本文選題：脂肪干細(xì)胞 + 支架��； 參考：《大連理工大學(xué)》2009年博士論文

【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,壞死的心肌組織即使得到再灌注治療也只能由無(wú)收縮功能的瘢痕組織替代,細(xì)胞移植治療只能修復(fù)小面積心肌損傷,而組織工程技術(shù)為大面積心肌缺損提供了一個(gè)較好的治療方法。組織工程包括種子細(xì)胞、支架材料和生物活性分子三大要素。 干細(xì)胞由于具有自我更新和多向分化潛能將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)的多潛能干細(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(xiǎn)性,而脂肪干細(xì)胞由于來(lái)源簡(jiǎn)便充足,容易大量提取獲得,移植后無(wú)免疫排斥反應(yīng),將成為組織工程比較有前景的種子細(xì)胞。因此我們用改進(jìn)的方法分離培養(yǎng)脂肪干細(xì)胞,并對(duì)其增殖能力和多向分化潛能進(jìn)行檢測(cè)。通過(guò)胰酶和膠原酶聯(lián)合分次消化和換液去除紅細(xì)胞,我們改進(jìn)了脂肪干細(xì)胞的分離方法,可從400～600mg脂肪組織收獲約5×10~5個(gè)脂肪組織來(lái)源的干細(xì)胞,并且細(xì)胞可以重疊生長(zhǎng)一個(gè)月以上,期間細(xì)胞表現(xiàn)出幾個(gè)對(duì)數(shù)增殖期;所有增殖的細(xì)胞其干細(xì)胞相關(guān)表面標(biāo)記(CD13,CD29,CD44,CD105和CD166)都呈陽(yáng)性表達(dá);多潛能細(xì)胞相關(guān)轉(zhuǎn)錄因子Nanog,Oct-4,Sox-2和Rex-1也呈強(qiáng)陽(yáng)性表達(dá);通過(guò)油紅、甲苯胺蘭、ALP、von Kossa及熒光染色證明脂肪組織來(lái)源的干細(xì)胞具有向多個(gè)胚層細(xì)胞分化的能力。此外,為獲得更多具有強(qiáng)增殖能力的細(xì)胞,根據(jù)生長(zhǎng)曲線,我們對(duì)細(xì)胞進(jìn)行每14天傳代而非常規(guī)的5天傳代,發(fā)現(xiàn)所得到的細(xì)胞仍保持強(qiáng)的增殖能力、干細(xì)胞表型以及更強(qiáng)的多向分化潛能。 制備適于種子細(xì)胞生長(zhǎng)的支架材料也是構(gòu)建工程化組織的關(guān)鍵。在各種生物材料中,天然生物材料膠原和殼聚糖都具有較好的生物相容性和生物可降解性,但膠原的機(jī)械強(qiáng)度差,而殼聚糖由于其晶體結(jié)構(gòu)而較堅(jiān)硬,因此結(jié)合膠原和殼聚糖來(lái)制備支架將可避免兩種材料的缺點(diǎn),從而制備出適于種子細(xì)胞生長(zhǎng)的支架。我們采用凍干法制備不同比例的膠原—?dú)ぞ厶侵Ъ?研究支架的孔徑、孔隙率、保水性、生物可降解性以及與脂肪干細(xì)胞的生物相容性。凍干后的支架內(nèi)部呈海綿狀多孔隙結(jié)構(gòu),其中以膠原/殼聚糖體積比為9:1的復(fù)合支架最為疏松,1:9的支架最致密。掃描電鏡下支架的膠原含量越高,支架內(nèi)的膠原絲越多,支架的孔與孔之間相互連通構(gòu)成了通孔。交聯(lián)前后支架的形態(tài)結(jié)構(gòu)無(wú)明顯改變。交聯(lián)后體積比為9:1,7:3和5:5的復(fù)合支架孔徑為50～200μm之間,可用于細(xì)胞的三維培養(yǎng)。體積比為5:5的復(fù)合支架的吸水性和含水量最高,7:3和3:7次之;多孔支架在水中未發(fā)生明顯的溶脹現(xiàn)象;支架的孔隙率均在90%以上。交聯(lián)后隨著膠原含量的減少,降解速率減慢,交聯(lián)后的復(fù)合支架降解速度較未交聯(lián)慢。脂肪干細(xì)胞在支架上培養(yǎng)5d后掃描電鏡和HE染色可見(jiàn)細(xì)胞在7:3的支架上爬行生長(zhǎng)并融合成片,而5:5支架上黏附生長(zhǎng)的細(xì)胞較少。 組織工程構(gòu)建過(guò)程中,提供適于種子細(xì)胞生長(zhǎng)的微環(huán)境也是很重要的,體外細(xì)胞生長(zhǎng)的環(huán)境要盡量模擬體內(nèi)的三維動(dòng)態(tài)微環(huán)境,而轉(zhuǎn)瓶生物反應(yīng)器可提供三維動(dòng)態(tài)微環(huán)境,促進(jìn)懸浮和貼壁細(xì)胞的增殖和分化。因此我們利用轉(zhuǎn)瓶生物反應(yīng)器提供三維動(dòng)態(tài)微環(huán)境,對(duì)支架內(nèi)的脂肪干細(xì)胞進(jìn)行擴(kuò)增,并檢測(cè)擴(kuò)增后干細(xì)胞的表型特性及多向分化潛能。脂肪干細(xì)胞在轉(zhuǎn)瓶生物反應(yīng)器中擴(kuò)增14天后,與靜態(tài)條件下相比,支架內(nèi)的細(xì)胞具有更強(qiáng)的增殖活性,擴(kuò)增倍數(shù)大約為26倍,而靜態(tài)條件下細(xì)胞擴(kuò)增倍數(shù)近20倍;所擴(kuò)增的細(xì)胞能夠保持原有的干細(xì)胞表型特性和多向分化潛能。 局部微環(huán)境在干細(xì)胞的定向分化中起著決定性作用,而心肌樣微環(huán)境可促進(jìn)骨髓干細(xì)胞向心肌細(xì)胞分化,因此心肌樣微環(huán)境也可能促進(jìn)脂肪干細(xì)胞向心肌細(xì)胞分化。我們分別對(duì)脂肪干細(xì)胞和心肌細(xì)胞進(jìn)行間接和直接共培養(yǎng)研究,并檢測(cè)共培養(yǎng)后脂肪干細(xì)胞超微結(jié)構(gòu)的變化以及心肌特異性蛋白和轉(zhuǎn)錄因子的表達(dá)。經(jīng)2周共培養(yǎng)后,分化的脂肪干細(xì)胞呈現(xiàn)心肌樣超微結(jié)構(gòu),并表達(dá)心肌特異性蛋白和轉(zhuǎn)錄因子,流式細(xì)胞儀分析結(jié)果顯示,間接共培養(yǎng)2周后約20%的細(xì)胞表達(dá)心肌特異性蛋白,而直接共培養(yǎng)2周后約30～40%的細(xì)胞表達(dá)心肌特異性蛋白,并且直接共培養(yǎng)體系中分化的脂肪干細(xì)胞的心肌特異性蛋白和轉(zhuǎn)錄因子的表達(dá)率明顯高于間接共培養(yǎng)體系中的表達(dá)率。 在各種生長(zhǎng)因子中,人胰島素樣生長(zhǎng)因子(IGF-1)在心臟發(fā)生和發(fā)育過(guò)程中起著重要的作用,能促進(jìn)早期心肌分化。因此我們利用IGF-1基因作為目的基因,整合到支架內(nèi)并轉(zhuǎn)染脂肪干細(xì)胞,應(yīng)用心肌細(xì)胞培養(yǎng)基作為誘導(dǎo)培養(yǎng)基,轉(zhuǎn)瓶生物反應(yīng)器提供三維動(dòng)態(tài)微環(huán)境,研究脂肪干細(xì)胞在膠原-殼聚糖支架內(nèi)向心肌細(xì)胞分化的情況。