【摘要】：背景:前期研究證實人胎盤胎兒側(cè)間充質(zhì)干細胞培養(yǎng)上清具有一定的清除活性氧自由基的能力,且本身具有一定的抗氧化酶活性。目的:探究人胎盤胎兒側(cè)間充質(zhì)干細胞在無血清培養(yǎng)條件下所得上清對氧化應(yīng)激損傷的肺臟上皮細胞的保護作用及其作用機制。方法:分別使用不同濃度的過氧化氫(200,400,500,600,800μmol/L)對肺臟上皮細胞A549細胞系進行6,12,24 h的氧化刺激,通過CCK-8法對肺臟上皮細胞存活率進行檢測,得出存活率為50%時的過氧化氫濃度作為氧化損傷模型的最適條件。用Hochest33258染色及Western Blot對模型有效性進行驗證。采用無血清培養(yǎng)基培養(yǎng)胎盤胎兒側(cè)間充質(zhì)干細胞,收集P3代細胞培養(yǎng)上清將其作用于氧化損傷的肺臟上皮細胞,培養(yǎng)24 h,即上清組。同時設(shè)立損傷組(僅進行氧化損傷)和維生素C組(培養(yǎng)基中添加100μmol/L的維生素C)。利用流式細胞術(shù)對各組細胞凋亡情況進行檢測以及Western Blot檢測凋亡相關(guān)蛋白及氧化應(yīng)激經(jīng)典信號通路Nrf2-Keap1-ARE信號通路相關(guān)蛋白的表達。結(jié)果與結(jié)論:(1)CCK-8法檢測得出,采用600μmol/L的過氧化氫對肺臟上皮細胞進行24 h的氧化刺激,A549細胞存活率為(56.41±3.31)%。Hochest33258染色及Western Blot證實模型的可靠性;(2)流式細胞術(shù)結(jié)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,其中上清組與損傷組相比差異有統(tǒng)計學(xué)意義(P0.05);(3)Western Blot對凋亡相關(guān)蛋白的檢測顯示,與損傷組對比,維生素C組和上清組凋亡促進基因Bax蛋白表達減弱,凋亡抑制基因Bcl-2蛋白表達增強,且差異有統(tǒng)計學(xué)意義(P0.05)。Nrf2-Keap1-ARE信號通路相關(guān)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分子表達減弱且差異有統(tǒng)計學(xué)意義(P0.05);(4)上述結(jié)果提示,實驗成功構(gòu)建了肺臟上皮細胞損傷模型,且人胎盤胎兒側(cè)間充質(zhì)干細胞培養(yǎng)上清具有一定的抗氧化能力,能夠起到減弱氧化損傷,抑制細胞凋亡的作用,其作用機制可能與激活Nrf2-Keap1-ARE信號通路有關(guān)。
[Abstract]:Background: previous studies have confirmed that the culture supernatant of human placental fetal lateral mesenchymal stem cells has the ability of scavenging reactive oxygen free radicals and has certain antioxidant enzyme activity. Aim: to investigate the protective effect and mechanism of the supernatant of human placental fetal lateral mesenchymal stem cells in serum-free culture on lung epithelial cells injured by oxidative stress. Methods: different concentrations of hydrogen peroxide (200400500600800 渭 mol/L) were used for oxidative stimulation of lung epithelial cell line A549 for 24 h. The survival rate of lung epithelial cells was detected by CCK-8 method. The hydrogen peroxide concentration at the survival rate of 50 is the best condition for the oxidative damage model. The validity of the model was verified by Hochest33258 staining and Western Blot. The placental fetal lateral mesenchymal stem cells were cultured in serum-free medium, and the supernatants of P3 generation cells were collected and cultured on the oxidized lung epithelial cells. The supernatants were cultured for 24 hours, that is, the supernatant group. The injury group (oxidative injury only) and vitamin C group (adding 100 渭 mol/L vitamin C to the medium) were set up at the same time. Flow cytometry was used to detect apoptosis and Western Blot was used to detect the expression of apoptosis-related protein and Nrf2-Keap1-ARE signal pathway associated with oxidative stress. Results and conclusions: (1) CCK-8 method was used to detect, The survival rate of A549 cells was (56.41 鹵3.31)% after 24 h oxidative stimulation with 600 渭 mol/L hydrogen peroxide. Hochest33258 staining and Western Blot confirmed the reliability of the model. (2) flow cytometry showed oxidative damage cells in vitamin C group and supernatant group. The rate of apoptosis was decreased in different degree compared with that of the injured cells. There was significant difference between the supernatant group and the injury group (P0.05); (3) Western Blot showed that compared with the injury group, the expression of Bax protein of apoptosis promoter gene in vitamin C group and supernatant group was lower than that in the injured group, and the expression of apoptosis-promoting gene Bax protein was decreased in vitamin C group and supernatant group. The expression of apoptosis suppressor gene Bcl-2 protein was increased, and the difference was statistically significant (P0.05). The expression of Nrf2-Keap1-ARE signal pathway related protein was compared with that of the injured group. In vitamin C group and supernatant group, the expression of Nrf2 protein was increased, the expression of Keap1 molecule was decreased and the difference was statistically significant (P0.05); (4). The above results suggested that the model of lung epithelial cell injury was successfully constructed in Vitamin C group and supernatant group. The culture supernatant of human placental fetal lateral mesenchymal stem cells has a certain antioxidant capacity, which can attenuate oxidative damage and inhibit apoptosis. The mechanism may be related to the activation of Nrf2-Keap1-ARE signaling pathway.
【作者單位】： 寧夏醫(yī)科大學(xué)臨床醫(yī)學(xué)院;寧夏醫(yī)科大學(xué)總醫(yī)院人類干細胞研究所;
【基金】：國家自然科學(xué)基金資助項目(81460247) 寧夏醫(yī)科大學(xué)臨床醫(yī)學(xué)一流學(xué)科建設(shè)項目 2017年寧夏“研究生教育創(chuàng)新計劃”學(xué)位點建設(shè)項目(YXW2017014)~~
【分類號】：R563.8

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