胎盘间充质干细胞对肺脏上皮细胞抗氧化损伤的作用机制研究(硕士)

胎盘间充质干细胞对肺脏上皮细胞抗氧化损伤的作用机制研究(硕士)(论文23000字)
摘要
目的：本课题旨在通过研究间充质干细胞（Mesenchyma stem cell, MSCs）的抗氧化能力以及其可能的作用机制。证实MSCs培养上清的抗氧化能力；构建肺脏上皮细胞氧化损伤模型并验证模型的有效性；探究MSCs培养上清对损伤的肺脏上皮细胞的修复能力；最后建立间充质干细胞与氧化损伤的肺脏上皮细胞的共培养模型，探究MSCs对氧化损伤的肺脏上皮细胞的保护及修复能力，以及其可能的作用机制。
方法：复苏并使用无血清培养基培养胎盘胎儿侧间充质干细胞，各代次培养48h后收集其培养上清，并置于负八十度保存，以含有100 μmol/L的维生素C的空白培养基为阳性对照，检测P2-P6代细胞的总抗氧化能力、活性氧自由基的清除能力以及抗氧化酶活性。采用不同浓度过氧化氢对肺脏上皮细胞A549细胞系进行不同时长的氧化刺激，确定最适浓度及刺激时间，建立氧化损伤模型并验证模型的有效性。采用无血清培养基培养胎盘胎儿侧间充质干细胞，收集P3代细胞培养上清将其作用于氧化损伤的肺脏上皮细胞的培养24h，即上清组；同时设立损伤组-阴性对照组(仅进行氧化损伤)和维生素C组-阳性对照组(培养基中添加100μmol/L的维生素C)。利用流式细胞术对各组细胞凋亡情况进行检测以及Western Blot检测凋亡相关蛋白及氧化应激经典信号通路Nrf2-Keap1-ARE信号通路相关蛋白的表达。培养肺脏上皮细胞A549细胞系，设置A、B两个实验组，其中实验组A先使用上述既定条件的过氧化氢进行刺激，刺激结束后A1组不进行任何处理，A2组培养基中添加100μmol/L的维生素C，A3组插入Transwell并在上层接种胎盘胎儿侧间充质干细胞；而实验组B，B1不进行任何处理，B2组培养基中添加100μmol/L的维生素C，B3组插入Transwell并在上层接种胎盘胎儿侧间充质干细胞，三组共培养结束后，再使用既定浓度的过氧化氢进行刺激；最后利用流式细胞术对六组细胞凋亡情况进行检测以及Western Blot检测凋亡相关蛋白及氧化应激经典信号通路Nrf2-Keap1-ARE信号通路相关蛋白的表达。
结果：各代次人胎盘胎儿侧间充质干细胞培养上清具有一定的抗氧化能力，且不同代次间的上清具有一定的差异，其中总抗氧化能力与40-80 μmol/L 的维生素C相当。各代次上清均具有一定的清除1,1-二苯基-2-苦肼基自由基（DPPH）、羟自由基（OH）、超氧阴离子自由基（O2-）等自由基清除能力；各代次间的人胎盘胎儿侧间充质干细胞培养上清中均可以检测到一定水平的超氧化物歧化酶和谷胱甘肽过氧化物酶活性且不同代次间的上清具有一定的差异。采用600μmol/L的过氧化氢对肺脏上皮细胞进行24h的氧化刺激，A549细胞存活率为(56.41±3.31)%，为氧化应激发生的最适条件，Hochest33258染色及Western Blot可以证实模型的可靠性；流式细胞术结果显示维生素C组和上清组氧化损伤细胞的凋亡率与损伤组细胞相比有不同程度的降低，其中上清组与损伤相比差异具有统计学意义(P < 0.05)；Western Blot的实验中，对凋亡相关蛋白的检测显示，与实验的阴性对照，单纯损伤组对比，维生素C组和上清组促凋亡基因Bax蛋白表达减弱，抑制凋亡基因Bcl-2蛋白表达增强，且差异均具有统计学意义(P<0.05)。Nrf2-Keap1-ARE信号通路相关蛋白显示与损伤组对比，维生素C组和上清组Nrf2蛋白表达增强，Keap1分子表达减低，差异具有统计学意义(P<0.05)；在共培养实验中，实验组A中，流式细胞术检测A2及A3细胞存活率高于A1组，Western Blot对凋亡相关蛋白的检测显示A2及A3细胞促凋亡基因BAX，Caspsae3表达减弱，凋亡抑制基因Bcl-2、MCL-1蛋白表达增强，抗氧化酶HO-1表达增强：差异均具有统计学意义(P<0.05)。对抗氧化信号通路Nrf2-Keap1-ARE上相关蛋白的检测显示：A2及A3细胞Nrf2蛋白表达增强；Keap1分子表达减弱，差异均具有统计学意义(P<0.05)；实验组B与A组相比呈现相似结果。
结论：1.在无血清条件下培养胎儿侧胎盘间充质干细胞的上清具有一定的抗氧化能力和抗氧化酶的活性。人胎盘胎儿侧间充质干细胞的抗氧化能力与其旁分泌机制有关，但其中的抗氧化成分及分子机制有待进一步的研究。
2. 实验成功构建了肺脏上皮细胞损伤模型,使用600μmol/L的过氧化氢进行24h的刺激时，A549细胞存活率为(56.41±3.31)%，为氧化应激发生的最适条件。
3.人胎盘胎儿侧间充质干细胞及其培养上清具有一定的抗氧化能力，可以起到减弱氧化损伤，抑制细胞凋亡，增强抗氧化酶表达的作用，且其作用机制可能与Nrf2-Keap1-ARE信号通路的激活有关。
关键词间充质干细胞；胎盘；过氧化氢；氧化应激；无血清培养；培养上清；抗氧化能力；Nrf2-Keap1-ARE信号通路
 
Mechanism of placental mesenchymal stem cells on anti-oxidative damage of lung epithelial cells
ABSTRACT
Objective: The aim of this study was to explore the antioxidant capacity of Mesenchymal stem cell (MSCs) and its possible mechanism. To confirm the antioxidant capacity of MSCs culture supernatant; to construct an oxidative damage model of lung epithelial cells and to verify the effectiveness of the model; to explore the ability of the MSCs culture supernatant to repair the injured lung epithelial cells. Finally, to establish a co culture model of mesenchymal stem cells and oxidative damaged lung epithelial cells, explore the protective and repair ability of MSCs on oxidative damaged lung epithelial cells and its possible mechanism.
