【摘要】： 組織工程學(xué)是20世紀(jì)80年代提出的一個(gè)概念,是指遵循發(fā)育生物學(xué)的規(guī)律,應(yīng)用細(xì)胞生物學(xué)和工程學(xué)原理,將人體某部分的組織細(xì)胞種植在某種生物支架上人工培養(yǎng),然后移植到人體所需部位,從而達(dá)到器官修復(fù)或再造的治療目的的一種技術(shù)。它的三要素包括種子細(xì)胞、支架材料和細(xì)胞因子。間充質(zhì)干細(xì)胞(mesenchymal stem cells,MSCs)體外增殖能力強(qiáng)、具有多向分化潛能,成為了組織工程的首選種子細(xì)胞。目前,人們已從多種組織(骨髓、脂肪、臍血等)中分離到了間充質(zhì)干細(xì)胞,但普遍存在來源受限、年齡的限制、對(duì)機(jī)體的損傷、倫理爭(zhēng)議、免疫排斥反應(yīng)和潛在致瘤性等問題,限制了其應(yīng)用。新近研究表明,毛囊中也存在間充質(zhì)樣干細(xì)胞,且其具有組織來源豐富,取材方便,對(duì)機(jī)體無損害,無倫理問題,免疫原性低,未見致瘤性等優(yōu)點(diǎn),可能成為再生醫(yī)學(xué)中理想的種子細(xì)胞。 本實(shí)驗(yàn)中,我們?cè)噲D建立一種簡(jiǎn)單、方便的人毛囊間充質(zhì)樣干細(xì)胞(hHF-MSCs,human hair follicle-derived mesenchymal-like stem cells)的分離方法,并對(duì)分離的細(xì)胞進(jìn)行鑒定,從而為組織工程和再生醫(yī)學(xué)提供理想的種子細(xì)胞。我們通過拔取成人毛發(fā),并進(jìn)行組織塊培養(yǎng),獲得了成纖維樣的細(xì)胞,經(jīng)流式細(xì)胞儀分析顯示,這些細(xì)胞高表達(dá)間充質(zhì)干細(xì)胞表面標(biāo)志物CD29、CD44、CD73、CD90、CD105,陽性率依次是97.8%、88.9%、97%、58.2%、57.8%;細(xì)胞周期分析顯示,85.909 %的細(xì)胞處于G.0/G1期(靜息期), 8.461%的細(xì)胞處于S期,5.630%的細(xì)胞處于G2期;對(duì)細(xì)胞進(jìn)行成脂誘導(dǎo)后,經(jīng)油紅O染色可見脂肪細(xì)胞的形成;對(duì)細(xì)胞進(jìn)行成骨誘導(dǎo)后,經(jīng)硝酸銀染色和茜素紅染色顯示,可見鈣鹽沉積和鈣結(jié)節(jié)的形成,經(jīng)堿性磷酸酶染色顯示,誘導(dǎo)組堿性磷酸酶活性顯著增高,RT-PCR檢測(cè)成骨細(xì)胞標(biāo)志物OCN的mRNA表達(dá)也顯示,誘導(dǎo)組OCN的mRNA表達(dá)也明顯高于對(duì)照組。結(jié)果表明,此方法獲得的細(xì)胞含有高純度的間充質(zhì)樣干細(xì)胞,具有慢周期性和多向分化潛能,可誘導(dǎo)分化為脂肪細(xì)胞和成骨細(xì)胞。 本研究建立了一種通過直接拔取成人毛發(fā)獲取人成體毛囊間充質(zhì)樣干細(xì)胞的方法,此方法簡(jiǎn)單、方便、對(duì)機(jī)體無損傷,這為毛囊干細(xì)胞的深入研究和臨床應(yīng)用奠定了基礎(chǔ),使得毛囊間充質(zhì)干細(xì)胞可能成為再生醫(yī)學(xué)中理想的種子細(xì)胞。
[Abstract]:Tissue engineering is a concept proposed in the 1980s. It refers to the application of cell biology and engineering principles to the artificial cultivation of tissue cells in a certain part of the human body on a biological scaffold. A technique that is then transplanted to the desired site of the human body for the purpose of organ repair or reconstruction. Its three elements include seed cells, scaffold materials and cytokines. Mesenchymal stem cell (mesenchymal stem cells,MSCs) has the ability to proliferate in vitro and has the potential to differentiate into many directions, so it has become the first choice of seed cells in tissue engineering. At present, mesenchymal stem cells have been isolated from various tissues (bone marrow, fat, umbilical cord blood, etc.). Immune rejection and potential tumorigenicity limit its application. Recent studies have shown that mesenchymal stem cells also exist in hair follicles, and they have the advantages of abundant tissue sources, convenient material collection, no damage to the body, no ethical problems, low immunogenicity, no tumorigenicity, etc. It may be an ideal seed cell in regenerative medicine. In this experiment, we try to establish a simple and convenient method for the isolation of human hair follicle mesenchymal stem cells (hHF-MSCs,human hair follicle-derived mesenchymal-like stem cells) and identify the isolated cells, so as to provide ideal seed cells for tissue engineering and regenerative medicine. We extracted adult hair and cultured tissue blocks to obtain fibroblast cells, which were analyzed by flow cytometry. The positive rate of CD29,CD44,CD73,CD90,CD105, on the surface of mesenchymal stem cells was 97.88% and 88.9% and 58.2% and 57.8% respectively. Cell cycle analysis showed that 85.909% of the cells were in the G.0/G1 phase (resting phase), 8.461% in S phase, 5.630% in G2 phase, and that after fat-forming induction, 85.909% of the cells were in the phase of G.0/G1 (resting phase), 8.461% of the cells were in the phase of S, and 5.630% of the cells were in the phase of G2. The formation of adipocytes was observed by oil red O staining. After osteogenic induction, calcium salt deposition and calcium nodule formation were observed by silver nitrate staining and alizarin red staining. The activity of alkaline phosphatase in the induction group was significantly higher than that in the control group. The mRNA expression of osteoblast marker OCN was also detected by RT-PCR, and the mRNA expression of OCN in the induced group was significantly higher than that in the control group. The results showed that the cells obtained by this method contained high purity mesenchymal stem cells, which had the potential of slow cycle and multiple differentiation, and could induce adipocytes and osteoblasts to differentiate into adipocytes and osteoblasts. This study established a method to obtain human adult hair follicle mesenchymal stem cells by direct extraction of adult hair. This method is simple, convenient and harmless to the body, which lays a foundation for the further study and clinical application of hair follicle stem cells. Hair follicle mesenchymal stem cells may be the ideal seed cells in regenerative medicine.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R329

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