結(jié)果顯示動(dòng)態(tài)微環(huán)境能促進(jìn)質(zhì)粒DNA的釋放和轉(zhuǎn)染;IGF-1可促進(jìn)脂肪干細(xì)胞在膠原-殼聚糖支架內(nèi)增殖以及向心肌細(xì)胞分化;動(dòng)態(tài)微環(huán)境可加強(qiáng)IGF-1的促進(jìn)脂肪干細(xì)胞增殖分化作用。 本研究采用改進(jìn)的方法可較容易的獲得大量脂肪干細(xì)胞,并發(fā)現(xiàn)脂肪干細(xì)胞具有較強(qiáng)的增殖能力和多向分化潛能;所制備的膠原-殼聚糖多孔支架具有較好生物相容性和生物可降解性,適于脂肪干細(xì)胞的三維培養(yǎng);所設(shè)計(jì)的支架—轉(zhuǎn)瓶培養(yǎng)系統(tǒng)是一個(gè)簡(jiǎn)便有效的擴(kuò)增脂肪干細(xì)胞的方法;心肌樣微環(huán)境可促進(jìn)脂肪干細(xì)胞向心肌細(xì)胞分化;將細(xì)胞生長(zhǎng)的支架內(nèi)的陽(yáng)離子多聚物作為IGF-1基因轉(zhuǎn)染的載體,這種轉(zhuǎn)染方法較傳統(tǒng)的方法更簡(jiǎn)易,效率更高;聯(lián)合IGF-1基因、心肌細(xì)胞培養(yǎng)基和動(dòng)態(tài)微環(huán)境多因素刺激,可促進(jìn)脂肪干細(xì)胞在支架內(nèi)向心肌細(xì)胞分化。此研究對(duì)體外構(gòu)建工程化心肌樣組織進(jìn)行心肌再生有著重要的指導(dǎo)意義。
[Abstract]:At present, myocardial infarction is still one of the diseases with high morbidity and mortality. Due to the limited regenerative ability of cardiac myocytes, necrotic myocardial tissue can only be replaced by non contractile scar tissue even by reperfusion therapy. Cell transplantation can only repair small area of myocardial injury, and tissue engineering technique is a large area of myocardial defect. It provides a better method of treatment. Tissue engineering includes three elements: seed cells, scaffold materials and bioactive molecules.
Stem cells, due to their self renewal and multidirectional differentiation potential, will become important tissue engineering seed cells. However, because of embryonic stem cells, bone marrow stem cells and induced pluripotent stem cells, there are ethical or immune rejection and even the risk of tumorigenesis, and fat stem cells are easily raised because of their simple and convenient sources. There is no immune rejection after transplantation, and it will become a promising seed cell in tissue engineering. Therefore, we use an improved method to isolate and culture fat stem cells and detect their proliferation and pluripotent differentiation potential. We have improved the fat dry through the combined digestion of trypsin and collagenase and the removal of red blood cells. The cell separation method can harvest about 5 * 10~5 adipose tissue derived stem cells from 400 to 600mg adipose tissue, and the cells can overlap and grow for more than a month. During the period, the cells show several logarithmic proliferation periods; all the proliferating cells have positive expression of the stem cell related surface markers (CD13, CD29, CD44, CD105 and CD166); multipotential Cell related transcription factors, Nanog, Oct-4, Sox-2 and Rex-1, are also strongly positive; the stem cells derived from adipose tissue have the ability to differentiate into multiple germ cells through oil red, methaniline, ALP, von Kossa and fluorescent staining. In addition, to obtain more cells with strong proliferative ability, we carry out the cells according to the growth curve. After 14 days of passage instead of conventional 5 days passage, it was found that the cells still maintained strong proliferative capacity, stem cell phenotype and stronger multidirectional differentiation potential.
