發(fā)布時(shí)間：2018-12-14 23:57

【摘要】：目的:采用中藥黃芩苷(baicalin)改變體外培養(yǎng)的小鼠胚胎干細(xì)胞(embryonic stem cell-D3)mES-D3微環(huán)境,探討黃芩苷對mES-D3定向分化為心肌樣細(xì)胞的影響。 方法:在無白血病抑制因子(leukemia inhibitory factor, LIF)存在的條件下,96孔板中體外培養(yǎng)mES-D3細(xì)胞8天,MTT法檢測不同濃度的黃芩苷對ES細(xì)胞增殖和活性的影響,篩選合適的濃度用于分化誘導(dǎo)。采用經(jīng)典的“懸滴-懸浮-貼壁”三步法,用三種濃度的黃芩苷誘導(dǎo)ES細(xì)胞向心肌樣細(xì)胞分化,倒置顯微鏡下觀察細(xì)胞形態(tài);在不同貼壁時(shí)間點(diǎn)計(jì)數(shù)跳動的EB(embryoid body )個(gè)數(shù),計(jì)算跳動EB的百分率;免疫細(xì)胞化學(xué)方法檢測心肌肌小節(jié)特異性α-輔肌動蛋白(α-actinin)的表達(dá);RT-PCR檢測心肌特異性α-肌球蛋白重鏈(α-MHC)的表達(dá);用流式細(xì)胞技術(shù)檢測心肌細(xì)胞分化比例。 結(jié)果: MTT結(jié)果顯示,1μmol/L,10μmol/L,102μmol/L,103μmol/L的黃芩苷顯著抑制了mES-D3細(xì)胞的增殖,在所選濃度中10-1μmo/L的黃芩苷對細(xì)胞增殖的影響最小。通過對分化EB形態(tài)的觀察,我們發(fā)現(xiàn)中藥誘導(dǎo)組EB個(gè)數(shù)較空白組少,EB大小較空白組小,但是流式細(xì)胞技術(shù)結(jié)果顯示1μmo/L黃芩苷組明顯提高了心肌樣細(xì)胞分化率。而且免疫細(xì)胞化學(xué)和RT-PCR分別檢測到α-actinin和α-MHC的表達(dá)。 結(jié)論:高濃度的黃芩苷顯著了抑制mES-D3細(xì)胞的增殖,1μmol/L的黃芩苷可提高mES-D3細(xì)胞向心肌樣細(xì)胞的分化率,其誘導(dǎo)分化率高達(dá)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 偽 -coactin (偽 -actinin) was detected by immunocytochemistry. The expression of myocardial specific 偽 -myosin heavy chain (偽-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 偽-actinin and 偽-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%.
【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】：2009
【分類號】：R329

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