緩激肽對氧化應(yīng)激誘導(dǎo)的心肌細(xì)胞衰老的影響及其機(jī)制
發(fā)布時間：2019-05-12 02:29
【摘要】：研究目的:探討緩激肽對氧化應(yīng)激誘導(dǎo)的心肌細(xì)胞衰老的影響及其可能的分子機(jī)制。 實驗方法: 1. H9C2細(xì)胞的傳代培養(yǎng)。H9C2細(xì)胞購置于美國模式培養(yǎng)集存庫(American type culture collection,ATCC)。按常規(guī)培養(yǎng)于含10%FBS的DMEM(含有青霉素和鏈霉素)培養(yǎng)基中,將H9C2細(xì)胞放入37℃,含5%CO2的培養(yǎng)箱中培養(yǎng)。當(dāng)H9C2細(xì)胞長到一定密度后進(jìn)行1:3傳代。實驗采用4-12代細(xì)胞。 2. H_2O_2誘導(dǎo)H9C2細(xì)胞衰老。H9C2細(xì)胞在6孔板長到每孔大約5000個細(xì)胞時,便加入各種濃度的H_2O_2 ,誘導(dǎo)DNA損傷和衰老。2小時后,更換培養(yǎng)基,再培養(yǎng) 3天。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(xì)胞數(shù)目。用Caspase-3活性檢測方法來檢測H_2O_2誘導(dǎo)的H9C2細(xì)胞凋亡情況。確定合適的干預(yù)濃度。 3. H9C2細(xì)胞衰老的檢測。用Sa-β-gal染色法檢測衰老細(xì)胞數(shù)目。用Western-blot方法來檢測P21在H9C2細(xì)胞中的表達(dá)。用彗星實驗檢測H_2O_2誘導(dǎo)的H9C2細(xì)胞DNA損傷情況。 4. BK對H_2O_2誘導(dǎo)的H9C2細(xì)胞衰老的作用及可能機(jī)制。各種濃度的BK在誘導(dǎo)衰老前30分鐘加入。而B2受體拮抗劑HOE-140和NO合酶抑制劑硝基左旋精氨酸甲酯(Nitro levorotatory arginine methyl ester ,L-NAME)干預(yù)時間要比BK提前5分鐘。然后用Sa-β-gal染色法檢測衰老細(xì)胞數(shù)目。 實驗結(jié)果: 1.在H_2O_2濃度為20-100 u mol/L時,衰老細(xì)胞數(shù)目增加和H_2O_2濃度呈劑量依賴性。當(dāng)H_2O_2濃度大于100 u mol/L時就會導(dǎo)致凋亡。但是當(dāng)H_2O_2濃度為30 u mol/L時并不會引起Caspase-3活性增高。因此,選擇30 u mol/L作為干預(yù)濃度。 2. BK與H_2O_2誘導(dǎo)的衰老細(xì)胞數(shù)目呈劑量依賴性減少,但是在沒有H_2O_2誘導(dǎo)的組中并不具有此作用。 3. Western blot檢測結(jié)果顯示:BK能夠顯著地減少P21表達(dá)水平(P0.05),表明BK能夠保護(hù)H9C2細(xì)胞衰老。 4.彗星實驗結(jié)果顯示:H_2O_2能顯著地增加olive尾含量(P0.05),即能顯著增加DNA損傷。BK能顯著地減少olive尾含量(P0.05),表明BK能夠保護(hù)DNA免受損傷。 5.結(jié)果顯示:B2受體拮抗劑HOE-140和NO合酶抑制劑L-NAME均能顯著增加衰老細(xì)胞數(shù)目(P0.05),表明HOE-140和L-NAME均能拮抗BK的保護(hù)作用,認(rèn)為B2受體和NO合酶途徑共同參與了此作用。 結(jié)論:BK抑制氧化應(yīng)激誘導(dǎo)的心肌細(xì)胞衰老,其機(jī)制可能是BK通過B2受體激活NO合酶途徑抑制DNA損傷。
[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
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R363
本文編號：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
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R363
【引證文獻(xiàn)】
中國期刊全文數(shù)據(jù)庫 前1條
1 黃琳;;緩激肽對氧化應(yīng)激反應(yīng)誘導(dǎo)的心肌細(xì)胞衰老影響的臨床研究[J];醫(yī)學(xué)綜述;2015年03期
，本文編號：2475031
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