【摘要】：目的探討可溶性Fractalkine對(duì)于H9C2心肌細(xì)胞的增殖的影響;進(jìn)一步探討可溶性Fractalkine對(duì)H9C2心肌細(xì)胞凋亡的影響及黃芪甲苷的干預(yù)作用。方法首先分別選用 Ong/ml、100ng/ml、200ng/ml、300ng/ml、400ng/ml、500ng/ml的可溶性Fractalkine刺激H9C2心肌細(xì)胞,分別于0、12、24、36、48小時(shí)采用MTT法檢測(cè)可溶性Fractalkine對(duì)心肌細(xì)胞增殖的作用;計(jì)算出可溶性Fractalkine作用的最佳時(shí)間及最佳作用劑量;然后分別采用Ong/ml(A組)、200ng/ml(B組)、200ng/ml可溶性Fractalkine+50mg/L黃芪甲苷(C組)刺激H9C2心肌細(xì)胞,通過熒光染色檢測(cè)細(xì)胞的凋亡情況;并進(jìn)一步應(yīng)用流式細(xì)胞儀測(cè)定細(xì)胞凋亡百分?jǐn)?shù),通過蛋白免疫印跡(western blotting)及實(shí)時(shí)定量聚合酶鏈?zhǔn)椒磻?yīng)(RT-qPCR)測(cè)定可溶性Fractalkine對(duì)H9C2心肌細(xì)胞凋亡的影響。計(jì)量資料采用均數(shù)±標(biāo)準(zhǔn)差((?)±s)表示,采用非配對(duì)t檢驗(yàn)或單因素方差分析,按P0.05的檢驗(yàn)水準(zhǔn),具有統(tǒng)計(jì)學(xué)意義。結(jié)果1.低劑量的可溶性的Fractalkine就能夠抑制H9C2心肌細(xì)胞的增殖;而且此抑制作用隨著可溶性Fractalkine濃度的增加及作用時(shí)間的延長而增加,各組間有統(tǒng)計(jì)學(xué)差異;得出最佳作用時(shí)間為24小時(shí),半數(shù)抑制劑量為277.5ng/ml;2.通過熒光染色發(fā)現(xiàn)可溶性Fractalkine可誘導(dǎo)心肌細(xì)胞凋亡;應(yīng)用黃芪甲苷后可見凋亡細(xì)胞減少;3.Annexin-FITC檢測(cè)細(xì)胞凋亡率,顯示A組、B組、C組凋亡率分別為4.13 ±0.07%,41.64±0.75%,34.07±0.55%;提示可溶性 Fractalkine 可誘導(dǎo) H9C2 心肌細(xì)胞凋亡(P0.001),而黃芪甲苷可以抑制這種作用(P0.01);4.Western blotting結(jié)果顯示,B組Bcl-2的表達(dá)減少,Bax的表達(dá)增加(P0.05),應(yīng)用黃芪甲苷后可抑制這種作用(P0.05);5.RT-qPCR結(jié)果顯示:B組Bcl-2mRNA的表達(dá)量明顯減少,而BaxmRNA的表達(dá)量明顯增加(P0.001),應(yīng)用黃芪甲苷后可抑制這種作用(P0.01)。結(jié)論1.可溶性Fractalkine可抑制H9C2心肌細(xì)胞增殖,此作用隨著可溶性Fractalkine濃度的增加及作用時(shí)間的延長而增大;2.可溶性Fractalkine可誘導(dǎo)H9C2心肌細(xì)胞發(fā)生凋亡;3.黃芪甲苷可減少可溶性Fractalkine誘導(dǎo)的H9C2心肌細(xì)胞凋亡。
[Abstract]:Objective to investigate the effect of soluble Fractalkine on the proliferation of H9C2 cardiomyocytes and the effect of soluble Fractalkine on apoptosis of H9C2 cardiomyocytes and the intervention of astragaloside A. Methods H9C2 cardiomyocytes were stimulated by soluble Fractalkine of Ong/ml,100ng/ml,200ng/ml,300ng/ml,400ng/ml,500ng/ml, and the effect of soluble Fractalkine on cardiomyocyte proliferation was detected by MTT assay at 0, 12, 24, 36 and 48 hours, respectively. The optimum time and dose of soluble Fractalkine were calculated. Then Ong/ml (group A), 200ng/ml (group B) and 200ng/ml soluble Fractalkine 50mg/L astragaloside A (group C) were used to stimulate H9C2 cardiomyocytes, and the apoptosis of H9C2 cardiomyocytes was detected by fluorescence staining. The percentage of apoptosis was measured by flow cytometry, and the effect of soluble Fractalkine on apoptosis of H9C2 cardiomyocytes was measured by Western imprinting (western blotting) and real-time quantitative polymerase chain reaction (RT-qPCR). The measurement data were expressed by mean 鹵standard deviation (?) 鹵s). Unmatched t test or single factor ANOVA analysis were used to show that it was statistically significant according to the test level of P05. Result 1. Low dose of soluble Fractalkine could inhibit the proliferation of H9C2 cardiomyocytes, and the inhibitory effect increased with the increase of soluble Fractalkine concentration and the prolongation of action time, and there was significant difference among the three groups. The optimum action time was 24 hours, and the half inhibitory dose was 277.5 ng 路ml ~ (- 2). It was found that soluble Fractalkine could induce cardiomyocyte apoptosis by fluorescence staining, and apoptotic cells decreased after astragalosides were used. The apoptosis rate of group A, group B and group C was 4.13 鹵0.07%, 41.64 鹵0.75% and 34.07 鹵0.55%, respectively. It is suggested that soluble Fractalkine can induce apoptosis of H9C2 cardiomyocytes (P0.001), while astragalosides can inhibit this effect (P0.01). The results of 4.Western blotting showed that the expression of Bcl-2 decreased and the expression of Bax increased in group B (P 0.05). Astragaloside A could inhibit this effect (P 0.05). The results of 5.RT-qPCR showed that the expression of Bcl-2mRNA in group B was significantly decreased, while the expression of BaxmRNA was significantly increased (P0.001). Astragaloside A could inhibit this effect (P 0.01). Conclusion 1. Soluble Fractalkine could inhibit the proliferation of H9C2 cardiomyocytes, which increased with the increase of soluble Fractalkine concentration and the prolongation of action time. Soluble Fractalkine could induce apoptosis of H9C2 cardiomyocytes. Astragaloside A can reduce the apoptosis of H9C2 cardiomyocytes induced by soluble Fractalkine.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R54

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