小鼠胎肝干细胞经化学试剂诱导向心肌细胞方向分化的研究

发布时间：2018-06-06 11:26

本文选题：胎肝间充质干细胞 + 诱导分化　；参考：《重庆医科大学》2009年硕士论文

【摘要】： 研究背景: 冠状动脉粥样硬化性心脏病,特别是急性心肌梗死,是严重危害人类健康的常见病,多发病。目前虽然能在发病4-6小时内通过及时溶栓或介入治疗以挽救濒临死亡的心肌组织,但由于绝大部分患者就诊时缺血心肌已经坏死。此时再灌注治疗只能防止梗死面积的扩大,而无法挽救已经坏死的心肌。如何更有效地治疗冠心病,成为医学研究的焦点。而新近的研究结果,为患者带来了希望。传统观点认为,心肌细胞出生后即向终末分化,不能够复制,并且成年心肌组织中没有储存的心肌祖细胞,因此心肌受损后心肌细胞不能再生,被疤痕组织代替,最终导致心脏的收缩功能受损[1 ]。然而目前很多研究结果[2]给我们带来观念上的更新:其一,心脏不是终末分化器官,心肌中有75%的细胞是单核细胞,,25%是双核细胞,这一比例不受疾病、年龄、性别、心肌肥大及心肌缺血等影响。其二,急性心肌损伤促进心肌细胞代偿性增殖。虽然有研究显示:急性心肌梗死后也有少量心肌细胞发生分裂增生[3]。但分裂的细胞数量很少,修复心肌组织的能力有限。因此,细胞移植,这一旨在增加心肌细胞数目的方法可能是有效治疗心肌损伤的最佳选择。目前研究多集中在胚胎干细胞、骨骼肌干细胞、骨髓间充质干细胞等各类干细胞的心肌移植,并屡有移植细胞在体内存活分化,修复梗死心肌,改善心脏功能的研究报道。然而要将上述研究最终用于临床,却面临诸多问题。胚胎干细胞是来源于哺乳动物早期胚胎的内细胞团或桑椹胚的二倍体细胞,从植入子宫内膜前的囊胚中提取,在体外分离培养而建立的细胞系。其显著特征是可以在体外长期保持不分化状态下的增殖能力,并仍具备分化为三胚层细胞的稳定发育潜能,有“万能细胞”之称[4]。近年有些国家已解除胚胎干细胞研究的禁令,为其扫除了社会和伦理方面的障碍。但获取受限和移植排斥反应是限制其发展的瓶颈。骨骼肌干细胞具有能耐受缺血环境;移植后很快分化成具有收缩性的骨骼肌等特点。但其数量极少,且与年龄增长负相关,分离纯化的技术难度大。而最大的缺陷在于骨骼肌干细胞并不能与受体心肌细胞形成闰盘连接,不能整合到受体心肌细胞中形成生理性功能合胞体,导致骨骼肌干细胞移植后往往发生心室节律紊乱,使其应用价值受到不少学者质疑。骨髓间充质干细胞具有取材方便、体外分离纯化相对简单、易扩增等特点;同时由于它们存在独特的免疫耐受性(其原因可能是缺乏Ⅱ型HLA和免疫共刺激因子B7[5]),能够在异种异体环境中存活,而不被受体免疫系统所排斥。所以骨髓间充质干细胞是用于心肌移植的优势细胞之一,被广泛关注[6,7]。胎肝干细胞(Embryonic hepatic stem cells,EHSCs)是近年逐渐被关注的一类间充质干细胞细胞。有学者[8]认为胚胎肝脏发育早,胎肝干细胞可能具有比骨髓干细胞更强的增殖分化能力和更低的免疫原性和免疫活性。有研究者已就其向类心肌细胞方向分化做出了初步探讨[9,10]。胎肝干细胞有望用于心肌移植的优势细胞之一。研究目的: 1.优化小鼠EHSCs的分离、纯化、扩增的方法;2.比较不同胎龄的EHSCs生物学性状,遴选出获得小鼠EHSCs的合适时期;3.通过体外诱导分化实验了解EHSCs是否具有分化为类心肌细胞的潜能;4.比较不同的诱导条件下,EHSCs向类心肌细胞方向的分化情况。方法与结论: 第一部分:探讨小鼠EHSCs分离、纯化、扩增方法,比较不同胎龄的小鼠EHSCs的生物学性状。采用胶原酶加EDTA消化法和差速贴壁法分离不同胎龄的小鼠EHSCs,用含15%优等胎牛血清的L-DMEM培养液培养,通过反复传代对EHSCs进行纯化和扩增培养。结果提示:13.5d胎龄组EHSCs形态均一,生长状态良好,干细胞特性明显,是相对原始的干细胞。随胎龄增加,16.5d胎龄组和19.5d胎龄组EHSCs形态差异渐大,生长状态渐次,逐渐向具有肝细胞和胆管细胞标志的双显型干细胞方向过渡。结论:1. 13.5d胎龄的EHSCs,是相对原始的干细胞,可能具有更广泛的分化潜能。2.通过上述方法,可获得数量可观,性质稳定的EHSCs。第二部分:探讨不同的诱导条件下,EHSCs向心肌细胞方向的分化情况。取3-4代细胞以1.5×10~4 /cm~2密度接种于培养板上。当细胞接近80%-90%融合时,用不同浓度的诱导剂诱导细胞24h,然后置于37OC,5%CO_2, 20%O_2 ,饱和湿度的孵箱中培养。倒置显微镜下观察细胞形态变化,发现在下述条件下细胞发生了向心肌细胞方向的分化:诱导剂为5-aza 5μmol/L+DMSO 0.8%,孵育时间为24h,培养液是含有15%FCS、1%非必需氨基酸、1%左旋谷氨酸的L-DMEM培养基。培养条件是37OC,5%CO_2,20%O_2,饱和湿度的孵箱。结果提示:诱导3周后,分化细胞呈小圆形,具有相互聚集形成球形细胞团结构的趋势;诱导后第4周,细胞免疫组化染色提示转化细胞表达心肌特异性肌钙蛋白T ( troponin T, Tn T)和α-肌动蛋白(α-actin)。结论:1.小鼠EHSCs具有向心肌细胞方向分化的潜能;2.不同种属来源的细胞发生分化的时间和过程并不完全相同,转化为成熟心肌细胞所需时间也不同。总结: 1.利用改良的差速贴壁法,可以从胎肝中分离出具有良好贴壁能力的干细胞群,方法较为简单易行。 2.胎龄为13.5 d的小鼠胎肝,单位重量的胎肝中含有更多的具有形成集落能力的干细胞。 3.胎龄为13.5 d的小鼠胎肝干细胞处于相对原始的未分化阶段。 4.小鼠EHSCs在5-aza和DMSO联合诱导的情况下在体外可向类心肌细胞方向分化。 5.诱导剂组合5-aza 5μmol/L+ DMSO 0.8%能诱导EHSCs在体外向心肌细胞方向分化,加大诱导剂剂量并不能提高诱导效率。 6.诱导剂组合5-aza 5μmol/L+ DMSO 0.8%诱导EHSCs在体外向心肌细胞方向分化的合理作用时间为24h,延长诱导剂的作用时间,并不能提高诱导效率。
[Abstract]:Research background:
