本文關鍵詞：山萘酚和芒柄花黃素對缺氧條件下H9c2心肌細胞內活性氧水平的影響及其機制研究　出處：《北京中醫(yī)藥大學》2017年碩士論文　論文類型：學位論文

  更多相關文章： 活性氧 芒柄花黃素 NAPDH氧化酶 山萘酚

【摘要】：心力衰竭是多種心血管疾病的終末階段,發(fā)病率高,死亡率高,預后差,成為世界醫(yī)學難題。氧化應激是心衰的主要病理環(huán)節(jié)。前期研究發(fā)現(xiàn),芪參顆粒能夠有效的抗氧化應激,調控NADPH氧化酶活性,從而改善心功能。山萘酚和芒柄花黃素是其調控NADPH氧化酶、抗氧化應激的潛在效應成分。研究目的從氧化應激入手,采用體外培養(yǎng)的H9c2心肌細胞系,揭示山萘酚和芒柄花黃素改善心功能、降低活性氧水平的主要作用環(huán)節(jié)。為研發(fā)新的、可用于臨床心力衰竭防治的、低毒的、高效的NADPH氧化酶抑制劑提供可用的實驗依據(jù)。研究方法與內容1MTT法檢測細胞活性:體外培養(yǎng)H9c2心肌細胞,以細胞密度1×105個/ml接種于細胞培養(yǎng)板中,適應性培養(yǎng)至孔底80%-90%時,通過加入不同濃度梯度(10-4-10-8M)的山萘酚和芒柄花黃素,比較對細胞生長活性的影響;同樣的方法比較同時給藥和預給藥的不同。細胞缺氧造模,即正常組不變,其余組均轉入Earle's平衡鹽溶液中,并放入缺氧小室中培養(yǎng),確定最佳造模時間,確定山萘酚和芒柄花黃素的有效作用濃度。2 DCFH-DA法觀察活性氧水平:選取缺氧造模的H9c2細胞,用DCFH-DA進行染色。倒置熒光顯微鏡隨機選取5個不重復區(qū)域進行拍片記錄。比較各實驗組之間的差別。3流式細胞儀檢測細胞內活性氧水平:選取缺氧造模的H9c2細胞,用0.05%胰蛋白酶消化細胞,10μM DCFH-DA重懸細胞進行染色。清洗后,用200μl PBS緩沖液重懸細胞,200目-400目尼龍網過濾,上機檢測。4 Real time PCR 檢測 NADPH(nicotinamide adenine dinucleotide phosphate)氧化酶相關亞基Nox4、p67phox、p47phox、p22phox的mRNA變化:收取造模完成的H9c2細胞,Trizol裂解,提取RNA進行反轉錄,得到cDNA,實時熒光定量PCR儀上檢測基因含量。5Western blot檢測NADPH氧化酶相關亞基Nox4、p67phox、p47phox、p22phox的蛋白表達量的變化:RIPA裂解液裂解并用細胞刮刀收取造模完成的H9c2細胞,超聲裂解提取蛋白,進行電泳、電轉、顯色等操作,對蛋白條帶進行拍照分析。研究結果1MTT實驗結果顯示:與正常組相比,給予山萘酚和芒柄花黃素10-5M及以上的濃度對細胞的生長有抑制作用(P0.01);預給藥24h的細胞生長狀態(tài)優(yōu)于同時給藥組,二組細胞的生長活性之間有統(tǒng)計學差異(P0.05);缺氧造模隨著時間的增長,細胞生長狀態(tài)受到抑制,正常組與缺氧造模8h、12h、24h及其各組之間的都有顯著的統(tǒng)計學差異(P0.01);相同的缺氧造模時間,不同給藥濃度作用于細胞,確定山萘酚和芒柄花黃素濃度在10-6M對細胞的生長狀態(tài)最有益。2DCFH-DA法觀察活性氧水平結果顯示:H9c2細胞經缺氧造模8h后,倒置熒光顯微鏡下觀察到模型組細胞內有高強度綠色熒光,胞質內部黑顆粒增多,胞體四周卷曲變亮,細胞狀態(tài)較正常組有明顯改變,細胞數(shù)量明顯減少;經山萘酚和芒柄花黃素預給藥處理的細胞與模型組相比有明顯好轉,由此可見山萘酚和芒柄花黃素對心肌細胞有一定的保護作用,能夠改善H9c2心肌細胞的生長狀態(tài),增加細胞存活率和貼壁程度。3流式細胞儀結果顯示:與正常組相比,模型組熒光強度顯著增強,有顯著的統(tǒng)計學差異(P0.01)。與模型組相比,山萘酚組、芒柄花黃素組、陽性藥夾竹桃組熒光強度顯著降低(P0.01),說明山萘酚、芒柄花黃素能降低細胞內活性氧水平。4 Real time PCR結果顯示:H9c2細胞經缺氧造模4h,與正常組相比,模型組的NOX4、p67phox、p47phoxmRNA 顯著增加(P0.01),經山萘酚干預后,NOX4、p67phox的mRNA的量較模型組分別下降了 33%(P0.05)、70%(P0.01),經芒柄花黃素干預后,NOX4、p67phox的mRNA的量較模型組分別下降了 13%(P0.05)、69%(P0.01),說明山萘酚和芒柄花黃素能夠下調NOX4、p67phox的mRNA的表達量。另外一個時間點,經缺氧造模8h,與正常組相比,模型組的NOX4、p67phox、p47phoxmRNA 顯著增加(P0.01),經山萘酚干預后,NOX4、p67phox、p47phox 的mRNA 的量較模型組分別下降了 80%(P0.01)、53%(P0.01)、45%(P0.05),經芒柄花黃素干預后,NOX4、p67phox、p47phox的mRNA的量較模型組分別下降了 80%(P0.01)、18%(P0.05)、55%(P0.05),說明山萘酚和芒柄花黃素能夠下調 NOX4、p67phox、p47phox 的 mRNA 的表達量。5 Western blot結果顯示:經缺氧造模4h,與正常組相比,模型組的p67phox、p47phox蛋白表達量顯著增加(P0.01),經山萘酚干預后,NOX4、p67phox、p47phox的蛋白量較模型組分別下降了 21%(P0.05)、19%(P0.05)、26%(P0.05),經芒柄花黃素干預后,NOX4、p67phox的蛋白量較模型組分別下降了 9%(P0.05)、55%(P0.01),說明山萘酚和芒柄花黃素能夠下調NOX4、p67phox的蛋白表達量。另一個時間點,經缺氧造模8h,與正常組相比,模型組的NOX4、p67phox、p47phox蛋白表達量顯著增加(P0.01),經山萘酚干預后,NOX4、p67phox、p47phox的蛋白量較模型組分別下降了 32%(P0.05)、22%(P0.05)、30%(P0.05),經芒柄花黃素干預后,NOX4、p67phox、p47phox的蛋白量較模型組分別下降了 35%(P0.01)、65%(P0.01)、24%(P0.05),說明山萘酚和芒柄花黃素能夠下調NOX4、p67phox、p47phox的蛋白表達量。結論經缺氧誘導的H9c2心肌細胞細胞活力下降,細胞內活性氧水平顯著升高,缺氧8h時表現(xiàn)最明顯。山萘酚、芒柄花黃素濃度為10-6M,預給藥24h處理,可明顯改善細胞的生長和增殖狀態(tài),可以明顯降低細胞內活性氧水平;山萘酚可以下調Nox4、p67phox、p47phox的mRNA和蛋白水平,芒柄花黃素能夠下調p67phox、p47phox的mRNA,明顯下調Nox4、p67phox蛋白的水平。結果提示山萘酚和芒柄花黃素可降低細胞內活性氧水平,抗氧化應激,保護心肌細胞,其作用機制與下調Nox4、p67phox、p47phox的表達水平相關。本研究為臨床調控氧化應激治療心力衰竭提供了實驗依據(jù)和新的思路。
