催产素联合乳鼠心肌细胞条件培养液诱导胚胎干细胞分化的作用
发布时间：2018-10-17 11:58
【摘要】： 实验背景和研究目的 背景 心力衰竭是全世界范围内发生率最高的心脏疾病,其主要特征为正常心肌细胞的死亡而被无生理功能的纤维瘢痕组织所替代。因为成体心肌细胞几乎无法再生,所以目前最有效的治疗方案就是进行心脏移植。近10年的体外及体内的研究表明:对于晚期的心力衰竭进行干细胞移植治疗或许是另外一种有效的治疗方案。胚胎干细胞(embryonic stem cells,ESCs)具有无限增殖能力及多向分化潜能,在体内及体外环境中都可以分化成包括心肌细胞在内的各种类型的细胞。如何更加高效的诱导ESCs定向分化为心肌细胞引起了研究者们的极大兴趣。 目的 本研究旨在优化胚胎干细胞的扩增、培养方法;观察小鼠ESCs(CGR8系)能否在体外分化成具有功能性的心肌细胞;比较体外多种因素诱导ESCs的定向分化的效率,探讨一种安全有效的途径用于体外诱导ESCs向心肌细胞分化。 方法和结果 取乳鼠心肌组织按差速贴壁分离法,纯化乳鼠的心肌细胞,收集心肌细胞培养液,以1 000 r/min离心5 min。取上清,与完全培养基按1∶1的比例混合制备乳鼠心肌细胞条件培养液。复苏本实验室冻存的ESCs并扩增培养。经三步法形成拟胚体(embryoid bodies,EBs):(1)悬滴培养2d;(2)悬浮培养5d;(3)贴壁培养:5d。按培养基添加的诱导剂的不同分为随机等分四组:乳鼠CMs条件培养液诱导组;催产素(oxytocin,OT)诱导组;混合添加诱导组(乳鼠CMs条件培养液加OT);对照组(无任何添加)。 观察ESCs分化后的搏动性发现试验各组8d后肉眼可见EBs上出现节律性自发收缩的区域,随着培养时间的延长,逐渐整个EBs出现一致性搏动,但对照组未观察到此现象。混合添加诱导组细胞的搏动率分别和OT诱导组和乳鼠CMs条件培养液诱导组比较差异均显著(P0.01)。随着心肌细胞的发育成熟,细胞的搏动频率也会逐渐下降。通过免疫组化染色观察发现混合添加诱导组分化成CMs的效率明显高于另外两组。分别于各组跳动的细胞团使用下列药物:异丙肾上腺、乙酰胆碱和维拉帕米。结果ESCs经OT联合CMs的条件培养液诱导后,在分化的早期对药物的反应性均显著高于其他实验组;而分化晚期因各组CMs均逐渐发育成熟,三组细胞对于药物的反应性均有所增强。提示CMs的条件培养液可能有促进上述3种受体成熟的作用。接着通过透射电镜观察到三组ESCs分化的CMs出现了类似原代CMs的肌原纤维、肌节、Z线及缝隙连接,桥粒连接结构。细胞基因表达的测定过程中发现,作为ESCs标志的OCT-4仅仅在细胞分化的早期表达。心肌特异性因子cTnT及ANF在三组细胞分化的全程均有表达,但早期cTnT的表达量混合添加诱导组显著高于乳鼠CMs条件培养液诱导组及OT诱导组(P0.05);分化中、晚期心肌特异性因子的表达量无明显差异。这些都证实了乳鼠CMs条件培养液添加OT后诱导ESCs成CMs的高效性。 结论 小鼠ESCs(CGR8系)在体外的培养过程中可以被诱导分化成心肌细胞;ESCs诱导分化成的心肌细胞具有收缩功能、具有药理反应性并且可以表达心肌特异性结构蛋白;与以前的研究结果相同,悬滴、悬浮、贴壁三步培养法是一种可靠的培养ESCs成EBs的方法;ESCs诱导而成的CMs随着细胞发育的成熟,细胞的搏动频率也会逐渐下降,这个可能与成熟心肌细胞内较少含有窦房结细胞有关;在体外环境中OT、乳鼠CMs条件培养液、OT添加乳鼠CMs的条件培养液均具有诱导ESCs分化为CMs的作用,且后者的诱导更具有高效性和安全性。
[Abstract]:Experimental Background and Study Purpose Background Heart Failure is the highest incidence of heart disease worldwide, characterized by the absence of physiological functions for the death of normal cardiomyocytes The fibrous tissue is replaced. Because the body cardiomyocytes are almost impossible to regenerate, the most effective healers are currently The case is a heart transplant. In vitro and in vivo studies in nearly 10 years suggest stem cell transplantation for late heart failure may be another Embryonic stem cells (ESCs) have unlimited proliferation ability and multi-directional differentiation potential, and can differentiate into cardiomyocytes in vivo and in vitro. Various types of cells in the cells. How to induce ESCs to differentiate into cardiomyocytes more efficiently? Study The aim of this study was to optimize the amplification and culture of embryonic stem cells, and to observe whether mouse ESCs (CGR8 system) could differentiate into functional cardiac myocytes in vitro; External factors induce the efficiency of directional differentiation of ESCs and explore a safe and effective method pathway for body The method and the results of the method and the results of the method and the results of the method are as follows: the myocardial tissue of the milk mouse is separated by the differential adherence method to purify the heart muscle of the milk mouse; Cells were collected and cultured for 5 min at 1,000 r/ min. and mixing with the complete culture medium according to the ratio of 1: 1 to prepare the milk The rat cardiomyocytes conditioned medium were cultured. ESCs frozen in this laboratory were recovered and cultured. 1) suspension culture 2d; (2) suspension culture 5d; (3) adherent culture: 5d. The different groups of inducers added according to culture medium were randomly divided into four groups: milk rat CMs conditioned medium induction group; oxytocin (OT) induction group; mixed addition Induction group (CMMs conditioned medium with OT); control group (without any addition). Observation of the beat after ESCs differentiation showed that there was a rhythmic spontaneous contraction in the EBs after eight days of test. There was a consistent beat of the whole EBs, but this phenomenon was not observed in the control group. The beat rate of the mixed addition-induced group cells was Compared with OT-induced group and CMs condition culture medium-induced group, the difference was significant. (P 0.01). With the development of cardiac myocytes, the frequency of beating of cells gradually decreased. The results showed that the efficiency of the mixed addition induced component formation was significantly higher than that in the other two groups. Results ESCs were significantly higher in the early stage of differentiation than in other experimental groups. Due to the mature development of CMs in each group, three groups of cells respond to the drug. The results suggested that CMs conditioned medium might promote the maturation of the above three receptors, and then the CM of three groups of ESCs were observed by transmission electron microscope. S is similar to primary CMs of myofibrils, muscle segments, Z-rays and gap junctions, bridging structures, and cellular gene expression. The results showed that OCT-4, which was an ESCs marker, was only expressed early in the differentiation of cells. In T-induced group (P0.05), there was no significant difference in the expression of specific factor of late cardiac muscle in differentiation. It's all. Conclusion In vitro culture, ESCs (CR8) can be induced to differentiate into cardiomyocytes. The contractile function has pharmacological reactivity and can express the myocardial specific structural protein; the three-step culture method is the same as the previous research results; the suspension, suspension and adherent three-step culture method is a reliable method for culturing the ESCs into ESCs; and the Cs induced by the ESCs are mature and the cells are developed with the development of the cells. The frequency of beating may also decrease gradually, which may be related to the smaller cells in mature myocardial cells; in vitro, OT, CMCs conditioned medium, OT may be added to the mice.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R329
