本文選題：衰老 + H9c2心肌細胞��； 參考：《青島大學(xué)》2017年碩士論文

【摘要】：目的:通過研究心肌細胞衰老相關(guān)炎癥指標的變化,探討hUC-MSCs對H_2O_2誘導(dǎo)的H9c2衰老心肌細胞的保護作用及可能機制。方法:本實驗用終濃度為30μmol/L,50μmol/L,100μmol/L,150μmol/L,200μmol/L的H_2O_2溶液干預(yù)H9c2心肌細胞,應(yīng)用衰老相關(guān)性β-半乳糖苷酶(Senescence-associatedβ-galactosidase,Sa-β-gal)染色法檢測衰老細胞比例,并應(yīng)用流式細胞術(shù)檢測凋亡細胞比例,以確定最適的H_2O_2濃度,構(gòu)建H9c2心肌細胞衰老模型。在此基礎(chǔ)上建立hUC-MSCs與H9c2衰老心肌細胞共培養(yǎng)體系,通過Sa-β-gal染色法檢測衰老陽性細胞所占比例的變化,酶聯(lián)免疫吸附測定法(ELISA)檢測心肌細胞上清液中促炎癥因子(TNF-α、IL-1β)和抗炎癥因子(IL-10)的表達水平。結(jié)果:1.H_2O_2干預(yù)誘導(dǎo)H9c2心肌細胞衰老模型的建立。1.1β-半乳糖苷酶染色法結(jié)果顯示:與Control組相比較,β-半乳糖苷酶染色陽性心肌細胞比例隨著H_2O_2濃度的增大而增加,當H_2O_2濃度為30μmol/L時,β-半乳糖苷酶染色陽性細胞數(shù)增加不明顯,差異無統(tǒng)計學(xué)意義(P0.05);當H_2O_2濃度為50μmol/L,100μmol/L,150μmol/L,200μmol/L時,β-半乳糖苷酶染色陽性細胞比例顯著增加,差異具有統(tǒng)計學(xué)意義(P0.05)。1.2流式細胞儀檢測結(jié)果顯示:與Control組相比較,H_2O_2濃度的越大,心肌細胞凋亡比例越高,當H_2O_2濃度為30μmol/L,50μmol/L,100μmol/L時,凋亡的心肌細胞比例差別不大,差異無統(tǒng)計學(xué)意義(P0.05);當H_2O_2濃度為150μmol/L,200μmol/L時,心肌細胞凋亡比例顯著增加,差異具有統(tǒng)計學(xué)意義(P0.05)。2.建立hUC-MSCs與H9c2衰老心肌細胞共培養(yǎng)體系,實驗分四組:Control組、H_2O_2組、H_2O_2+hUC-MSCs培養(yǎng)基組、H_2O_2+hUC-MSCs組。2.1β-半乳糖苷酶染色法結(jié)果顯示:與Control相比較,H_2O_2組β-半乳糖苷酶染色陽性心肌細胞比例明顯增多,差異具有統(tǒng)計學(xué)意義(P0.05);與H_2O_2組相比較,H_2O_2+hUC-MSCs組β-半乳糖苷酶染色陽性心肌細胞比例顯著降低(P0.05)。2.2 ELISA結(jié)果顯示:與Control相比較,H_2O_2組心肌細胞上清液中促炎因子TNF-α、IL-1β表達水平顯著增加,抗炎因子IL-10表達水平顯著降低,差異具有統(tǒng)計學(xué)意義(P0.05);與H_2O_2組相比較,H_2O_2+hUC-MSCs組心肌細胞上清液中促炎因子TNF-α、IL-1β表達水平顯著降低,抗炎因子IL-10表達水平顯著增加(P0.05)。結(jié)論:當應(yīng)用100μmol/L的H_2O_2誘導(dǎo)H9c2心肌細胞時,H9c2心肌細胞衰老模型構(gòu)建成功。hUC-MSCs與H9c2衰老心肌細胞共培養(yǎng)體系建立后,可見hUC-MSCs共培養(yǎng)可以顯著延緩H_2O_2所誘導(dǎo)的心肌細胞衰老程度;并可使心肌細胞上清液中促炎因子TNF-α、IL-1β表達水平顯著降低,抗炎因子IL-10表達水平顯著增加;證實了人臍帶間充質(zhì)干細胞對H_2O_2誘導(dǎo)的H9c2心肌細胞衰老性損傷具有保護作用,其機制可能與降低促炎癥因子表達水平、增加抗炎因子的表達水平有關(guān)。
[Abstract]:Aim: to investigate the protective effect and possible mechanism of hUC-MSCs on H _ 2O _ 2 induced cardiac myocyte senescence. Methods: 30 渭 mol 路L ~ (50) 渭 mol 路L ~ (-1) 50 渭 mol 路L ~ (-1) / L ~ (100) 渭 mol / L ~ 100 渭 mol 路L ~ (-1) H _ 2O _ 2 solution was used to interfere with H9c2 cardiomyocytes. Senescence-associated 尾 -galactosidase Sa- 尾 -galal staining was used to detect the percentage of senescence-associated 尾 -galactosidase Sa- 尾 -galactosidase, and flow cytometry was used to detect the proportion of apoptotic cells in order to determine the optimal concentration of H2O2. The aging model of H 9 c 2 cardiomyocytes was established. On this basis, the co-culture system of hUC-MSCs and H9c2 aging cardiomyocytes was established, and the percentage of aging positive cells was detected by Sa- 尾 -gal staining. Enzyme linked immunosorbent assay (Elisa) was used to detect the expression of pro-inflammatory factor TNF- 偽 (IL-1 尾) and anti-inflammatory factor (IL-10) in the supernatant of cardiomyocytes. Results 1. Compared with the control group, the proportion of 尾 -galactosidase positive myocardial cells increased with the increase of H _ 2O _ 2 concentration. When the number of positive cells for 尾 -galactosidase staining was not significantly increased at 30 渭 mol / L, there was no significant difference in the number of