發(fā)布時間：2018-03-08 05:09

  本文選題：脂肪干細(xì)胞　切入點：心肌樣細(xì)胞　出處：《大連理工大學(xué)》2010年碩士論文　論文類型：學(xué)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(xì)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(shù)治療均不能代替壞死的心肌,而組織工程技術(shù)則為心肌梗死提供了一個較好的治療方法。此外,由于干細(xì)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(xì)胞,但由于胚胎干細(xì)胞、骨髓干細(xì)胞以及誘導(dǎo)性多潛能干細(xì)胞存在著倫理道德或免疫排斥反應(yīng),甚至有致瘤的危險性,而脂肪干細(xì)胞由于來源廣泛,容易大量獲取,移植后無免疫排斥反應(yīng),因此成為組織工程比較有前景的種子細(xì)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(xì)胞,通過換液的方法去除紅細(xì)胞,減小了紅細(xì)胞裂解液對細(xì)胞的損傷。體外培養(yǎng)的脂肪干細(xì)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(lán)染色,證明了我們分離的脂肪干細(xì)胞有多向分化潛能,流式細(xì)胞儀檢測細(xì)胞表面抗原陽性表達(dá)CD44, CD 105,陰性表達(dá)CD34, CD45及HLA-DR。 隨后,本文對脂肪干細(xì)胞向心肌樣細(xì)胞的分化能力進(jìn)行了研究,采用血管緊張素Ⅱ誘導(dǎo)人脂肪干細(xì)胞向心肌樣細(xì)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(xì)胞化學(xué)染色鑒定,光鏡下計數(shù)細(xì)胞總數(shù)及熒光激發(fā)下呈陽性的細(xì)胞數(shù),得到血管緊張素Ⅱ和5-氮胞苷分別誘導(dǎo)脂肪干細(xì)胞向心肌樣細(xì)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(xué)誘導(dǎo)劑5-氮胞苷。 在上述實驗的基礎(chǔ)上,本文進(jìn)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(dǎo)脂肪干細(xì)胞向心肌樣細(xì)胞分化,誘導(dǎo)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(jìn)行免疫細(xì)胞化學(xué)染色(組1),流式細(xì)胞儀(組2)和Western blot(組3)分析。組1誘導(dǎo)3周后細(xì)胞出現(xiàn)聚集,4周后形成球形細(xì)胞團(tuán),肌球蛋白重鏈(MHC)染色呈陽性表達(dá),但由于細(xì)胞聚集成團(tuán),無法估計其誘導(dǎo)分化率,同時對照組不表達(dá)肌球蛋白重鏈,而采用流式細(xì)胞術(shù)和Western blot也都未能檢測出蛋白的含量。 脂肪干細(xì)胞具有間充質(zhì)干細(xì)胞的特征,在一定條件下能向心肌樣細(xì)胞分化,是一種有潛力的治療心肌梗死的細(xì)胞源。血管緊張素Ⅱ能誘導(dǎo)人脂肪干細(xì)胞向心肌樣細(xì)胞分化,可以用來替代傳統(tǒng)的誘導(dǎo)劑5-氮胞苷。血管緊張素Ⅱ誘導(dǎo)人脂肪干細(xì)胞向心肌樣細(xì)胞分化可能與細(xì)胞接種密度以及細(xì)胞培養(yǎng)的材質(zhì)有重要關(guān)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