[Abstract]:Heart failure is the end stage of many cardiovascular diseases, high incidence, high mortality and poor prognosis, become the world medical problem. Oxidative stress is the main pathological process of heart failure. The preliminary study found that Shenqi granule can effectively regulate oxidative stress, NADPH oxidase activity, so as to improve the cardiac function of kaempferol and Ononis. The regulation of NADPH oxidase flavin, potential effects of oxidative stress. Objective to study components from oxidative stress by H9c2 of myocardial cells were cultured in vitro, revealing the kaempferol and formononetin improve heart function, the main role low level of reactive oxygen species. For the development of new, can be used for clinical prevention and treatment of heart failure. Low toxicity, and provide experimental evidence available NADPH oxidase inhibitor. Efficient cell activity detection research method and content 1MTT method: H9c2 myocardial cells were cultured in vitro, the cell density of 1 * 105 / Ml cells seeded in culture plate, adaptability cultivation to the bottom of hole 80%-90%, by adding different concentration gradient (10-4-10-8M) of kaempferol and formononetin, compare the effects on cell growth activity; the same method is also administered and pre administration. Different cell hypoxia model, normal group constant, the other groups were transferred into Earle's balanced salt solution, and placed in the hypoxic chamber culture, determine the best modeling time, determine the level of reactive oxygen species observed effective concentrations of.2 DCFH-DA kaempferol and formononetin: H9c2 cells from hypoxia model, using DCFH-DA staining. Fluorescence microscope randomly selected 5 do not repeat regional shooting record. The comparison between the experimental group and the difference of.3 flow cytometry was used to detect intracellular ROS levels: select hypoxia model of H9c2 cells with 0.05% trypsin digestion cells, 10 M DCFH-DA heavy suspension The cells were stained. After cleaning, the cells with 200 L PBS buffer suspension, 200 -400 mesh nylon net filter, the detection of.4 Real time (nicotinamide adenine dinucleotide NADPH PCR detection phosphate) oxidase related subunit Nox4, p67phox, p47phox, mRNA, p22phox changes: charge modeling of H9c2 cells, Trizol cracking, the