Coronary atherosclerotic heart disease, especially acute myocardial infarction, is a common disease that seriously endangers human health. It is often possible to save the dying myocardium by timely thrombolytic or interventional therapy within 4-6 hours of the disease, but the ischemic myocardium has been necrotic at the time of most of the patients. Treatment can only prevent the expansion of the infarct area and can not save the necrotic myocardium. How to treat coronary heart disease more effectively has become the focus of medical research, and recent results have brought hope to the patients.
The traditional point of view is that cardiac myocytes are differentiated into terminal cells after birth and can not be replicated, and there is no stored cardiac progenitor cells in adult myocardium, so myocardial cells can not regenerate after myocardial damage and are replaced by scar tissue, resulting in impaired cardiac contractile function [1).
However, a lot of current research results [2] bring us a conceptual update: first, the heart is not a terminal differentiation organ, 75% of the cells in the myocardium are mononuclear cells, and 25% are binuclear cells. This proportion is not affected by disease, age, sex, myocardial hypertrophy and myocardial ischemia.
Although studies have shown that a small number of cardiomyocytes have split proliferative [3]. after acute myocardial infarction, the number of divided cells is small and the ability to repair myocardial tissue is limited. Therefore, cell transplantation, a method aimed at increasing the number of cardiomyocytes, may be the best choice for effective treatment of myocardial injury.
At present, many studies have focused on the transplantation of stem cells, such as embryonic stem cells, skeletal muscle stem cells, bone marrow mesenchymal stem cells and other kinds of stem cells, and the research reports on the survival and differentiation of the transplanted cells in the body, repair the infarcted myocardium and improve the heart function. However, the above research should be finally used in clinical practice, but many problems are faced.
The embryonic stem cell is a diploid cell derived from the inner cell mass or morula of the early mammalian embryo, extracted from the blastocyst before the implantation of the endometrium and isolated and cultured in vitro, which is characterized by the ability to maintain the proliferation in an undifferentiated state for a long time in vitro, and still have a differentiation of three germ cells. In recent years, some countries have lifted the prohibition of embryonic stem cell research and removed the social and ethical barriers in [4].. However, access to restriction and graft rejection is the bottleneck to restrict its development.
