本文選題：高氧 + AECⅡ��； 參考：《重慶醫(yī)科大學》2011年博士論文

【摘要】：第一部分高氧暴露對肺泡Ⅱ型上皮細胞存活的影響 目的研究高氧誘導的AECⅡ損傷、死亡情況及高氧暴露后細胞增殖的變化,從而了解高氧對AECⅡ存活的影響。 方法將MLE-12細胞暴露于90%體積分數(shù)的氧制備高氧損傷模型,在高氧暴露不同時點利用Annexin V/PI雙標記流式細胞儀檢測細胞早期凋亡、晚期凋亡及壞死,酶標儀檢測凋亡相關蛋白caspase-3活性,DAPI染色熒光顯微鏡觀察細胞核及核內染色質形態(tài),透射電鏡觀察線粒體超微結構的改變、流式細胞儀檢測線粒體膜電位變化以了解高氧對線粒體結構及功能的損害, MTT法及細胞周期檢測反映高氧狀態(tài)下的MLE-12細胞增殖情況。 結果Annexin V/PI雙標記流式細胞儀檢測結果顯示高氧暴露0.5 h,1 h,2 h,4 h細胞早期凋亡、晚期凋亡及壞死率與空氣對照組相比無顯著差異,高氧暴露后6 h細胞早期凋亡、晚期凋亡及壞死率明顯增加(p 0.01),高氧暴露后12 h、24 h更多細胞發(fā)生早期凋亡、晚期凋亡及壞死(p 0.01),且發(fā)生死亡的細胞大部分集中于代表晚期凋亡及壞死的右上象限。酶標儀檢測caspase-3活性結果顯示與空氣對照組比較,高氧暴露后0.5 h,1 h,2 h,4 h,6 h,12 h,24 h各組OD_(405nm)無顯著差異。DAPI染色熒光顯微鏡觀察,與空氣對照組比較,高氧暴露0.5 h,1 h,2 h,4 h細胞核及染色質形態(tài)未見明顯變化,胞核呈圓形,邊緣清晰,染色均勻,高氧暴露6 h,12 h,24 h細胞核腫脹,體積變大,未見核固縮及染色質聚集現(xiàn)象。JC-1染色流式細胞儀分析及熒光顯微鏡觀察,與空氣對照組相比,高氧暴露0.5 h,1 h,2 h,4 h線粒體膜電位無明顯變化,高氧暴露后6 h,12 h,24 h,隨暴露時間延長,線粒體膜電位下降趨勢愈發(fā)明顯(p 0.01)。透射電鏡觀察發(fā)現(xiàn),高氧暴露后線粒體發(fā)生腫脹,內膜及外膜均遭到破壞。MTT結果顯示,與同時點空氣對照組相比,高氧暴露0.5 h,1 h,2 h,4 h細胞OD_(490nm)值無顯著變化,高氧暴露6 h,12 h,24 h隨暴露時間延長OD_(490nm)值降低趨勢越為明顯(p0.01)。細胞周期分析顯示高氧暴露0.5 h,1 h,2 h,4 h G)_0/G_1、S、G2/M各期細胞比例與空氣對照組相比無顯著差異,大部分細胞分布于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期細胞比例明顯增加(p0.01),而S期、G_2/M比例顯著降低(p0.01),大部分細胞分布于G_0/G_1期即靜止期及DNA合成前期。 結論高氧可以時間依賴性的方式誘導AECⅡ損傷及死亡,抑制AECⅡ增殖,從而使得AECⅡ存活減少。90%氧體積分數(shù)的高氧暴露后,AECⅡ表現(xiàn)為細胞膜的破壞、細胞核及線粒體的腫脹等脹亡的特征,而磷脂酰絲氨酸外翻、核固縮、染色質聚集、凋亡相關蛋白酶活化等凋亡特征不明顯。 第二部分血管活性腸肽對高氧損傷肺泡Ⅱ型上皮細胞再生修復的影響 目的研究VIP干預后高氧暴露的AECⅡ損傷、死亡情況及細胞增殖的變化,從而了解VIP對高氧損傷后AECⅡ再生修復的影響。 方法MLE-12細胞進行高氧暴露及VIP干預,MTT法及細胞周期檢測細胞增殖,了解VIP對高氧暴露細胞增殖的影響,流式細胞儀檢測線粒體膜電位的變化,Annexin V/PI雙標記流式細胞學檢測細胞早期凋亡、晚期凋亡及壞死,DAPI染色熒光顯微鏡觀察細胞核及核內染色質形態(tài),了解VIP對高氧誘導的細胞死亡的影響。 結果MTT結果顯示,與空氣對照組相比,高氧組氧暴露后24 h,MLE-12細胞OD_(490nm)值顯著降低(p0.05),高氧VIP各濃度組與高氧組比較,隨加入的VIP終濃度由10~(-9)至10~(-6) M升高,OD_(490nm)值亦漸升高,高氧VIP10~(-7)M組與高氧VIP10~(-10)M組、高氧VIP10~(-9)M組、高氧VIP10~(-8)M組比較,OD_(490nm)值差異有顯著性(p0.01)。Annexin-V/PI雙標記流式細胞儀檢測結果顯示,高氧VIP10~(-7)M組細胞早期凋亡率、晚期凋亡及壞死率與單純高氧暴露組相比明顯降低( p 0.01),但仍高于空氣對照組,VIP干預后死亡的細胞仍大部分分布于代表晚期凋亡及壞死的右上象限。