本文编号：2276582
[Abstract]:Experimental Background and Study Purpose Background Heart Failure is the highest incidence of heart disease worldwide, characterized by the absence of physiological functions for the death of normal cardiomyocytes The fibrous tissue is replaced. Because the body cardiomyocytes are almost impossible to regenerate, the most effective healers are currently The case is a heart transplant. In vitro and in vivo studies in nearly 10 years suggest stem cell transplantation for late heart failure may be another Embryonic stem cells (ESCs) have unlimited proliferation ability and multi-directional differentiation potential, and can differentiate into cardiomyocytes in vivo and in vitro. Various types of cells in the cells. How to induce ESCs to differentiate into cardiomyocytes more efficiently? Study The aim of this study was to optimize the amplification and culture of embryonic stem cells, and to observe whether mouse ESCs (CGR8 system) could differentiate into functional cardiac myocytes in vitro; External factors induce the efficiency of directional differentiation of ESCs and explore a safe and effective method pathway for body The method and the results of the method and the results of the method and the results of the method are as follows: the myocardial tissue of the milk mouse is separated by the differential adherence method to purify the heart muscle of the milk mouse; Cells were collected and cultured for 5 min at 1,000 r/ min. and mixing with the complete culture medium according to the ratio of 1: 1 to prepare the milk The rat cardiomyocytes conditioned medium were cultured. ESCs frozen in this laboratory were recovered and cultured. 1) suspension culture 2d; (2) suspension culture 5d; (3) adherent culture: 5d. The different groups of inducers added according to culture medium were randomly divided into four groups: milk rat CMs conditioned medium induction group; oxytocin (OT) induction group; mixed addition Induction group (CMMs conditioned medium with OT); control group (without any addition). Observation of the beat after ESCs differentiation showed that there was a rhythmic spontaneous contraction in the EBs after eight days of test. There was a consistent beat of the whole EBs, but this phenomenon was not observed in the control group. The beat rate of the mixed addition-induced group cells was Compared with OT-induced group and CMs condition culture medium-induced group, the difference was significant. (P 0.01). With the development of cardiac myocytes, the frequency of beating of cells gradually decreased. The results showed that the efficiency of the mixed addition induced component formation was significantly higher than that in the other two groups. Results ESCs were significantly higher in the early stage of differentiation than in other experimental groups. Due to the mature development of CMs in each group, three groups of cells respond to the drug. The results suggested that CMs conditioned medium might promote the maturation of the above three receptors, and then the CM of three groups of ESCs were observed by transmission electron microscope. S is similar to primary CMs of myofibrils, muscle segments, Z-rays and gap junctions, bridging structures, and cellular gene expression. The results showed that OCT-4, which was an ESCs marker, was only expressed early in the differentiation of cells. In T-induced group (P0.05), there was no significant difference in the expression of specific factor of late cardiac muscle in differentiation. It's all. Conclusion In vitro culture, ESCs (CR8) can be induced to differentiate into cardiomyocytes. The contractile function has pharmacological reactivity and can express the myocardial specific structural protein; the three-step culture method is the same as the previous research results; the suspension, suspension and adherent three-step culture method is a reliable method for culturing the ESCs into ESCs; and the Cs induced by the ESCs are mature and the cells are developed with the development of the cells. The frequency of beating may also decrease gradually, which may be related to the smaller cells in mature myocardial cells; in vitro, OT, CMCs conditioned medium, OT may be added to the mice.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R329
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