胎盤間充質干細胞對肺臟上皮細胞抗氧化損傷的作用機制研究(碩士)

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胎盤間充質干細胞對肺臟上皮細胞抗氧化損傷的作用機制研究(碩士)(論文23000字)
摘要
目的:本課題旨在通過研究間充質干細胞(Mesenchyma stem cell, MSCs)的抗氧化能力以及其可能的作用機制。證實MSCs培養上清的抗氧化能力;構建肺臟上皮細胞氧化損傷模型并驗證模型的有效性;探究MSCs培養上清對損傷的肺臟上皮細胞的修復能力;最后建立間充質干細胞與氧化損傷的肺臟上皮細胞的共培養模型,探究MSCs對氧化損傷的肺臟上皮細胞的保護及修復能力,以及其可能的作用機制。
方法:復蘇并使用無血清培養基培養胎盤胎兒側間充質干細胞,各代次培養48h后收集其培養上清,并置于負八十度保存,以含有100 μmol/L的維生素C的空白培養基為陽性對照,檢測P2-P6代細胞的總抗氧化能力、活性氧自由基的清除能力以及抗氧化酶活性。采用不同濃度過氧化氫對肺臟上皮細胞A549細胞系進行不同時長的氧化刺激,確定最適濃度及刺激時間,建立氧化損傷模型并驗證模型的有效性。采用無血清培養基培養胎盤胎兒側間充質干細胞,收集P3代細胞培養上清將其作用于氧化損傷的肺臟上皮細胞的培養24h,即上清組;同時設立損傷組-陰性對照組(僅進行氧化損傷)和維生素C組-陽性對照組(培養基中添加100μmol/L的維生素C)。利用流式細胞術對各組細胞凋亡情況進行檢測以及Western Blot檢測凋亡相關蛋白及氧化應激經典信號通路Nrf2-Keap1-ARE信號通路相關蛋白的表達。培養肺臟上皮細胞A549細胞系,設置A、B兩個實驗組,其中實驗組A先使用上述既定條件的過氧化氫進行刺激,刺激結束后A1組不進行任何處理,A2組培養基中添加100μmol/L的維生素C,A3組插入Transwell并在上層接種胎盤胎兒側間充質干細胞;而實驗組B,B1不進行任何處理,B2組培養基中添加100μmol/L的維生素C,B3組插入Transwell并在上層接種胎盤胎兒側間充質干細胞,三組共培養結束后,再使用既定濃度的過氧化氫進行刺激;最后利用流式細胞術對六組細胞凋亡情況進行檢測以及Western Blot檢測凋亡相關蛋白及氧化應激經典信號通路Nrf2-Keap1-ARE信號通路相關蛋白的表達。
結果:各代次人胎盤胎兒側間充質干細胞培養上清具有一定的抗氧化能力,且不同代次間的上清具有一定的差異,其中總抗氧化能力與40-80 μmol/L 的維生素C相當。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)、羥自由基(OH)、超氧陰離子自由基(O2-)等自由基清除能力;各代次間的人胎盤胎兒側間充質干細胞培養上清中均可以檢測到一定水平的超氧化物歧化酶和谷胱甘肽過氧化物酶活性且不同代次間的上清具有一定的差異。采用600μmol/L的過氧化氫對肺臟上皮細胞進行24h的氧化刺激,A549細胞存活率為(56.41±3.31)%,為氧化應激發生的最適條件,Hochest33258染色及Western Blot可以證實模型的可靠性;流式細胞術結果顯示維生素C組和上清組氧化損傷細胞的凋亡率與損傷組細胞相比有不同程度的降低,其中上清組與損傷相比差異具有統計學意義(P < 0.05);Western Blot的實驗中,對凋亡相關蛋白的檢測顯示,與實驗的陰性對照,單純損傷組對比,維生素C組和上清組促凋亡基因Bax蛋白表達減弱,抑制凋亡基因Bcl-2蛋白表達增強,且差異均具有統計學意義(P<0.05)。Nrf2-Keap1-ARE信號通路相關蛋白顯示與損傷組對比,維生素C組和上清組Nrf2蛋白表達增強,Keap1分子表達減低,差異具有統計學意義(P<0.05);在共培養實驗中,實驗組A中,流式細胞術檢測A2及A3細胞存活率高于A1組,Western Blot對凋亡相關蛋白的檢測顯示A2及A3細胞促凋亡基因BAX,Caspsae3表達減弱,凋亡抑制基因Bcl-2、MCL-1蛋白表達增強,抗氧化酶HO-1表達增強:差異均具有統計學意義(P<0.05)。對抗氧化信號通路Nrf2-Keap1-ARE上相關蛋白的檢測顯示:A2及A3細胞Nrf2蛋白表達增強;Keap1分子表達減弱,差異均具有統計學意義(P<0.05);實驗組B與A組相比呈現相似結果。
結論:1.在無血清條件下培養胎兒側胎盤間充質干細胞的上清具有一定的抗氧化能力和抗氧化酶的活性。人胎盤胎兒側間充質干細胞的抗氧化能力與其旁分泌機制有關,但其中的抗氧化成分及分子機制有待進一步的研究。
2. 實驗成功構建了肺臟上皮細胞損傷模型,使用600μmol/L的過氧化氫進行24h的刺激時,A549細胞存活率為(56.41±3.31)%,為氧化應激發生的最適條件。
3.人胎盤胎兒側間充質干細胞及其培養上清具有一定的抗氧化能力,可以起到減弱氧化損傷,抑制細胞凋亡,增強抗氧化酶表達的作用,且其作用機制可能與Nrf2-Keap1-ARE信號通路的激活有關。
關鍵詞間充質干細胞;胎盤;過氧化氫;氧化應激;無血清培養;培養上清;抗氧化能力;Nrf2-Keap1-ARE信號通路
 