Methods: fetal placenta derived mesenchymal stem cells were cultured in serum-free medium and cultured for 48h after each generation. The culture supernatants were collected and stored at eighty degrees below zero.The total antioxidant capacity, scavenging capacity and antioxidant enzyme activity of P2-P6 generation cells were detected with a blank medium containing 100 μmol/L as a positive control.Different concentrations of hydrogen peroxide were used to stimulate the lung epithelial -A549 cell line at different time. The optimal concentration and stimulation time were determined, and the oxidative damage model was established, and the validity of the model was verified. Using serum-free culture medium placenta fetal mesenchymal stem cells, cell culture supernatant from the P3 generation of 24 h culture and its role in the oxidative damage of lung epithelial cells, the supernatant group; at the same time - injury group, negative control group (only the establishment of oxidative damage) and vitamin C group positive control group (culture add 100 mol/L medium vitamin C).Flow cytometry was used to detect apoptosis in each group. Western Blot was used to detect apoptosis related proteins and expression of Nrf2-Keap1-ARE signaling pathway related proteins in oxidative stress classical signaling pathway.The first training of lung epithelial cell line A549, set up two experimental groups of A and B, the experimental group A first set, using the established conditions of hydrogen peroxide was stimulated after stimulation of A1 group without any treatment, group A2 was added to the culture medium with 100 mol/L of vitamin C, A3 and Transwell was inserted in the upper inoculation of placenta fetal mesenchymal stem cells;In the experimental group B ,B1 did not take any treatment. Group B2 was added with 100 mol/L of vitamin C, B3 group was inserted into Transwell and placenta fetal mesenchymal stem cells were inoculated at the top. After three groups were co cultured, they were stimulated by hydrogen peroxide. Finally, the apoptosis of the six groups was detected by flow cytometry, and Western Blot was used to detect the expression of apoptosis related proteins and Nrf2-Keap1-ARE signaling pathway related proteins.
