【作者】 赵周婷；

【导师】 胡大海； 朱雄翔；

【作者基本信息】 第四军医大学 ， 外科学， 2010， 硕士

【摘要】 【研究背景】皮肤缺损是外科中的常见问题。尽早封闭创面,减少机体消耗和内环境紊乱是治疗皮肤缺损的关键。烧伤外科中大面积烧伤病人皮源缺乏,自体皮肤移植是目前临床治疗全层皮肤缺损最有效的方法。但是自体刃厚皮移植皮片易收缩,不耐摩擦,中厚皮有瘢痕挛缩等问题。随着组织工程技术的发展,皮肤替代物的研究取得了实质性的进展。种子细胞的体外培养、扩增是组织工程学研究中最基础的工作。2001年,Zuk [1]等首次分离培养出能够自我复制、稳定增殖,并具有多向分化潜能的脂肪间充质干细胞。ADSCs来自中胚层,在特定条件下可分化为脂肪、骨、软骨、心肌细胞、肝细胞、神经细胞等[1‐4]。ADSCs具有来源丰富、方便取材、低免疫原性、容易体外扩增等优点,在组织工程、创面修复及基因治疗方面具有良好的应用前景。近年来研究表明,ADSCs在体外培养过程中加入不同的刺激因子,模拟皮肤的微环境,能诱导成为具有各种表型的细胞。Ramos等[5]证实,将分选纯化后的ADSCs植入裸鼠体内,可显著促进创面愈合,且具有表皮细胞再生的能力。人角质形成细胞是皮肤创面修复的重要细胞,而表皮生长因子(epithelial growth factor,EGF)是创面修复过程中的关键因子。EGF能促进细胞增殖、表皮再生、血管形成,同时能刺激表皮细胞合成分泌胶原、透明质酸等细胞外基质,促进结缔组织细胞的生长[6]。Shikiji等[7]成功地应用EGF诱导体外培养于立体胶原表面的人表皮细胞向胶原内生长并形成汗腺管结构。因此,本实验研究利用pcDNA3.1(+)/SP+EGF质粒经脂质体介导转染的HaCat细胞诱导人ADSCs体外转化为表皮细胞表型的可行性,为其在组织工程中的应用提供理论依据。【研究目的】1、探索高效分离和扩增人脂肪干细胞的方法,观察其生物学特性,并通过形态学、免疫表型及多向分化能力进行鉴定。2、探索人脂肪间充质干细胞向表皮细胞表型转化的方法。【研究内容】1、胶原酶消化法分离培养ADSCs,利用形态学及MTT比色法观察细胞生物学特性。2、通过流式细胞仪及诱导多向分化鉴定ADSCs。3、利用转染EGF的HaCat细胞,与ADSCs共培养,诱导ADSCs向表皮细胞分化。4、利用外源性EGF诱导ADSCs向表皮细胞分化。【研究结果】1、原代和传代细胞呈梭形外观,生长增殖能力良好。细胞传代后2天内处于潜伏期,第3天进入生长期,5天后进入平台期。2、流式细胞仪检测CD29、CD31、CD34、CD45、CD90、CD105阳性率分别为73.4%、3.6%、4.5%、2.0%、97.3%、80.4%。经定向诱导分化后,细胞分别呈现脂肪细胞、骨细胞的表型特征。3、ADSCs与转染EGF的HaCat细胞共培养后ADSCs表达表皮细胞特征性表面抗原,提示利用此方法可诱导ADSCs向表皮细胞分化。4、加入EGF刺激后ADSCs中表皮细胞表面抗原CK19、integrin-β的mRNA水平随时间增加而上升,诱导4w后时表达最高。【研究结论】1、酶消化法能有效分离纯化人ADSCs,细胞生长稳定,增殖能力活跃,具有ADSCs的一般生物学特性。2、转染EGF的HaCat细胞可诱导ADSCs向表皮细胞表型分化,从而为其成为组织工程理想的种子细胞提供了进一步的支持。3、EGF在诱导ADSCs向表皮细胞分化的过程中起着决定性作用,4、合理有效地利用ADSCs在创面愈合的研究与治疗方面具有广阔的前景。更多还原

