人胚胎干细胞体外诱导分化为心肌细胞的实验研究

发布时间：2018-01-26 13:19

本文关键词： 骨髓间充质干细胞胚胎干细胞心肌细胞 5-氮胞苷肝细胞生长因子细胞分化　出处：《青岛大学》2007年硕士论文　论文类型：学位论文

【摘要】： 第一部分研究目的：观察大鼠骨髓间充质干细胞(Mesenchymal stem cells MSCs)的分离和培养，，通过不同浓度的5-氮胞苷(5-aza-dC)诱导大鼠MSCs分化为心肌细胞的比较，确定合适的的诱导浓度。研究方法：抽取大鼠骨髓，采用贴壁培养法分离MSCs，进行培养、传代，在第4代MSCs中添加诱导剂5-氮胞苷(5-aza)对其进行诱导分化，其浓度分别为：0μmol／L、10μmol／L、30μmol／L，观察细胞形态学的变化，荧光免疫组织化学方法进行鉴定。研究结果： 5-aza-dC作用24h后，随着时间的延长，加入终浓度为10μmol／L 5-aza-dC的大鼠MSCs，10d后细胞之间出现连接，排列方向渐趋一致，有肌管形成，部分细胞则死亡。诱导28d后，细胞变稀，周围伸出多个长的突起，细胞形态大小不一。而加入终浓度为30μmol／L 5-aza-dC的MSCs，在诱导后第5d，细胞则全部死亡。结论：大鼠MSCs体外在5-氮胞苷作用下可定向分化为心肌样细胞。第二部分研究目的：探讨5-氮胞苷(5-aza-dC)、肝细胞生长因子(Hepatocyte growth factor HGF)作为诱导剂，体外诱导人类胚胎干细胞(Human embryonic stem cell hESC)分化为心肌细胞的最佳诱导分化浓度。研究方法： 1．将传代的hESC(PKU-1-1)接种到低粘附六孔板中进行悬浮培养形成拟胚体(Embryonic Bodies EBs)。7d后，分别将EBs接种到四孔板中贴壁培养，分别以5-aza-dC、HGF为分化诱导剂加入分化培养基中，组合成几种分化培养基，分别为无诱导物组；5-aza-dC组，其浓度分别为：2.5μmol／L、5μmol／L、10μmol／L、20μmol／L；HGF组，其浓度分别为：1ng/ml、5ng/ml、10ng/ml、30ng/ml。通过观察出现的自发搏动的EBs的数量，计算各组分化成功的EBs数。 2．光镜动态观察细胞形态学的变化，荧光免疫组织化学，RT-PCR以及电镜方法对分化细胞进行鉴定。组间比较用方差分析及t检验，以p＜0.05，为差异有显著性，比较各组之间的分化比率。研究结果： 1．5-aza-dC终浓度为0-10μmol／L时均能在体外诱导hESC向心肌细胞分化，2.5-5μmol／L为最佳诱导浓度。Troponin T及Connexin43的表达呈阳性，并可见清晰肌小节。RT-PCR检测MLC-2A、MLC-2V、hANP、β-Actin mRNA表达阳性。电镜下可见细胞内有肌节样结构。细胞分化率比较：2.5μmol／L、5μmol／L组之间无明显差异(p＞0.05)，均明显高于0μmol／L、10μmol／L(p＜0.05)，此外0μmol／L、10μmol／L组之间也没有明显差异(p＞0.05)。而20μmol／L组的细胞大部分出现死亡。 2．HGF终浓度为1ng/ml、5ng/ml、10ng／ml、30ng／ml时均能在体外诱导hESC向心肌细胞分化，Troponin T及Connexin43的表达呈阳性，并可见清晰肌小节。RT-PCR检测MLC-2A、MLC-2V、hANP、NKx2.5、GATA-4、a-MHC、β-Actin mRNA表达阳性。细胞分化率比较：5ng／ml、10ng／ml组之间无明显差异(p＞0.05)，均明显高于1ng／ml、30ng／ml组(p＜0.05)。结论： 5-aza-dC、HGF均能诱导人类胚胎干细胞向心肌细胞分化，2.5μmol／L-5μmol／L的5-aza-dC效果最理想；5ng／ml-10ng／ml的HGF为最佳浓度区间。
[Abstract]:Part one
The purpose of the study is:
Objective To observe the isolation and culture of rat bone marrow mesenchymal stem cells (Mesenchymal stem cells MSCs), and compare the induction of MSCs differentiation into cardiomyocytes by 5- azacytidine (5-aza-dC) at different concentrations, and determine the appropriate induced concentration.
Research methods:
From rat bone marrow by adherent culture method isolated MSCs were cultured and passaged, adding inducer of 5- azacytidine in the fourth generation of MSCs (5-aza) to induce differentiation, the concentrations were 0 mol / L 10 mol / L 30 mol / L, to observe the change of cell the morphology, immunohistochemistry methods were identified.
The results of the study:
The role of 5-aza-dC 24h, with the extension of time, with a final concentration of 10 mol / L 5-aza-dC rat MSCs, appeared a connection between 10d cells, orientation is consistent, a part of myotube formation, cell death induced by 28d cells. After thinning, a plurality of protrusions around the protruding length, cell morphology is not the same size and adding a final concentration of 30 mol / L 5-aza-dC MSCs in 5D after induction, cells all died.
Conclusion:
Rat MSCs can differentiate into cardiomyocytes in vitro under the action of 5- azacytidine.