The key to the preparation of scaffold materials suitable for seed cell growth is also the key to the construction of engineered tissue. In various biological materials, natural biomaterials collagen and chitosan have good biocompatibility and biodegradability, but the mechanical strength of collagen is poor, and chitosan is hard because of its crystal structure, so it is combined with collagen and chitosan. Sugar scaffolds will avoid the shortcomings of the two materials and prepare a scaffold suitable for seed cell growth. We use a freeze-drying method to prepare different proportions of collagen chitosan scaffolds to study the pore size, porosity, water retention, biodegradability and biocompatibility with fat stem cells. The composite scaffold with collagen / chitosan volume ratio of 9:1 was most loose and the 1:9 scaffold was the densest. The higher the collagen content of the stent under scanning electron microscope, the more collagenes in the scaffold and the interconnected pores between the scaffolds and the pores. The pore size of the composite scaffold with a ratio of 9:1,7:3 and 5:5 is 50~200 u m, which can be used for three-dimensional culture of cells. The water absorption and water content of the composite scaffold with a volume ratio of 5:5 is the highest, 7:3 and 3:7 are the second. The porous scaffold has no obvious swelling phenomenon in the water; the porosity of the scaffold is above 90%. After cross linking, the content of collagen decreases and decreases. The rate of degradation was slow and the degradation rate of the composite scaffold after crosslinking was slower than that of non crosslinking. After the 5D was cultured on the scaffold, the cells grew and fused on the 7:3 scaffold, while the cells attached to the 5:5 scaffold were less.
During the construction of tissue engineering, it is also important to provide a microenvironment suitable for the growth of seed cells. The environment in vitro cell growth is to simulate three-dimensional dynamic microenvironment in vivo, and the vase bioreactor can provide three-dimensional dynamic microenvironment to promote the proliferation and differentiation of suspended and adherent cells. Therefore, we use the bioreaction of the vase. The apparatus provides a three-dimensional dynamic microenvironment to amplify the fat stem cells in the scaffold and detect the phenotypic characteristics and pluripotent differentiation potential of the expanded stem cells. The cells in the stents have a stronger proliferation activity compared with the static conditions for 14 days after the amplification of the stem cells in a vase bioreactor, and the multiplier of the expansion is about 26 times. Under the condition, the amplification times of cells were nearly 20 times, and the expanded cells could maintain the phenotypic and pluripotent differentiation potential of stem cells.
