人毛囊源性间充质样干细胞的分离培养与成骨诱导

发布时间：2018-10-25 10:44

【摘要】： 组织工程学是20世纪80年代提出的一个概念,是指遵循发育生物学的规律,应用细胞生物学和工程学原理,将人体某部分的组织细胞种植在某种生物支架上人工培养,然后移植到人体所需部位,从而达到器官修复或再造的治疗目的的一种技术。它的三要素包括种子细胞、支架材料和细胞因子。间充质干细胞(mesenchymal stem cells,MSCs)体外增殖能力强、具有多向分化潜能,成为了组织工程的首选种子细胞。目前,人们已从多种组织(骨髓、脂肪、脐血等)中分离到了间充质干细胞,但普遍存在来源受限、年龄的限制、对机体的损伤、伦理争议、免疫排斥反应和潜在致瘤性等问题,限制了其应用。新近研究表明,毛囊中也存在间充质样干细胞,且其具有组织来源丰富,取材方便,对机体无损害,无伦理问题,免疫原性低,未见致瘤性等优点,可能成为再生医学中理想的种子细胞。本实验中,我们试图建立一种简单、方便的人毛囊间充质样干细胞(hHF-MSCs,human hair follicle-derived mesenchymal-like stem cells)的分离方法,并对分离的细胞进行鉴定,从而为组织工程和再生医学提供理想的种子细胞。我们通过拔取成人毛发,并进行组织块培养,获得了成纤维样的细胞,经流式细胞仪分析显示,这些细胞高表达间充质干细胞表面标志物CD29、CD44、CD73、CD90、CD105,阳性率依次是97.8%、88.9%、97%、58.2%、57.8%;细胞周期分析显示,85.909 %的细胞处于G.0/G1期(静息期), 8.461%的细胞处于S期,5.630%的细胞处于G2期;对细胞进行成脂诱导后,经油红O染色可见脂肪细胞的形成;对细胞进行成骨诱导后,经硝酸银染色和茜素红染色显示,可见钙盐沉积和钙结节的形成,经碱性磷酸酶染色显示,诱导组碱性磷酸酶活性显著增高,RT-PCR检测成骨细胞标志物OCN的mRNA表达也显示,诱导组OCN的mRNA表达也明显高于对照组。结果表明,此方法获得的细胞含有高纯度的间充质样干细胞,具有慢周期性和多向分化潜能,可诱导分化为脂肪细胞和成骨细胞。本研究建立了一种通过直接拔取成人毛发获取人成体毛囊间充质样干细胞的方法,此方法简单、方便、对机体无损伤,这为毛囊干细胞的深入研究和临床应用奠定了基础,使得毛囊间充质干细胞可能成为再生医学中理想的种子细胞。
[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.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R329

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