The second part
The purpose of the study is:
Objective to investigate the optimal differentiation concentration of 5- 5-aza-dC, Hepatocyte growth factor HGF as an inducer to induce the differentiation of human embryonic stem cells (Human embryonic stem cell hESC) into cardiomyocytes in vitro.
Research methods:
1.. HESC (PKU-1-1) was inoculated into the culture of embryoid bodies were suspended in low adhesion plate (Embryonic Bodies EBs).7d, respectively, EBs were inoculated into four well plate adherent culture, respectively by 5-aza-dC, HGF induced differentiation agent into the differentiation medium, combined into several differentiation medium. Respectively no inducer group; group 5-aza-dC, the concentrations were 2.5 mol / L 5 mol / L 10 mol / L 20 mol / L; group HGF, the concentrations were 1ng/ml, 5ng/ml, 10ng/ml, the number of spontaneous beating 30ng/ml. by observing the EBs and calculate the number of EBs groups successfully differentiated.
2., the morphological changes of cells were observed dynamically under light microscope. The differentiated cells were identified by fluorescence immunohistochemistry, RT-PCR and electron microscopy. The difference between groups was statistically significant with P < 0.05, and the ratio of differentiation between groups was compared by T analysis.
The results of the study:
HESC can be induced to differentiation into cardiomyocytes in vitro 1.5-aza-dC final concentration of 0-10 mol / L, mol / 2.5-5 L for the expression of.Troponin T and optimal concentration of Connexin43 was positive, and clear sarcomere.RT-PCR detection of MLC-2A, MLC-2V, hANP, -Actin expression of mRNA positive cells under electron microscope. Sarcomere structure. The rate of cell differentiation: 2.5 mol / L, no significant difference between the 5 mol / L group (P > 0.05), were significantly higher than that of 0 mol / L 10 mol / L (P < 0.05), in addition to 0 mol / L, there is no obvious difference between the 10 mol / L group (P > 0.05). And 20 mol / L group most of the cells died.
The final concentration of 2.HGF is 1ng/ml, 5ng/ml, 10NG / ml, were able to induce hESC differentiation into cardiomyocytes in vitro by 30ng / ml, the expression of Troponin T and Connexin43 were positive and clear sarcomere.RT-PCR detection of MLC-2A, MLC-2V, hANP, NKx2.5, GATA-4, a-MHC, -Actin. MRNA positive expression of beta cell rate differentiation: 5ng / ml, there is no significant difference between 10NG / ml group (P > 0.05), were significantly higher than 1ng / ml, 30ng / ml group (P < 0.05).
Conclusion:
5-aza-dC and HGF can induce human embryonic stem cells to differentiate into cardiomyocytes. The 5-aza-dC effect of 2.5 mol / L-5 mol / L is the best, and 5ng / ml-10ng / ml HGF is the best concentration range.

【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R329

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