缓激肽对氧化应激诱导的心肌细胞衰老的影响及其机制
发布时间：2019-05-12 02:29
【摘要】：研究目的:探讨缓激肽对氧化应激诱导的心肌细胞衰老的影响及其可能的分子机制。 实验方法: 1. H9C2细胞的传代培养。H9C2细胞购置于美国模式培养集存库(American type culture collection,ATCC)。按常规培养于含10%FBS的DMEM(含有青霉素和链霉素)培养基中,将H9C2细胞放入37℃,含5%CO2的培养箱中培养。当H9C2细胞长到一定密度后进行1:3传代。实验采用4-12代细胞。 2. H_2O_2诱导H9C2细胞衰老。H9C2细胞在6孔板长到每孔大约5000个细胞时,便加入各种浓度的H_2O_2 ,诱导DNA损伤和衰老。2小时后,更换培养基,再培养 3天。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数目。用Caspase-3活性检测方法来检测H_2O_2诱导的H9C2细胞凋亡情况。确定合适的干预浓度。 3. H9C2细胞衰老的检测。用Sa-β-gal染色法检测衰老细胞数目。用Western-blot方法来检测P21在H9C2细胞中的表达。用彗星实验检测H_2O_2诱导的H9C2细胞DNA损伤情况。 4. BK对H_2O_2诱导的H9C2细胞衰老的作用及可能机制。各种浓度的BK在诱导衰老前30分钟加入。而B2受体拮抗剂HOE-140和NO合酶抑制剂硝基左旋精氨酸甲酯(Nitro levorotatory arginine methyl ester ,L-NAME)干预时间要比BK提前5分钟。然后用Sa-β-gal染色法检测衰老细胞数目。 实验结果: 1.在H_2O_2浓度为20-100 u mol/L时,衰老细胞数目增加和H_2O_2浓度呈剂量依赖性。当H_2O_2浓度大于100 u mol/L时就会导致凋亡。但是当H_2O_2浓度为30 u mol/L时并不会引起Caspase-3活性增高。因此,选择30 u mol/L作为干预浓度。 2. BK与H_2O_2诱导的衰老细胞数目呈剂量依赖性减少,但是在没有H_2O_2诱导的组中并不具有此作用。 3. Western blot检测结果显示:BK能够显著地减少P21表达水平(P0.05),表明BK能够保护H9C2细胞衰老。 4.彗星实验结果显示:H_2O_2能显著地增加olive尾含量(P0.05),即能显著增加DNA损伤。BK能显著地减少olive尾含量(P0.05),表明BK能够保护DNA免受损伤。 5.结果显示:B2受体拮抗剂HOE-140和NO合酶抑制剂L-NAME均能显著增加衰老细胞数目(P0.05),表明HOE-140和L-NAME均能拮抗BK的保护作用,认为B2受体和NO合酶途径共同参与了此作用。 结论:BK抑制氧化应激诱导的心肌细胞衰老,其机制可能是BK通过B2受体激活NO合酶途径抑制DNA损伤。
[Abstract]:Objective: To study the effect of bradykinin on the aging of myocardial cells induced by oxidative stress and its possible molecular mechanism. experimental party Method:1. The transmission of H9C2 cells Generation culture. H9C2 cells are purchased in American type culture collection, AT CC). H9C2 cells were placed in an incubator at 37 & deg; C, containing 5% CO2, in DMEM containing 10% FBS (containing penicillin and streptomycin). Medium culture. After the H9C2 cells grow to a certain density, perform 1: 3. Passage 4-12 H _ 2O _ 2-induced H9C 2. Cell senescence. H9C2 cells were cultured in 6-well plates to about 5000 cells per well, and various concentrations of H _ 2O _ 2 were added to induce DNA damage and aging. After 2 hours, the cells were replaced. a base, 3 days of further culture, and the Senescence-associated antigen-galactosidase (Sa-I-gal) staining method was used to test. Determination of the number of senescent cells. H9C induced by H _ 2O _ 2 was detected by the method of Caspase-3 activity. 2. Cell apoptosis. suitable concentration of intervention.3. H9 The detection of cell senescence of C2 cells. The number of senescent cells was detected by Western-blot. Expression in 9 C2 cells. H9C2 induced by H _ 2O _ 2 was detected by comet assay The damage of cell DNA.4. The H9C2 induced by BK against H _ 2O _ 2 The role and possible mechanism of cellular senescence. BK at various concentrations 2 receptor antagonists HOE-140 and NO synthase inhibitor nitro-L-arginine methyl ester (L-NAME) were added for 30 minutes prior to the induction of aging. Time to be 5 minutes earlier than BK. Then use Sa-1-gal staining test The results of experiment:1. The number of aging cells increased when the concentration of H _ 2O _ 2 was 20-100u mol/ L. The concentration of H _ 2O _ 2 is in a dose-dependent manner. When the concentration of H _ 2O _ 2 is greater than 1 When the concentration of H _ 2O _ 2 is 30u mol/ L, it does not And the activity of the Caspase-3 is increased. 2. The number of aging cells induced by BK and H _ 2O _ 2 decreased in a dose-dependent manner, but in the absence of H 3. The results of Western blot showed that BK was able to significantly reduce the expression level of P21 (P0.05). 5) It is shown that BK can protect the aging of H9C2 cells. Content (P0.05) can significantly increase the DNA damage. BK can significantly reduce the content of the molive tail (P The results showed that both HOE-140 and NO synthase inhibitor L-NAME could significantly increase the number of senescent cells (P0.05), indicating that both HOE-140 and L-NAME could antagonize the protection of BK. Conclusion: BK inhibits the senescence of myocardial cells induced by oxidative stress, and its mechanism can