positive cells for 尾 -galactosidase staining. When the concentration of H2O2 was 50 渭 mol / L, 100 渭 mol 路L ~ (-1) or 100 渭 mol 路L ~ (-1) or 200 渭 mol 路L ~ (-1), the proportion of 尾 -galactosidase positive cells increased significantly. The results of flow cytometry showed that the higher the concentration of H _ 2O _ 2 was, the higher the proportion of cardiomyocyte apoptosis was. When the concentration of H _ 2O _ 2 was 30 渭 mol 路L ~ (-1) or 50 渭 mol / L ~ (100 渭 mol / L), there was no significant difference in the proportion of apoptotic cardiomyocytes. When the concentration of H2O2 was 150 渭 mol / L or 200 渭 mol / L, the ratio of cardiomyocyte apoptosis was significantly increased, and the difference was statistically significant. The co-culture system of hUC-MSCs and H9c2 senescent cardiomyocytes was established. The results showed that the proportion of 尾 -galactosidase positive cardiomyocytes in H2O-2 group was significantly higher than that in H2O2 group, and that in H2O2-hUC-MSCs culture medium group was 2.1 尾 -galactosidase staining method. The ratio of 尾 -galactosidase staining positive cardiomyocytes in the H2O-2 UC-MSCs group was significantly lower than that in the control group. The results showed that the expression of TNF- 偽 IL-1 尾 in the cardiomyocyte supernatant of the H2O-2 group was significantly higher than that in the control group, and the expression of TNF- 偽 IL-1 尾 in the cardiomyocyte supernatant of the H2O-MSCs group was significantly lower than that in the control group, the results showed that the expression of TNF- 偽 IL-1 尾 in the supernatant of cardiomyocytes in the H2O-MSCs group was significantly higher than that in the control group. The expression of anti-inflammatory factor IL-10 was significantly decreased (P 0.05), and the expression of proinflammatory factor TNF- 偽 (IL-1 尾) was significantly decreased in the myocardial supernatant of group H _ 2O _ 2 compared with that of group H _ 2O _ 2O _ 2, and the expression level of anti-inflammatory factor (IL-10) increased significantly (P _ (0.05) in the supernatant of cardiac myocytes in group H _ 2O _ 2O _ 2 compared with that in group H _ 2O _ 2O _ 2. Conclusion: when H9c2 cardiomyocytes were induced by 100 渭 mol / L H2O2, the co-culture system of hUC-MSCs and H9c2 senescent cardiomyocytes was successfully established. The results showed that the co-culture of hUC-MSCs could significantly delay the senescence of cardiac myocytes induced by H _ 2O _ 2. The expression of pro-inflammatory factor TNF- 偽 and anti-inflammatory factor IL-10 in cardiomyocyte supernatant was significantly decreased, and the expression of anti-inflammatory factor IL-10 was significantly increased, which confirmed that human umbilical cord mesenchymal stem cells had protective effect on aging injury induced by H _ 2O _ 2 in cardiac myocytes. The mechanism may be related to the decrease of proinflammatory factor expression and the increase of anti-inflammatory factor expression.
【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R54

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