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

 
縮略詞與中英文對照
英文縮寫    英文全稱    中文全稱
fPMSCs    human fetal placenta mesenchymal stem cells    人胎盤胎兒側間充質干細胞
MSCs    mesenchymal stem cells    間充質干細胞
H202    hydrogen peroxide    過氧化氫
OS    oxidative stress    氧化應激
ROS    Reactive oxygen free radicals    活性氧自由基
T-AOC    Total antioxidant capacity    總抗氧化能力
DPPH    1,1-diphenyl-2-picrylhydrazyl    1,1-二苯基-2-三硝基苯肼
•OH    hydroxyl free radical    羥自由基
•O2    superoxide anion    超氧陰離子自由基
SOD    Superoxide Dismutase    超氧化物歧化酶
GSH-Px    Glutathione peroxidase,    谷胱甘肽過氧化物酶
VC    Vitamin C    維生素C
HO-1    heme oxygenase-1    血紅素加氧酶1
bcl-2    B-cell lymphoma-2    B淋巴細胞瘤-2基因
bax    BCL2-Associated X    Bcl2相關的X基因
ml    milliliter    毫升
μl    microlitre    微升
 
目錄
摘要    I
ABSTRACT    IV
縮略詞與中英文對照    VIII
目錄    IX
前言    1
一、活性氧自由基的定義及研究現狀    1
二、間充質干細胞的研究進展    1
三、Nrf2-Keap1-ARE信號通路    3
第一章 不同代次間充質干細胞培養上清抗氧化能力的檢測    6
1.材料與方法    6
2.結果    9
3.討論    12
第二章 肺臟上皮細胞氧化損傷模型的建立及驗證    14
1.材料與方法    14
2.結果    16
3.討論    16
第三章 fPMSCs培養上清對氧化損傷的肺臟上皮細胞的修復作用    18
1.材料與方法    18
2.結果    20
3.討論    21
第四章 共培養條件下fPMSCs對氧化損傷的肺臟上皮細胞的保護及修復作用    23
1.材料與方法    23
2.結果    25
3.討論    26
參考文獻(正文部分)    29
綜述 • 間充質干細胞抗氧化能力的研究進展    32
參考文獻(綜述部分)    38

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