人脂肪干细胞诱导分化为心肌样细胞的实验研究

发布时间：2018-03-08 05:09

  本文选题：脂肪干细胞　切入点：心肌样细胞　出处：《大连理工大学》2010年硕士论文　论文类型：学位论文

【摘要】： 目前心肌梗死仍是发病率和死亡率较高的疾病之一。由于心肌细胞再生能力有限,坏死的心肌组织即由无收缩功能的疤痕组织替代,中药或西药治疗、介入治疗和手术治疗均不能代替坏死的心肌,而组织工程技术则为心肌梗死提供了一个较好的治疗方法。此外,由于干细胞具有自我更新和多向分化潜能,已成为重要的组织工程种子细胞,但由于胚胎干细胞、骨髓干细胞以及诱导性多潜能干细胞存在着伦理道德或免疫排斥反应,甚至有致瘤的危险性,而脂肪干细胞由于来源广泛,容易大量获取,移植后无免疫排斥反应,因此成为组织工程比较有前景的种子细胞。 本文首先用多次联合消化脂肪组织的方法分离培养脂肪干细胞,通过换液的方法去除红细胞,减小了红细胞裂解液对细胞的损伤。体外培养的脂肪干细胞生长迅速,冻存后仍具有较好的增殖能力。之后,我们采用油红、碱性磷酸酶、von Kossa和甲苯胺蓝染色,证明了我们分离的脂肪干细胞有多向分化潜能,流式细胞仪检测细胞表面抗原阳性表达CD44, CD 105,阴性表达CD34, CD45及HLA-DR。 随后,本文对脂肪干细胞向心肌样细胞的分化能力进行了研究,采用血管紧张素Ⅱ诱导人脂肪干细胞向心肌样细胞分化,通过心肌肌钙蛋白(cTn-I)、肌球蛋白重链(MHC)以及缝隙连接蛋白43(Con43)的免疫细胞化学染色鉴定,光镜下计数细胞总数及荧光激发下呈阳性的细胞数,得到血管紧张素Ⅱ和5-氮胞苷分别诱导脂肪干细胞向心肌样细胞分化的百分比分别为18%和21%,相差不大,因此血管紧张素Ⅱ可以用来代替传统的化学诱导剂5-氮胞苷。 在上述实验的基础上,本文进一步采用血管紧张素Ⅱ和碱性成纤维生长因子联合诱导脂肪干细胞向心肌样细胞分化,诱导4周后对肌球蛋白重链、缝隙连接蛋白43以及心肌肌钙蛋白进行免疫细胞化学染色(组1),流式细胞仪(组2)和Western blot(组3)分析。组1诱导3周后细胞出现聚集,4周后形成球形细胞团,肌球蛋白重链(MHC)染色呈阳性表达,但由于细胞聚集成团,无法估计其诱导分化率,同时对照组不表达肌球蛋白重链,而采用流式细胞术和Western blot也都未能检测出蛋白的含量。 脂肪干细胞具有间充质干细胞的特征,在一定条件下能向心肌样细胞分化,是一种有潜力的治疗心肌梗死的细胞源。血管紧张素Ⅱ能诱导人脂肪干细胞向心肌样细胞分化,可以用来替代传统的诱导剂5-氮胞苷。血管紧张素Ⅱ诱导人脂肪干细胞向心肌样细胞分化可能与细胞接种密度以及细胞培养的材质有重要关系。
[Abstract]:Myocardial infarction is still one of the diseases with high morbidity and mortality. Due to the limited regeneration ability of myocardial cells, necrotic myocardial tissue is replaced by scar tissue without contractile function, and treated with traditional Chinese medicine or western medicine. Neither interventional therapy nor surgical treatment can replace necrotic myocardium, while tissue engineering provides a better treatment for myocardial infarction. In addition, stem cells have the potential of self-renewal and multi-differentiation. Has become an important seed cell for tissue engineering, but because of the ethical or immune rejection of embryonic stem cells, bone marrow stem cells and induced pluripotent stem cells, and even the risk of tumorigenesis, Adipose stem cells (ASCs) are widely available, easy to obtain, and have no immunological rejection after transplantation, so they are promising seed cells for tissue engineering. In this paper, adipose stem cells were isolated and cultured by multiple digestibility of adipose tissue, red blood cells were removed by liquid exchange method, and the damage caused by erythrocyte lysate was reduced. The adipose stem cells cultured in vitro grew rapidly. After cryopreservation, we used oil red, alkaline phosphatase von Kossa and toluidine blue staining to prove the differentiation potential of adipose stem cells. The positive expression of CD44, CD105, negative expression of CD34, CD45 and HLA-DR were detected by flow cytometry. Subsequently, the ability of adipose stem cells to differentiate into cardiomyocyte-like cells was studied. Angiotensin 鈪，

本文编号：1582512