【摘要】：心肌梗塞以及充血性心力衰竭等心臟類疾病一直有很高的死亡率,此類疾病都伴隨著大量的心肌細(xì)胞死亡以及心臟功能的減退,其他心臟疾病如心率不齊等癥狀、心臟藥物篩選等,都需要一個(gè)理想的體外組織模型,來(lái)了解細(xì)胞移植后心肌功能的改善和心機(jī)細(xì)胞肥大的減緩,以及細(xì)胞移植對(duì)干細(xì)胞心肌向分化、心肌成纖維細(xì)胞增殖分化的影響或者藥物的有效性等。心臟修復(fù)中應(yīng)用較廣泛的是細(xì)胞移植療法,此方法旨在使用細(xì)胞改善心臟功能或者是修復(fù)受損的心肌層。多種類型的細(xì)胞可以應(yīng)用于心臟修復(fù),并且都取得一定效果,但是這些方法都有不同的缺點(diǎn),比如細(xì)胞在宿主體內(nèi)的存活、分化以及與原組織的交聯(lián)。心肌干細(xì)胞(Cardiac Stem Cells,CSCs)具有成體干細(xì)胞特性,并且自體移植不會(huì)發(fā)生免疫排斥反應(yīng),因此,心肌干細(xì)胞是構(gòu)建工程化心肌組織的理想細(xì)胞來(lái)源。利用心肌干細(xì)胞、心肌細(xì)胞及心肌成纖維細(xì)胞作為種子細(xì)胞,發(fā)展一種體外的心肌組織模型,為完善心肌損傷的細(xì)胞移植療法、認(rèn)識(shí)組織力學(xué)環(huán)境對(duì)種子細(xì)胞的增殖分化的影響、藥物干預(yù)心臟的功能提供了可能的體外模型。Matrigel包含多種ECM蛋白以及生長(zhǎng)因子,可以作為構(gòu)建3D心肌組織工程的理想材料。本文主要探究在力學(xué)環(huán)境下,體外構(gòu)建3D工程化心肌組織。將Matrigel與DMEM培養(yǎng)基按照不同比例稀釋,將心肌干細(xì)胞,心肌細(xì)胞以及心肌成纖維細(xì)胞按照按一定的比例與Matrigel工作液混勻。使用FlexCell力學(xué)加載設(shè)備,首先將六孔組織培養(yǎng)版形成凹槽,然后將混合均勻的細(xì)胞加到凹槽中,待靜置40分鐘后,3D組織構(gòu)建成功,然后靜置培養(yǎng)2-3天,再使用FlexCell力學(xué)加載設(shè)備進(jìn)行拉力加載,體外構(gòu)建3D工程化心肌組織。通過(guò)合適的力學(xué)加載、細(xì)胞比例及數(shù)量、3D凝膠的稀釋比例,構(gòu)建3D微心肌組織,發(fā)展了在體外構(gòu)建3D心肌組織的方法,為構(gòu)建心肌組織用于體外研究心肌損傷等疾病的研究,奠定可用的組織模型。本文共分為四章進(jìn)行介紹,第一章為緒論,綜合介紹了心肌梗死以及當(dāng)前最主要的治療方案;第二章為種子細(xì)胞的分離純化培養(yǎng)及鑒定工作;第三章使用FlexCell力學(xué)加載系統(tǒng),構(gòu)建3D工程化微心肌組織,并對(duì)微心肌組織進(jìn)行心肌細(xì)胞檢測(cè);第四章為全文總結(jié)以及工作展望。
[Abstract]:Heart diseases such as myocardial infarction and congestive heart failure have a high mortality rate. These diseases are accompanied by a large number of cardiac cell death and cardiac dysfunction, other heart diseases such as heart rate disorders such as symptoms, The screening of cardiac drugs requires an ideal tissue model in vitro to understand the improvement of myocardial function and the slowdown of cardiomyocyte hypertrophy after cell transplantation, as well as the differentiation of stem cell myocardium by cell transplantation. Effects of myocardial fibroblasts on proliferation and differentiation or the availability of drugs. Cell transplantation is widely used in cardiac repair, which aims to improve heart function or repair damaged myocardial layer. Many types of cells can be applied to heart repair, and all of these methods have different disadvantages, such as cell survival, differentiation and cross-linking with original tissue. Myocardial stem cells (Cardiac Stem cells) have the characteristics of adult stem cells, and there is no immune rejection in autologous transplantation. Therefore, myocardial stem cells are the ideal cell source for the construction of engineering myocardial tissue. Myocardial stem cells, cardiomyocytes and myocardial fibroblasts were used as seed cells to develop a myocardial tissue model in vitro to improve cell transplantation therapy for myocardial injury. In order to understand the effect of tissue mechanics environment on the proliferation and differentiation of seed cells, drug intervention provides a possible in vitro model. Matrigel contains a variety of ECM proteins and growth factors, which can be used as an ideal material for the construction of 3D myocardial tissue engineering. The aim of this study was to construct 3 D engineered myocardial tissue in vitro under mechanical environment. The Matrigel and DMEM media were diluted in different proportions, and the myocardial stem cells, cardiomyocytes and myocardial fibroblasts were mixed with the working solution of Matrigel in a certain proportion. Using the FlexCell mechanical loading equipment, the six-hole tissue culture plate was first formed into a groove, and then the mixed cells were added to the grooves. After 40 minutes of statics, the 3D tissue was successfully constructed, and then the cells were statically cultured for 2-3 days. Then FlexCell mechanical loading equipment was used to construct 3D engineered myocardial tissue in vitro. Through proper mechanical loading, cell proportion and dilution ratio of 3D gel, 3D micromyocardial tissue was constructed, and the method of constructing 3D myocardial tissue in vitro was developed, which was used to study myocardial injury and other diseases in vitro. Establish an organization model that is available. This paper is divided into four chapters, the first chapter is the introduction, the second chapter is the isolation, purification, culture and identification of seed cells, the third chapter is the use of FlexCell mechanical loading system. The 3D engineered micromyocardial tissue was constructed and the cardiac myocytes were detected. Chapter 4 was the summary of the whole paper and the prospect of the work.
【學(xué)位授予單位】：山東師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R54
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本文編號(hào)：2155611