JC-1染色流式細胞儀分析顯示,與單純高氧組相比,高氧VIP10~(-7)M組線粒體膜電位下降幅度減少(p 0.01)。DAPI染色熒光顯微鏡下觀察,VIP干預后高氧暴露的MLE-12細胞仍可見增大的胞核,而無核固縮及染色質聚集。流式細胞學細胞周期檢測結果發(fā)現(xiàn),高氧VIP10~(-7)M組與單純高氧組比較,G_0/G_1期細胞比例明顯減少(p0.01),S期、G2/M期比例顯著增高(p0.01),但兩者均未達空氣對照組水平。結論VIP可以劑量依賴性的方式促進高氧暴露的AECⅡ的增殖,減輕高氧暴露對細胞增殖的抑制作用,同時VIP通過穩(wěn)定線粒體功能,減少細胞高氧性損傷及死亡,從而改善高氧暴露后AECⅡ的存活。VIP干預后高氧暴露細胞仍表現(xiàn)出胞膜破壞、胞核腫脹等脹亡的特征,提示VIP可減少高氧誘導的AECⅡ的脹亡,脹亡可能與凋亡一樣也是可調控的過程。 第三部分血管活性腸肽促進高氧損傷肺泡Ⅱ型上皮細胞再生修復的STAT3信號機制 目的研究高氧暴露及VIP干預后JAK2/STAT3的活化以及STAT3表達敲低后VIP對高氧暴露AECⅡ增殖、死亡的影響,探討VIP調控高氧損傷AECⅡ再生修復的可能信號機制。 方法Western Blot及凝膠遷移實驗(Electrophoretic Mobility Shift Assay, EMSA)檢測VIP干預前后高氧暴露的MLE-12細胞JAK2、磷酸化JAK2(p-JAK2),STAT3、磷酸化STAT3(p-STAT3)的表達以及STAT3與DNA結合活性,了解高氧損傷過程中JAK2/STAT3活化情況和VIP對JAK2/STAT3活化影響,RNAi技術敲低MLE-12細胞STAT3表達后采用Annexin V/PI雙標記流式細胞學檢測細胞死亡、MTT法檢測細胞增殖情況,了解STAT3是否介導了VIP對高氧損傷AECⅡ再生修復的調控。 結果Western Blot結果顯示,在高氧暴露后2 h,p-JAK2蛋白表達增加,4 h達峰值,高氧暴露后6 h表達有所下降,高氧暴露后12 h p-JAK2蛋白表達回復至未干預時水平,與此同時,高氧暴露24 h內的各時點JAK2總蛋白水平并無顯著變化,高氧暴露VIP 10~(-7)M組細胞與單純高氧暴露組細胞相比,24 h內的各檢測時間點JAK2總蛋白水平、JAK2發(fā)生磷酸化的時點及p-JAK2表達無顯著變化。p-STAT3蛋白表達水平在高氧暴露后2 h增加,4 h最強,6 h后有所下降,高氧暴露后12 h p-STAT3蛋白表達與未干預時水平無差異,在p-STAT3表達增強的同時,STAT3總蛋白表達在高氧暴露24 h內各時點無明顯差異,10~(-7)M VIP干預后,高氧暴露細胞STAT3總蛋白水平和STAT3磷酸化的時效性均無變化,但p-STAT3表達較單純高氧暴露組明顯增加(p0.05)。EMSA結果顯示,高氧暴露后2 h即可見STAT3 DNA結合活性增加,4 h達峰值,6 h減弱,12 h降至對照組水平,10~(-7)M VIP干預后,高氧暴露細胞中STAT3與DNA結合活性進一步增強(p0.01)。Annexin-V/PI雙標記流式細胞儀檢測結果顯示,與單純高氧暴露的未轉染組細胞比較,高氧STAT3 siRNA組早期凋亡細胞、晚期凋亡及壞死細胞顯著增多(p0.01),而高氧VIP未轉染組早期凋亡細胞及晚期凋亡和壞死細胞顯著降低(p0.01),與高氧VIP未轉染組相比,高氧VIP STAT3 siRNA組早期凋亡細胞、晚期凋亡及壞死細胞數(shù)明顯增加(p0.05)。MTT結果顯示,與高氧未轉染組相比,高氧STAT3 siRNA組OD_(490nm)值明顯降低(p0.01),高氧VIP未轉染組OD_(490nm)值顯著增高(p0.01),高氧VIP STAT3 siRNA組較之高氧VIP未轉染組,OD_(490nm)值明顯降低(p0.01)。 結論VIP可通過促進高氧誘導的STAT3的活化,減輕細胞損傷死亡,促進細胞增殖,改善細胞存活,但VIP活化STAT3的過程與經(jīng)典的JAK2/STAT3途徑激活不同,并不影響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.
【學位授予單位】：重慶醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2011
【分類號】：R363

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