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R363
本文编号：2475031
[Abstract]:Objective: To study the effect of bradykinin on the aging of myocardial cells induced by oxidative stress and its possible molecular mechanism. experimental party Method:1. The transmission of H9C2 cells Generation culture. H9C2 cells are purchased in American type culture collection, AT CC). H9C2 cells were placed in an incubator at 37 & deg; C, containing 5% CO2, in DMEM containing 10% FBS (containing penicillin and streptomycin). Medium culture. After the H9C2 cells grow to a certain density, perform 1: 3. Passage 4-12 H _ 2O _ 2-induced H9C 2. Cell senescence. H9C2 cells were cultured in 6-well plates to about 5000 cells per well, and various concentrations of H _ 2O _ 2 were added to induce DNA damage and aging. After 2 hours, the cells were replaced. a base, 3 days of further culture, and the Senescence-associated antigen-galactosidase (Sa-I-gal) staining method was used to test. Determination of the number of senescent cells. H9C induced by H _ 2O _ 2 was detected by the method of Caspase-3 activity. 2. Cell apoptosis. suitable concentration of intervention.3. H9 The detection of cell senescence of C2 cells. The number of senescent cells was detected by Western-blot. Expression in 9 C2 cells. H9C2 induced by H _ 2O _ 2 was detected by comet assay The damage of cell DNA.4. The H9C2 induced by BK against H _ 2O _ 2 The role and possible mechanism of cellular senescence. BK at various concentrations 2 receptor antagonists HOE-140 and NO synthase inhibitor nitro-L-arginine methyl ester (L-NAME) were added for 30 minutes prior to the induction of aging. Time to be 5 minutes earlier than BK. Then use Sa-1-gal staining test The results of experiment:1. The number of aging cells increased when the concentration of H _ 2O _ 2 was 20-100u mol/ L. The concentration of H _ 2O _ 2 is in a dose-dependent manner. When the concentration of H _ 2O _ 2 is greater than 1 When the concentration of H _ 2O _ 2 is 30u mol/ L, it does not And the activity of the Caspase-3 is increased. 2. The number of aging cells induced by BK and H _ 2O _ 2 decreased in a dose-dependent manner, but in the absence of H 3. The results of Western blot showed that BK was able to significantly reduce the expression level of P21 (P0.05). 5) It is shown that BK can protect the aging of H9C2 cells. Content (P0.05) can significantly increase the DNA damage. BK can significantly reduce the content of the molive tail (P The results showed that both HOE-140 and NO synthase inhibitor L-NAME could significantly increase the number of senescent cells (P0.05), indicating that both HOE-140 and L-NAME could antagonize the protection of BK. Conclusion: BK inhibits the senescence of myocardial cells induced by oxidative stress, and its mechanism can
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R363
【引证文献】
中国期刊全文数据库 前1条
1 黄琳;;缓激肽对氧化应激反应诱导的心肌细胞衰老影响的临床研究[J];医学综述;2015年03期
，本文编号：2475031
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