Skeletal muscle stem cells have the ability to tolerate ischemic environment and quickly differentiate into contractile skeletal muscles. But the number of skeletal muscle cells is very small and is negatively related to age growth. The technical difficulty of separating and purifying is very difficult. The biggest defect is that skeletal muscle stem cells do not form intercalated disc connections with receptor cardiac myocytes and can not be integrated into the receptor. The physiological function syncytial body is formed in the cardiac myocytes, which leads to the ventricular rhythm disorder after the transplantation of skeletal muscle stem cells, which has been questioned by many scholars.
Bone marrow mesenchymal stem cells (MSCs) have the advantages of convenient extraction, relatively simple separation and purification in vitro, and their unique immune tolerance (which may be due to the lack of type II HLA and immuno stimulating factor B7[5]) and can survive in xenoallogenic environment without rejection by the receptor immune system. Mesenchymal stem cells are one of the dominant cells for myocardial transplantation. They are widely concerned about [6,7]..
Embryonic hepatic stem cells (EHSCs) is a kind of mesenchymal stem cell cells which have been paid more attention in recent years. Some scholars believe that embryonic liver is early developed, and fetal liver stem cells may have stronger proliferation and differentiation ability and lower immunogenicity and immune activity than bone marrow stem cells. Cell differentiation has made a preliminary study. [9,10]. fetal liver stem cells are expected to be one of the dominant cells in myocardial transplantation.
The purpose of the study is:
1. to optimize the isolation, purification and amplification of EHSCs in mice; 2. to compare the EHSCs biological characters of different gestational ages and to select the appropriate time to obtain EHSCs in mice; 3. through the induction of differentiation in vitro to understand the potential of EHSCs to differentiate into the myocyte like cells; 4. compare the differentiation of EHSCs into the direction of the myocardial like cells under different induction conditions. Situation.
Methods and conclusions:
The first part: To explore the isolation, purification and amplification of EHSCs in mice, to compare the biological characters of EHSCs in mice of different gestational ages. Using collagenase, EDTA digestion and differential adherence to separate EHSCs in mice of different gestational ages, the L-DMEM culture medium containing 15% superior fetal bovine serum was cultured, and EHSCs was purified and amplified by repeated passages. The results showed that the EHSCs morphology of 13.5d gestational age group was uniform, the growth state was good, the stem cell characteristics were obvious, it was relatively primitive stem cells. As the gestational age increased, the difference of EHSCs morphology between the 16.5d gestational age group and the 19.5d fetal age group gradually increased, and the growth state gradually shifted to the direction of the double developing stem cells with the markers of hepatocytes and bile duct cells.
Conclusion: the EHSCs of 1. 13.5d gestational age is relatively primitive stem cells and may have a wider range of differentiation potential.2. through the above method, which can obtain a considerable quantity and stable EHSCs..
The second part: To investigate the differentiation of EHSCs to myocardial cells under different induction conditions. 3-4 generation cells were inoculated on the culture plate at 1.5 x 10~4 /cm~2 density. When the cells were close to 80%-90% fusion, the cells were induced with different concentrations of inducers to induce cell 24h, and then placed in the incubator of 37OC, 5%CO_2, 20%O_2, saturated humidity. Under the microscope, it was found that the cells were differentiated into cardiomyocyte direction under the following conditions: the inducer was 5-aza 5 mu mol/L+DMSO 0.8%, the incubation time was 24h, and the culture medium was the L-DMEM medium containing 15%FCS, 1% non essential amino acids and 1% L-glutamic acid. The culture condition was the incubator of 37OC, 5%CO_2,20%O_2, saturated humidity. The results suggest that after 3 weeks of induction, the differentiated cells are small round and have a tendency to form spherical cell clusters with each other. After fourth weeks of induction, cell immuno histochemical staining suggests that the transformed cells express cardiac specific troponin T (troponin T, Tn T) and alpha actin (alpha -actin).
Conclusion: 1. EHSCs in mice has the potential of centripetal muscle cell differentiation. 2. the time and process of differentiation of cells from different species are not exactly the same, and the time needed to transform into mature cardiomyocytes is also different.
Summary:
1. by using the modified differential adherence method, stem cells with good adherence ability can be isolated from fetal liver.
2. fetal liver with 13.5 gestational age of 13.5 D contains more stem cells with colony forming ability.
The fetal liver stem cells of 3. gestational age of 13.5 D were in relatively primitive undifferentiated stage.
4. mouse EHSCs can differentiate into cardiomyocytes in vitro under the combined induction of 5-aza and DMSO.
5. inducer combination 5-aza 5 mu mol/L+ DMSO 0.8% can induce EHSCs to differentiate into cardiomyocytes in vitro. Increasing the dose of inducer can not improve the induction efficiency.
6. inducer combination 5-aza 5 mol/L+ DMSO 0.8% induced the rational time of EHSCs to differentiate into cardiomyocytes in vitro, which is 24h, prolonging the action time of inducer, and not improving the induction efficiency.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R329

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