Local microenvironment plays a decisive role in the directional differentiation of stem cells, and myocardial microenvironment can promote the differentiation of bone marrow stem cells into cardiomyocytes. Therefore, myocardial microenvironment may also promote the differentiation of adipose stem cells into cardiomyocytes. We have conducted indirect and direct co culture studies on adipose stem cells and cardiomyocytes, respectively. The ultrastructural changes of adipose stem cells and the expression of specific protein and transcription factors were observed after co culture. After 2 weeks of co culture, the differentiated adipose stem cells showed myocardial ultrastructure and expressed the specific protein and transcription factors of the myocardium. The flow cytometry showed that about 20% of the cells were expressed after 2 weeks of indirect co culture. Myocardium specific protein was expressed in 30 to 40% cells after 2 weeks of direct co culture, and the expression rate of specific protein and transcription factors of the differentiated adipose stem cells in the direct co culture system was significantly higher than that in the indirect co culture system.
In various growth factors, human insulin-like growth factor (IGF-1) plays an important role in the process of cardiac development and development, which can promote early myocardial differentiation. Therefore, we use the IGF-1 gene as the target gene to integrate into the stent and transfect the adipose stem cells, use the cardiomyocyte culture medium as the inducible medium, and turn the vase biological reaction. A three-dimensional dynamic microenvironment was provided to study the differentiation of adipose stem cells into cardiomyocytes in collagen chitosan scaffold. The results showed that dynamic microenvironment could promote the release and transfection of plasmid DNA; IGF-1 could promote the proliferation of adipose stem cells in collagen chitosan scaffold and differentiate into cardiomyocytes; dynamic microenvironment can strengthen IGF-1 It promotes the proliferation and differentiation of adipose stem cells.
This study can easily obtain a large number of fat stem cells, and found that fat stem cells have strong proliferation ability and multidirectional differentiation potential. The prepared collagen chitosan porous scaffold has good biocompatibility and biodegradability, suitable for three-dimensional culture of fat stem cells; the designed scaffold - turn bottle The culture system is a simple and effective method for amplification of fat stem cells. Myocardial microenvironment can promote the differentiation of fat stem cells into cardiomyocytes, and the cationic polypeptides in the scaffold of the cells are used as the carrier of IGF-1 gene transfection. The transfection method is simpler and more efficient than the traditional method. Combined with the IGF-1 gene, the myocardium is fine. The multi factor stimulation of cell culture medium and dynamic microenvironment can promote the differentiation of adipose stem cells into cardiomyocytes in the scaffold. This study has important guiding significance for the construction of myocardium like tissue in vitro for construction of engineered myocardium.
【學(xué)位授予單位】：大連理工大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2009
【分類號(hào)】：R329;R542.22

【引證文獻(xiàn)】

相關(guān)博士學(xué)位論文 前1條

1 楊磊;新型溫敏膜的構(gòu)建及其培養(yǎng)收獲干細(xì)胞的研究[D];大連理工大學(xué);2012年

相關(guān)碩士學(xué)位論文 前1條

1 張文;人脂肪干細(xì)胞在水凝膠內(nèi)三維培養(yǎng)的研究[D];大連理工大學(xué);2010年

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本文編號(hào)：1941160