integrin β1对rMSCs增殖和分化影响的初步研究
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摘要
创面处理是烧伤救治的重要环节之一,而创面的修复又是一个十分复杂的生物学过程,包含着炎症反应、细胞增殖等过程。目前,烧伤创面修复方法主要是以局部治疗为主。大面积烧伤患者由于自体皮源不足,多年来运用网状皮、微粒植皮等技术来扩大皮肤覆盖面积,取得了一定的效果。但这些方法扩增面积有限,难以满足大面积皮肤缺损患者尽早封闭创面的需求,这些常规的方法显然还存在着很多的不足。组织工程学的兴起和发展,为临床解决这一难题提供了崭新的思路和途径。近年来,由于骨髓间充质干细胞(mesenchymal stem cells,MSCs)具有易取材、方便导入外源基因、自体移植不存在排斥反应等优点,在创伤修复方面的作用日益受到各国学者的关注。成体骨髓来源的MSCs是存在于骨髓基质内的非造血细胞来源的细胞亚群,它们可以在体外经诱导后最终分化为成骨细胞、软骨细胞、脂肪细胞、肌腱细胞、肌管、神经细胞与支持造血干细胞的基质。有学者将自体骨髓来源的细胞应用于经久不愈的皮肤慢性溃疡创面后观察到这些创面均顺利愈合并且能减轻瘢痕组织的产生,并有学者发现利用bFGF诱导MSCs向表皮细胞分化的过程中,MSCs的表面标记物integrinβ1明显下降并呈时间依赖性。由此可见MSCs有可能作为皮肤组织工程的种子细胞,参与烧伤创面的愈合,且integrinβ1与MSCs的分化具有一定的相关性。而本室致力于表皮干细胞的研究,先期已应用RNA干扰的方法,发现下调integrinβ1的表达是表皮干细胞走向分化的重要调控因素之一。
基于以上认识,本研究拟通过体外培养获得足够数量的MSCs,利用表皮生长因子(EGF)在培养表皮干细胞的FAD培养液环境下来探究诱导MSCs向表皮细胞分化的可能性,并探索integrinβ1的表达与rMSCs的增殖状态的相关性。
第一部分大鼠骨髓间充质干细胞的分离、纯化及鉴定
目的:利用直接贴壁法和percoll密度梯度离心法从大鼠骨髓中分离纯化MSCs。优化MSCs的分离方法,得到高纯度的MSCs,为深入研究MSCs创造条件。
方法:利用直接贴壁法和percoll密度梯度离心法从大鼠骨髓中分离纯化MSCs,培养、传代。同时观察两种方法在获得原代细胞的不同,测第1、3、5代细胞的生长曲
Background: Treatment of the wounds plays an important role in the management of burns. The process of wound repair is very sophistic, including inflammatory response, cell proliferation, etc. Up to now, the traditional approach for wound repair is autologous skin grafting, which often results in heavy pigmentation and scarring in both donor sites and recipient sites. In addition, limited area of repaired skin can’t meet the need of wound repair. Over the past 25 years, tissue engineering has developed rapidly.It is a new frontier science which combines knowledge from the biology, material and engineering science.The main objective of tissue engineering is to develop new approaches to repair tissue and to develop replacement for tissues.With the development of tissue engineering, it is possible to replace the defected or injured organs with tissue-engineered ones.
The seeding cells are the key point in skin tissue engineering.However, poor proliferative poitential of epidermal cells obtained from culture in vitro limits its further use.Most cells in cultured sheet in vitro had differentiated and lost most proliferative ability.And due to ethic and technical reasons, embryonic stem cells still has a very long way to clinic use.Therefore, recent research focuses on adult stem cells.
Recent evidence indicates that bone marrow contains stem cells (mesenchymal stem cells) with the potential to differentiate into various tissues, including bone, cartilage, fat, tendon, muscle, etc. As can be easily acquired and transfected with exogenous gene covenniently, much attention have been given to the cells in tissue engineering.Reasearch indicated that autologous cells from bone marrow can accelerate the healing of chronic skin ulcer and reduce scar.Some scholar induced MSCs to differentiate into epidermal cells by bFGF, and found that following the differentiation,surface marker integrinβ1 decreased.It indicates that MSCs can migrate into the wounds during the course of wound healing, and act as seeding cells of skin engineering. What’s more, there is correlation between integrin
基于以上认识,本研究拟通过体外培养获得足够数量的MSCs,利用表皮生长因子(EGF)在培养表皮干细胞的FAD培养液环境下来探究诱导MSCs向表皮细胞分化的可能性,并探索integrinβ1的表达与rMSCs的增殖状态的相关性。
第一部分大鼠骨髓间充质干细胞的分离、纯化及鉴定
目的:利用直接贴壁法和percoll密度梯度离心法从大鼠骨髓中分离纯化MSCs。优化MSCs的分离方法,得到高纯度的MSCs,为深入研究MSCs创造条件。
方法:利用直接贴壁法和percoll密度梯度离心法从大鼠骨髓中分离纯化MSCs,培养、传代。同时观察两种方法在获得原代细胞的不同,测第1、3、5代细胞的生长曲
Background: Treatment of the wounds plays an important role in the management of burns. The process of wound repair is very sophistic, including inflammatory response, cell proliferation, etc. Up to now, the traditional approach for wound repair is autologous skin grafting, which often results in heavy pigmentation and scarring in both donor sites and recipient sites. In addition, limited area of repaired skin can’t meet the need of wound repair. Over the past 25 years, tissue engineering has developed rapidly.It is a new frontier science which combines knowledge from the biology, material and engineering science.The main objective of tissue engineering is to develop new approaches to repair tissue and to develop replacement for tissues.With the development of tissue engineering, it is possible to replace the defected or injured organs with tissue-engineered ones.
The seeding cells are the key point in skin tissue engineering.However, poor proliferative poitential of epidermal cells obtained from culture in vitro limits its further use.Most cells in cultured sheet in vitro had differentiated and lost most proliferative ability.And due to ethic and technical reasons, embryonic stem cells still has a very long way to clinic use.Therefore, recent research focuses on adult stem cells.
Recent evidence indicates that bone marrow contains stem cells (mesenchymal stem cells) with the potential to differentiate into various tissues, including bone, cartilage, fat, tendon, muscle, etc. As can be easily acquired and transfected with exogenous gene covenniently, much attention have been given to the cells in tissue engineering.Reasearch indicated that autologous cells from bone marrow can accelerate the healing of chronic skin ulcer and reduce scar.Some scholar induced MSCs to differentiate into epidermal cells by bFGF, and found that following the differentiation,surface marker integrinβ1 decreased.It indicates that MSCs can migrate into the wounds during the course of wound healing, and act as seeding cells of skin engineering. What’s more, there is correlation between integrin
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