本文選題：脂肪干細胞　切入點：心肌樣細胞　出處：《大連理工大學》2010年碩士論文　論文類型：學位論文

【摘要】： 目前心肌梗死仍是發(fā)病率和死亡率較高的疾病之一。由于心肌細胞再生能力有限,壞死的心肌組織即由無收縮功能的疤痕組織替代,中藥或西藥治療、介入治療和手術治療均不能代替壞死的心肌,而組織工程技術則為心肌梗死提供了一個較好的治療方法。此外,由于干細胞具有自我更新和多向分化潛能,已成為重要的組織工程種子細胞,但由于胚胎干細胞、骨髓干細胞以及誘導性多潛能干細胞存在著倫理道德或免疫排斥反應,甚至有致瘤的危險性,而脂肪干細胞由于來源廣泛,容易大量獲取,移植后無免疫排斥反應,因此成為組織工程比較有前景的種子細胞。 本文首先用多次聯(lián)合消化脂肪組織的方法分離培養(yǎng)脂肪干細胞,通過換液的方法去除紅細胞,減小了紅細胞裂解液對細胞的損傷。體外培養(yǎng)的脂肪干細胞生長迅速,凍存后仍具有較好的增殖能力。之后,我們采用油紅、堿性磷酸酶、von Kossa和甲苯胺藍染色,證明了我們分離的脂肪干細胞有多向分化潛能,流式細胞儀檢測細胞表面抗原陽性表達CD44, CD 105,陰性表達CD34, CD45及HLA-DR。 隨后,本文對脂肪干細胞向心肌樣細胞的分化能力進行了研究,采用血管緊張素Ⅱ誘導人脂肪干細胞向心肌樣細胞分化,通過心肌肌鈣蛋白(cTn-I)、肌球蛋白重鏈(MHC)以及縫隙連接蛋白43(Con43)的免疫細胞化學染色鑒定,光鏡下計數(shù)細胞總數(shù)及熒光激發(fā)下呈陽性的細胞數(shù),得到血管緊張素Ⅱ和5-氮胞苷分別誘導脂肪干細胞向心肌樣細胞分化的百分比分別為18%和21%,相差不大,因此血管緊張素Ⅱ可以用來代替?zhèn)鹘y(tǒng)的化學誘導劑5-氮胞苷。 在上述實驗的基礎上,本文進一步采用血管緊張素Ⅱ和堿性成纖維生長因子聯(lián)合誘導脂肪干細胞向心肌樣細胞分化,誘導4周后對肌球蛋白重鏈、縫隙連接蛋白43以及心肌肌鈣蛋白進行免疫細胞化學染色(組1),流式細胞儀(組2)和Western blot(組3)分析。組1誘導3周后細胞出現(xiàn)聚集,4周后形成球形細胞團,肌球蛋白重鏈(MHC)染色呈陽性表達,但由于細胞聚集成團,無法估計其誘導分化率,同時對照組不表達肌球蛋白重鏈,而采用流式細胞術和Western blot也都未能檢測出蛋白的含量。 脂肪干細胞具有間充質(zhì)干細胞的特征,在一定條件下能向心肌樣細胞分化,是一種有潛力的治療心肌梗死的細胞源。血管緊張素Ⅱ能誘導人脂肪干細胞向心肌樣細胞分化,可以用來替代傳統(tǒng)的誘導劑5-氮胞苷。血管緊張素Ⅱ誘導人脂肪干細胞向心肌樣細胞分化可能與細胞接種密度以及細胞培養(yǎng)的材質(zhì)有重要關系。
[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，

本文編號：1582512