【摘要】：干細胞是體內(nèi)一類具有自我更新、高度增殖和多向分化潛能的細胞群體，在一定條件下，可以分化成多種功能細胞。干細胞是再生醫(yī)學的原基，可用于組織修復、器官再生、基因治療等研究和臨床治療。目前研究比較深入的有胚胎干細胞、骨髓間充質干細胞、臍帶血干細胞、毛囊間充質干細胞等。毛囊間充質干細胞，不僅自我更新，定向分化為脂肪、骨、軟骨細胞，更重要的是毛囊間充質干細胞表達胚胎干細胞標志物（SOX2和Nanog）。毛囊來源豐富、獲取方便、收獲不受年齡的限制，幾乎不會對機體造成任何損害，還可重復獲取，并且免疫原性地下，是組織修復、器官重建和基因治療重要的種子細胞來源。再生醫(yī)學研究與治療需要大量種子細胞，傳統(tǒng)的平面培養(yǎng)方法很難在短時間獲取大量種子細胞，而應用微載體、生物反應器技術立體培養(yǎng)就可以解決這一問題。微載體表面積/體積（S/V）大，單位體積培養(yǎng)液細胞產(chǎn)率高；立體培養(yǎng)兼懸浮和貼壁培養(yǎng)的優(yōu)點，簡化細胞生長環(huán)境因素的檢測和控制，重現(xiàn)性好，成本低，勞動強度小。通過立體培養(yǎng)可以獲得大量的毛囊間充質干細胞，然而其生長特性、生物學特性未知。 本實驗研究目的是建立體外大量擴增人毛囊間充質干細胞（human hair folliclemesenchymal stem cells，hHF-MSCs）的生物反應器技術，，從而為再生醫(yī)學研究與臨床治療提供未分化、大量的種子細胞。 拔取成人毛發(fā)，采用組織塊培養(yǎng)法，成功獲得了hHF-MSCs。我們將獲取的hHF-MSCs成功的接種至載體上，經(jīng)吖啶橙染色，共聚焦顯微鏡掃描，可以觀察到hHF-MSCs貼附于載體表面并伸展呈長梭形。hHF-MSCs以1000cells/cm~2或500cells/cm~2密度接種至載體上，細胞在接種第3d時進入對數(shù)生長期，增殖至15d時細胞進入平臺期；平面培養(yǎng)hHF-MSCs至第6天時進入對數(shù)生長期。間充質干細胞表面標志CD90、CD105免疫熒光染色，二種方法培養(yǎng)hHF-MSCs呈陽性表達；流式細胞術分析：立體培養(yǎng)hHF-MSCs CD90、CD105表達率分別為80.25%、45.36%，平面培養(yǎng)hHF-MSCsCD90、CD105表達率分別為85.65%、43.88%；這二種方法培養(yǎng)hHF-MSCs經(jīng)成脂誘導后，油紅O染色，脂滴呈紅色；成骨誘導后，茜素紅染色，可見鈣鹽沉積和鈣結節(jié)被染成紅色。 本研究成功獲取hHF-MSCs，建立體外大量擴增hHF-MSCs生物反應器技術，應用該技術培養(yǎng)的hHF-MSCs仍能保持MSCs表面標志，并具有多向分化潛能，這為后期建立hHF-MSCs銀行，構建組織工程化器官，移植修復病變的組織器官，作為基因治療的靶細胞等相關實驗研究及臨床應用提供了豐富的種子細胞來源。
[Abstract]:Stem cells are a group of cells with self-renewal, high proliferative and multidirectional differentiation potential in vivo. Under certain conditions, stem cells can be differentiated into a variety of functional cells. Stem cells are the primordium of regenerative medicine and can be used in tissue repair, organ regeneration, gene therapy and clinical therapy. At present, embryonic stem cells, bone marrow mesenchymal stem cells, umbilical cord blood stem cells, hair follicle mesenchymal stem cells and so on. Hair follicle mesenchymal stem cells, not only self-renewal, directional differentiation into fat, bone, chondrocytes, and, more importantly, hair follicle mesenchymal stem cells express embryonic stem cell markers (SOX2 and Nanog).) Hair follicles are rich in source, easy to obtain, and not restricted by age. They can be obtained repeatedly and can be obtained repeatedly. They are important seed cells for tissue repair, organ reconstruction and gene therapy. The research and treatment of regenerative medicine require a large number of seed cells. The traditional plane culture method is difficult to obtain a large number of seed cells in a short time, which can be solved by using microcarriers and bioreactor technology. The microcarriers have large surface area / volume (S / V), high cell yield per unit volume, and the advantages of stereoscopic culture and suspension and adherent culture, which simplify the detection and control of the environmental factors of cell growth, with good reproducibility, low cost and low labor intensity. A large number of hair follicle mesenchymal stem cells can be obtained by stereoscopic culture. However, the growth and biological characteristics of hair follicle mesenchymal stem cells are unknown. The purpose of this study was to establish a bioreactor for the proliferation of human hair follicle mesenchymal stem cells (human hair folliclemesenchymal stem cells hHF-MSCs) in vitro, and to provide undifferentiated and large numbers of seed cells for the study of regenerative medicine and clinical treatment. The adult hair was extracted and the hHF-MSCs was successfully obtained by tissue mass culture. We successfully inoculated the obtained hHF-MSCs onto the vector, stained with acridine orange, and scanned by confocal microscope. We observed that the hHF-MSCs was attached to the surface of the carrier and stretched in the form of fusiform. 1000cells/cm~2 or 500cells/cm~2 density was used to inoculate the hHF-MSCs onto the carrier. The cells entered the logarithmic growth phase on the 3rd day of inoculation, the cell proliferation reached the plateau stage on the 15th day, and the logarithmic growth phase on the sixth day after hHF-MSCs was cultured on the plane. Flow cytometry analysis showed that the expression rates of CD90 CD105 were 80.25% and 45.36%, respectively, and the expression rates of CD105 were 85.65% in plane culture, 85.65% in hHF-MSCs and 43.88% in plane culture, respectively. After induced by lipogenesis, oil red O was stained with lipid droplets, and alizarin red was stained with alizarin red after osteogenic induction. Calcium salt deposition and calcium nodules were stained red after osteogenesis. In this study, hHF-MSCs was successfully obtained, and a large amount of hHF-MSCs bioreactor was established in vitro. The hHF-MSCs cultured with this technique can still maintain the surface marker of MSCs and have the potential of multi-differentiation. This is the late stage of establishing hHF-MSCs bank and constructing tissue engineering organs. Transplantation and repair of diseased tissues and organs, as target cells for gene therapy and other related experimental studies and clinical applications provide abundant seed cell sources.
【學位授予單位】：吉林大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R329

【引證文獻】

相關碩士學位論文 前1條

1 郭黎黎;毛囊干細胞修復皮膚損傷的實驗研究[D];吉林大學;2013年

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