人脐带间充质干细胞对衰老心肌细胞炎症因子表达的影响

发布时间：2018-06-13 11:51

本文选题：衰老 + H9c2心肌细胞　；参考：《青岛大学》2017年硕士论文

【摘要】：目的:通过研究心肌细胞衰老相关炎症指标的变化,探讨hUC-MSCs对H_2O_2诱导的H9c2衰老心肌细胞的保护作用及可能机制。方法:本实验用终浓度为30μmol/L,50μmol/L,100μmol/L,150μmol/L,200μmol/L的H_2O_2溶液干预H9c2心肌细胞,应用衰老相关性β-半乳糖苷酶(Senescence-associatedβ-galactosidase,Sa-β-gal)染色法检测衰老细胞比例,并应用流式细胞术检测凋亡细胞比例,以确定最适的H_2O_2浓度,构建H9c2心肌细胞衰老模型。在此基础上建立hUC-MSCs与H9c2衰老心肌细胞共培养体系,通过Sa-β-gal染色法检测衰老阳性细胞所占比例的变化,酶联免疫吸附测定法(ELISA)检测心肌细胞上清液中促炎症因子(TNF-α、IL-1β)和抗炎症因子(IL-10)的表达水平。结果:1.H_2O_2干预诱导H9c2心肌细胞衰老模型的建立。1.1β-半乳糖苷酶染色法结果显示:与Control组相比较,β-半乳糖苷酶染色阳性心肌细胞比例随着H_2O_2浓度的增大而增加,当H_2O_2浓度为30μmol/L时,β-半乳糖苷酶染色阳性细胞数增加不明显,差异无统计学意义(P0.05);当H_2O_2浓度为50μmol/L,100μmol/L,150μmol/L,200μmol/L时,β-半乳糖苷酶染色阳性细胞比例显著增加,差异具有统计学意义(P0.05)。1.2流式细胞仪检测结果显示:与Control组相比较,H_2O_2浓度的越大,心肌细胞凋亡比例越高,当H_2O_2浓度为30μmol/L,50μmol/L,100μmol/L时,凋亡的心肌细胞比例差别不大,差异无统计学意义(P0.05);当H_2O_2浓度为150μmol/L,200μmol/L时,心肌细胞凋亡比例显著增加,差异具有统计学意义(P0.05)。2.建立hUC-MSCs与H9c2衰老心肌细胞共培养体系,实验分四组:Control组、H_2O_2组、H_2O_2+hUC-MSCs培养基组、H_2O_2+hUC-MSCs组。2.1β-半乳糖苷酶染色法结果显示:与Control相比较,H_2O_2组β-半乳糖苷酶染色阳性心肌细胞比例明显增多,差异具有统计学意义(P0.05);与H_2O_2组相比较,H_2O_2+hUC-MSCs组β-半乳糖苷酶染色阳性心肌细胞比例显著降低(P0.05)。2.2 ELISA结果显示:与Control相比较,H_2O_2组心肌细胞上清液中促炎因子TNF-α、IL-1β表达水平显著增加,抗炎因子IL-10表达水平显著降低,差异具有统计学意义(P0.05);与H_2O_2组相比较,H_2O_2+hUC-MSCs组心肌细胞上清液中促炎因子TNF-α、IL-1β表达水平显著降低,抗炎因子IL-10表达水平显著增加(P0.05)。结论:当应用100μmol/L的H_2O_2诱导H9c2心肌细胞时,H9c2心肌细胞衰老模型构建成功。hUC-MSCs与H9c2衰老心肌细胞共培养体系建立后,可见hUC-MSCs共培养可以显著延缓H_2O_2所诱导的心肌细胞衰老程度;并可使心肌细胞上清液中促炎因子TNF-α、IL-1β表达水平显著降低,抗炎因子IL-10表达水平显著增加;证实了人脐带间充质干细胞对H_2O_2诱导的H9c2心肌细胞衰老性损伤具有保护作用,其机制可能与降低促炎症因子表达水平、增加抗炎因子的表达水平有关。
[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- 伪 (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- 伪 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- 伪 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- 伪 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- 伪 (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- 伪 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.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R54

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