黄芩苷对小鼠胚胎干细胞分化成心肌样细胞的影响的研究
发布时间：2018-12-14 23:57
【摘要】：目的:采用中药黄芩苷(baicalin)改变体外培养的小鼠胚胎干细胞(embryonic stem cell-D3)mES-D3微环境,探讨黄芩苷对mES-D3定向分化为心肌样细胞的影响。 方法:在无白血病抑制因子(leukemia inhibitory factor, LIF)存在的条件下,96孔板中体外培养mES-D3细胞8天,MTT法检测不同浓度的黄芩苷对ES细胞增殖和活性的影响,筛选合适的浓度用于分化诱导。采用经典的“悬滴-悬浮-贴壁”三步法,用三种浓度的黄芩苷诱导ES细胞向心肌样细胞分化,倒置显微镜下观察细胞形态;在不同贴壁时间点计数跳动的EB(embryoid body )个数,计算跳动EB的百分率;免疫细胞化学方法检测心肌肌小节特异性α-辅肌动蛋白(α-actinin)的表达;RT-PCR检测心肌特异性α-肌球蛋白重链(α-MHC)的表达;用流式细胞技术检测心肌细胞分化比例。 结果: MTT结果显示,1μmol/L,10μmol/L,102μmol/L,103μmol/L的黄芩苷显著抑制了mES-D3细胞的增殖,在所选浓度中10-1μmo/L的黄芩苷对细胞增殖的影响最小。通过对分化EB形态的观察,我们发现中药诱导组EB个数较空白组少,EB大小较空白组小,但是流式细胞技术结果显示1μmo/L黄芩苷组明显提高了心肌样细胞分化率。而且免疫细胞化学和RT-PCR分别检测到α-actinin和α-MHC的表达。 结论:高浓度的黄芩苷显著了抑制mES-D3细胞的增殖,1μmol/L的黄芩苷可提高mES-D3细胞向心肌样细胞的分化率,其诱导分化率高达21.45 %。
[Abstract]:Aim: to investigate the effect of baicalin (baicalin) on the differentiation of mES-D3 into cardiomyocyte-like cells by changing the mES-D3 microenvironment of mouse embryonic stem cells (embryonic stem cell-D3) in vitro. Methods: mES-D3 cells were cultured in 96 well plates without leukemia inhibitor (leukemia inhibitory factor, LIF) for 8 days. The effects of baicalin at different concentrations on the proliferation and activity of ES cells were detected by MTT assay. Screening suitable concentration for differentiation induction. The classical three-step method of "suspension adherence" was used to induce the differentiation of ES cells into cardiomyocyte-like cells with three concentrations of baicalin, and the morphology of the cells was observed under inverted microscope. The number of beating EB (embryoid body) was counted at different attachment time points, the percentage of beating EB was calculated, and the expression of 伪 -coactin (伪 -actinin) was detected by immunocytochemistry. The expression of myocardial specific 伪 -myosin heavy chain (伪-MHC) was detected by RT-PCR and the differentiation ratio of cardiomyocytes was detected by flow cytometry. Results: the results of MTT showed that baicalin of 1 渭 mol/L,10 渭 mol/L,102 渭 mol/L,103 渭 mol/L significantly inhibited the proliferation of mES-D3 cells, and baicalin of 10-1 渭 mo/L had the least effect on the proliferation of mES-D3 cells. By observing the morphology of differentiated EB, we found that the number of EB was less and the size of EB was smaller in traditional Chinese medicine induced group than in blank group, but flow cytometry showed that 1 渭 mo/L baicalin group significantly increased the differentiation rate of cardiomyocyte like cells. The expression of 伪-actinin and 伪-MHC were detected by immunocytochemistry and RT-PCR, respectively. Conclusion: baicalin at high concentration can significantly inhibit the proliferation of mES-D3 cells, and 1 渭 mol/L baicalin can increase the differentiation rate of mES-D3 cells into cardiomyocyte-like cells, and the induced differentiation rate is as high as 21.45%.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R329
本文编号：2379547
[Abstract]:Aim: to investigate the effect of baicalin (baicalin) on the differentiation of mES-D3 into cardiomyocyte-like cells by changing the mES-D3 microenvironment of mouse embryonic stem cells (embryonic stem cell-D3) in vitro. Methods: mES-D3 cells were cultured in 96 well plates without leukemia inhibitor (leukemia inhibitory factor, LIF) for 8 days. The effects of baicalin at different concentrations on the proliferation and activity of ES cells were detected by MTT assay. Screening suitable concentration for differentiation induction. The classical three-step method of "suspension adherence" was used to induce the differentiation of ES cells into cardiomyocyte-like cells with three concentrations of baicalin, and the morphology of the cells was observed under inverted microscope. The number of beating EB (embryoid body) was counted at different attachment time points, the percentage of beating EB was calculated, and the expression of 伪 -coactin (伪 -actinin) was detected by immunocytochemistry. The expression of myocardial specific 伪 -myosin heavy chain (伪-MHC) was detected by RT-PCR and the differentiation ratio of cardiomyocytes was detected by flow cytometry. Results: the results of MTT showed that baicalin of 1 渭 mol/L,10 渭 mol/L,102 渭 mol/L,103 渭 mol/L significantly inhibited the proliferation of mES-D3 cells, and baicalin of 10-1 渭 mo/L had the least effect on the proliferation of mES-D3 cells. By observing the morphology of differentiated EB, we found that the number of EB was less and the size of EB was smaller in traditional Chinese medicine induced group than in blank group, but flow cytometry showed that 1 渭 mo/L baicalin group significantly increased the differentiation rate of cardiomyocyte like cells. The expression of 伪-actinin and 伪-MHC were detected by immunocytochemistry and RT-PCR, respectively. Conclusion: baicalin at high concentration can significantly inhibit the proliferation of mES-D3 cells, and 1 渭 mol/L baicalin can increase the differentiation rate of mES-D3 cells into cardiomyocyte-like cells, and the induced differentiation rate is as high as 21.45%.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R329
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