extraction of RNA by reverse transcription, cDNA, real time fluorescent quantitative PCR detection blot detection.5Western gene content related NADPH oxidase subunit Nox4, p67phox, p47phox, the changes of the expression of p22phox protein: RIPA lysate and cell lysis for modeling the scraper H9c2 cells, sonication extraction protein. By electrophoresis, electroporation, color and other operations, with a picture of protein analysis. The results showed that 1MTT test results: compared with normal group, kaempferol and formononetin was given yellow 10-5M and above concentration on Cell growth inhibition (P0.01); pre administration of 24h cell growth state is superior to the drug group at the same time, there were significant differences between the growth activity of cells in two groups (P0.05); hypoxia model with time increasing, the cell growth was inhibited, the normal group and the hypoxia model of 8h, 12h, 24h and each group had statistically significant difference (P0.01); the same time hypoxia model, different concentration on the cell, kaempferol and formononetin in concentrations of 10-6M on cell growth was most beneficial results.2DCFH-DA method to observe the level of reactive oxygen species determined: H9c2 cells were treated with hypoxia model after 8h. Cells were observed under inverted fluorescence microscope in the model group with high intensity of green fluorescence, cytoplasmic internal black particles increased, the cell body curled around the light, cells compared with normal group changed obviously, the number of cells was significantly reduced; the kaempferol and Ononis Emodin pretreatment treated cells compared with the model group has significantly improved, thus kaempferol and formononetin has protective effects on myocardial cells, the growth state of H9c2 can improve the myocardial cells, increase cell viability and attachment of.3 flow cytometry results showed: compared with normal group, model the fluorescence intensity of group increased significantly, there was statistically significant difference (P0.01). Compared with the model group, kaempferol group, formononetin group, positive drug group significantly reduced oleander fluorescence intensity (P0.01), shows that kaempferol, Ononis Huang Suneng reduced the level of ROS were.4 Real time PCR showed that H9c2 cells were treated with hypoxia model of 4h, compared with the normal group, model group, NOX4, p67phox, p47phoxmRNA increased significantly (P0.01), by kaempferol intervention, NOX4, p67phox mRNA were compared with the model group decreased by 33% (P0.05), 70% (P0.01), by Ononis 鑺遍粍绱犲共棰勫悗,NOX4,p67phox鐨刴RNA鐨勯噺杈冩ā鍨嬬粍鍒嗗埆涓嬮檷浜，

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