showed that the expression of Bcl-2 was decreased, and the expression of Bax and Caspase-3 were increased in the human pulmonary microvascular endothelial cells when 72h of simulated microgravity. The results by Western blot showed the concentration of Cytochrome C in the cytoplasm was higher than that in the mitochondrion and the expression of Caspase-9 and Caspase-3 were increased when 72h of simulated microgravity.
3. The results by Western blot showed that simulated microgravity significantly decreased the expression of PI3K and the phosphorylation level of Akt, meanwhile, increased the expression of NF-κB.
4. The results by phallodidin showed that the expression of F-actin was decreased and appeared depolymerization at 48h, and the change of that was more obvious at 72h.
Conclusion
1. Simulated microgravity by clinostat can induce human pulmonary microvascular endothelial cells prophase apoptosis and the optimal time was 72h.
2. The mitochondial signal transduction pathway played an important role in the apoptosis of human pulmonary microvascular endothelial cells by simulated microgravity.
3. PI3K-Akt and NF-κB took part in the apoptosis of human pulmonary microvascular endothelial cells by simulated microgravity.
4. The express of F-actin was decreased and appeared depolymerization in the human pulmonary microvascular endothelial cells by simulated microgravity, and this suggested that the change of F-actin can take part in the cell apoptosis.
引文
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