【Abstract】 【Background】Skin defects are common problems in surgery. Wound closure as soon as possible to reduce the body’s consumption and disorder within the environment is the key to the treatment of skin defects. Autologous skin graft is the most effective way for large area burns patients of full thickness skin defects in burns surgery clinical treatment. However, autologous thin skin graft show contraction and not rubbing, and the thick skin bring out scar and other problems. With the development of tissue engineering, skin substitutes made substantial progress in research. Seed cells amplification in vitro is the most basic tissue engineering work. In 2001, Zuk et al first isolated and cultured, such as self-replicating, stable and proliferation, and differentiation potential of multipotent mesenchymal stem cells in adipose tissue, called adipose derived stem cells. ADSCs from the mesoderm, under certain conditions can differentiate into fat, bone, cartilage, heart cells, liver cells, nerve cells, etc. ADSCs have the advantages of rich source, easily derived, low immunogenicity, so it has a good prospect in tissue engineering, wound healing and gene therapy. Recent studies show that, ADSCs can be induced to other cells with various phenotypes when cultured in vitro to stimulate the process of adding different factors to simulate the skin’s micro-environment.Ramos et al confirmed that implanted the purified ADSCs in nude mice, can significantly promote wound healing, and ADSCs has a capacity of epidermal cell regeneration. Human keratinocytes is important to wound healing, and epidermal growth factor (EGF) is the key factor of wound healing process. EGF can promote cell proliferation, epidermal regeneration, angiogenesis, and also could stimulate epithelial cells synthesis and secretion of collagen, hyaluronic acid and other extracellular matrix, promote the growth of connective tissue cells.Shikiji et al successfully applied EGF to induct human epithelial cells that were cultured on the surface of three-dimension collagen, and then formated tube structure of sweat glands. Therefore, our research use pcDNA3.1 (+) / SP + EGF plasmid liposome transfected HaCat cells induced ADSCs into epithelial cells in vitro phenotype, and find the feasibility of its application in tissue engineering provide a theoretical basis. 【Purposes】1、To explore a method for effective isolation and multiplication of human adipose derived mesenchymal stem cells, observe its biological characteristics and verification by morphology and multi-potential differentiation.2、To explore a method that inverts human adipose derived mesenchymal stem cells into epithelial cells.【Contents】1、ADSCs were isolated and cultured by collagenase digestion, and morphological and biological characteristics of cells were observed by inverse microscope and MTT assay.2、ADSCs were identified by flow cytometry and the induction of different cells types.3、ADSCs and HaCat cells transfected with EGF were co-cultured, then the former could be induced into epithelial cells types.4、Use of exogenous EGF to induce ADSCs into epithelial cells types.【Results】1、The ADSCs in both primary and passage culture were fusiform shapes, and stable in growth with active proliferation. ADSCs were in latency for 2 days, converted into growth period on the 3rd day and entered the stationary phase on the 5th day.2、FCM results indicated that the positive rate of CD29, CD31, CD34, CD45, CD90, CD105 were respectively 73.4%, 3.6%, 4.5%, 2.0%, 97.3%, 80.4%. After purposively induced, ADSCs had the phenotype characterisitics of adipocyte or osteocyte.3、HaCat cells transfected with EGF could induce ADSCs to invert to epithelial cells, which will further make ADSCs as ideal seed cells for tissue engineering.【Conclusions】1、The methods of stirring digestion within collagenase can be effectively used to isolate and purify human ADSCs. The cultured ADSCs are stable in growth with active proliferation and share the general biological characterisitics of ADSCs.2、HaCat cells transfected with EGF could induce ADSCs into the epithelial cells phenotypic, which will further make ADSCs as ideal seed cells for tissue engineering.3、After adding EGF, the mRNA expression level of the epidermal cells surface antigen such as CK19, integrin-βin ADSCs were increased with time up, and the expression level reach to the highest after 4 weeks.4、Reasonable and effective use of ADSCs in wound healing research and has broad prospects for treatment.更多还原