Results: the culture supernatants of human placental fetal mesenchymal stem cells had certain antioxidant capacity, and the supernatants of different generations had certain differences. The total antioxidant capacity was equivalent to 40-80 C vitamin mol/L.Each generation of supernatant have certain clearance of two diphenyl picryl hydrazyl free radical (DPPH), hydroxyl radical (OH), superoxide anion radical (O2-) and free radical scavenging ability; each generation between the human placenta fetal mesenchymal stem cell culture supernatants were detected in a certain level superoxide dismutase and glutathione peroxidase activity and different passges supernatant has certain difference.Oxidative stress of lung epithelial cells was stimulated by 600 μmol / L hydrogen peroxide for 24 h. The survival rate of A549 cells was (56.41 ± 3.31)%, which was the optimal condition for oxidative stress. Hochest33258 staining and Western Blot showed that the results of flow cytometry showed that the apoptotic rates of oxidative damage cells in vitamin C group and supernatant group decreased to some extent compared with those in injured group, and the difference between the supernatant group and the injury group was statistically significant (P <0.05). In Western Blot, the detection of apoptosis-related proteins showed that compared with the negative control group and the simple injury group, the expression of Bax protein and the apoptosis-inhibiting gene in thevitamin C group and the supernatant group were weakened Bcl-2 protein expression increased, and the differences were statistically significant (P <0.05).The protein expression of Nrf2-Keap1-ARE signaling pathway showed that compared with the injury group, the expression of Nrf2 protein and Keap1 protein in the vitamin C group and the supernatant group were significantly decreased (P <0.05). In the co-culture experiment, In group A, the survival rate of A2 and A3 cells detected by flow cytometry was higher than that of A1 group. The detection of apoptosis-related proteins by Western Blot showed that the expression of apoptosis-promoting genes BAX and Caspsae3 decreased in A2 and A3 cells, 2, MCL-1 protein expression increased, the expression of antioxidant enzyme HO-1 increased: the difference was statistically significant (P <0.05). Detection of related proteins in the antioxidant signaling pathway Nrf2-Keap1-ARE showed that the expression of Nrf2 protein was enhanced in A2 and A3 cells, and the expression of Keap1 was decreased, the differences were statistically significant (P <0.05). Compared with group A, experimental group B showing the similar results.
Conclusion:1. The supernatant of fetal lateral placental mesenchymal stem cells under serum free conditions has a certain antioxidant capacity and antioxidant enzyme activity. The antioxidant capacity of human placenta derived mesenchymal stem cells is related to its paracrine mechanism, but the antioxidant components and molecular mechanisms need further study.
2.We successfully constructed a lung epithelial cell injury model. When using 600 mol/L of hydrogen peroxide to stimulate 24h, the survival rate of A549 cells was (56.41 + 3.31)%, which is the most suitable condition for oxidative stress.
3. Human placenta fetal mesenchymal stem cells and its culture supernatant has certain antioxidant capacity, can weakenor oxidative damage, inhibition of apoptosis, enhance the expression of anti-oxidative enzymes, and its mechanism may be related to the activation of Nrf2-Keap1-ARE signaling pathway related.
KEYWORDS:Mesenchymal stem cells; placenta; hydrogen peroxide; oxidative stress; serum-free culture; culture supernatant; antioxidant capacity; Nrf2-Keap1-ARE signaling pathway

 
缩略词与中英文对照
英文缩写    英文全称    中文全称
fPMSCs    human fetal placenta mesenchymal stem cells    人胎盘胎儿侧间充质干细胞
MSCs    mesenchymal stem cells    间充质干细胞
H202    hydrogen peroxide    过氧化氢
OS    oxidative stress    氧化应激
ROS    Reactive oxygen free radicals    活性氧自由基
T-AOC    Total antioxidant capacity    总抗氧化能力
DPPH    1,1-diphenyl-2-picrylhydrazyl    1,1-二苯基-2-三硝基苯肼
•OH    hydroxyl free radical    羟自由基
•O2    superoxide anion    超氧阴离子自由基
SOD    Superoxide Dismutase    超氧化物歧化酶
GSH-Px    Glutathione peroxidase，    谷胱甘肽过氧化物酶
VC    Vitamin C    维生素C
HO-1    heme oxygenase-1    血红素加氧酶1
bcl-2    B-cell lymphoma-2    B淋巴细胞瘤-2基因
bax    BCL2-Associated X    Bcl2相关的X基因
ml    milliliter    毫升
μl    microlitre    微升
 
目录
摘要    I
ABSTRACT    IV
缩略词与中英文对照    VIII
目录    IX
前言    1
一、活性氧自由基的定义及研究现状    1
二、间充质干细胞的研究进展    1
三、Nrf2-Keap1-ARE信号通路    3
第一章 不同代次间充质干细胞培养上清抗氧化能力的检测    6
1.材料与方法    6
2.结果    9
3.讨论    12
第二章 肺脏上皮细胞氧化损伤模型的建立及验证    14
1.材料与方法    14
2.结果    16
3.讨论    16
第三章 fPMSCs培养上清对氧化损伤的肺脏上皮细胞的修复作用    18
1.材料与方法    18
2.结果    20
3.讨论    21
第四章 共培养条件下fPMSCs对氧化损伤的肺脏上皮细胞的保护及修复作用    23
1.材料与方法    23
2.结果    25
3.讨论    26
参考文献（正文部分）    29
综述 • 间充质干细胞抗氧化能力的研究进展    32
参考文献（综述部分）    38