【摘要】： 一、研究背景 各種損傷特別是大面積燒傷導(dǎo)致皮膚缺損的治療,是盡快封閉創(chuàng)面,由于自體皮源的不足,常導(dǎo)致治療的失敗。組織工程化皮膚研究一直是當(dāng)前研究的熱點(diǎn),目前尚無(wú)特別有效的皮膚替代物應(yīng)用于臨床。而表皮干細(xì)胞(keratinocyte stem cell, KSC)具有強(qiáng)大的增殖能力,又可向各個(gè)階段表皮細(xì)胞分化以維持皮膚新陳代謝,因此KSC作為皮膚重建的“種子細(xì)胞”成為研究的熱點(diǎn)。如何在體外培養(yǎng)獲得足夠數(shù)量的KSC用于研究及移植,如何在體內(nèi)外誘導(dǎo)KSC定向分化,對(duì)于組織工程皮膚具有重要意義。解決這些問(wèn)題的關(guān)鍵在于研究KSC增殖與分化的調(diào)控機(jī)制。整合素β1是KSC未分化的分子標(biāo)記之一,Jones等發(fā)現(xiàn)高表達(dá)整合素β1的表皮細(xì)胞具有更高的克隆形成能力,傳代次數(shù)顯著多于低表達(dá)的細(xì)胞[1]。。本室前期通過(guò)構(gòu)建整合素β1小RNA干擾載體,轉(zhuǎn)染KSC,發(fā)現(xiàn)下調(diào)表達(dá)整合素β1蛋白,實(shí)驗(yàn)組克隆形成率比對(duì)照組降低,促進(jìn)KSC的分化[2]。提示整合素β1參與KSC的增殖分化的調(diào)控。目前雖然對(duì)整合素β1參與KSC增殖與分化的調(diào)控研究比較多,但是整合素β1的表達(dá)調(diào)控的機(jī)制尚不清楚。 體外實(shí)驗(yàn)研究中,KSC通常從不同的個(gè)體分離和純化,存在個(gè)體差異和不穩(wěn)定性,因此尋找一種穩(wěn)定細(xì)胞模型對(duì)KSC進(jìn)行分化與增殖調(diào)控機(jī)制的研究具有重要意義。HaCaT是來(lái)源于正常成人上皮細(xì)胞的表皮細(xì)胞株,具有無(wú)限的增殖能力。研究表明,將HaCaT移植到裸鼠上,HaCaT可以形成分化上皮組織,表達(dá)特異性分化蛋白標(biāo)志,因此HaCaT可以為研究人表皮細(xì)胞分化的調(diào)控提供一個(gè)非常理想的模型[3]。研究擬應(yīng)用整合素β1啟動(dòng)子熒光素酶報(bào)告基因系統(tǒng),以HaCaT細(xì)胞為模型,探討表皮細(xì)胞中整合素β1對(duì)增殖分化的影響,以及整合素β1的表達(dá)調(diào)節(jié)機(jī)制,為進(jìn)一步研究表皮干細(xì)胞的增殖分化機(jī)制奠定基礎(chǔ)。 二、研究目的 1、下調(diào)人表皮細(xì)胞株HaCaT整合素β1,觀察其增殖的情況。 2、研究整合素β1啟動(dòng)子在HaCaT中的活性,為研究干細(xì)胞分化與增殖調(diào)控機(jī)制提供理論基礎(chǔ)。
[Abstract]:Background: the treatment of skin defect caused by various kinds of injuries, especially large area burn, is to close the wound as soon as possible, because of the insufficiency of autogenous skin source, which often leads to the failure of treatment. Tissue engineered skin research has been the focus of current research, and there is no especially effective skin substitute for clinical application. The epidermal stem cell (keratinocyte stem cell, KSC) has strong proliferative ability and can differentiate into epidermal cells to maintain skin metabolism. Therefore, KSC as a "seed cell" for skin reconstruction has become a hot research topic. How to obtain enough KSC in vitro for research and transplantation, and how to induce directional differentiation of KSC in vitro and in vivo are of great significance for tissue engineering skin. The key to solve these problems is to study the regulation mechanism of KSC proliferation and differentiation. Integrin 尾 1 is one of the undifferentiated molecular markers of KSC. Jones et al found that the epidermal cells with high integrin 尾 1 expression had higher clone forming ability, and the number of passages was significantly higher than that of low expression cells [1]. In the early stage of this study, integrin 尾 1 small RNA interference vector was constructed, and down-regulated expression of integrin 尾 1 protein was found in KSC, transfection. The clone formation rate of the experimental group was lower than that of the control group, and the differentiation of KSC was promoted [2]. These results suggest that integrin 尾 1 is involved in the regulation of KSC proliferation and differentiation. Although there are many studies on integrin 尾 1 involved in KSC proliferation and differentiation, the mechanism of integrin 尾 1 expression regulation is not clear. In vitro, KSC is usually isolated and purified from different individuals. Therefore, it is of great significance to study the mechanism of differentiation and proliferation of KSC in a stable cell model. HaCaT is an epidermal cell line derived from normal adult epithelial cells and has unlimited proliferative ability. The results showed that HaCaT could form differentiated epithelial tissue and express specific differentiation protein markers by transplanting HaCaT to nude mice. Therefore, HaCaT could provide a very ideal model for studying the regulation of differentiation of human epidermal cells [3]. To study the effect of integrin 尾 1 on proliferation and differentiation and the regulation mechanism of integrin 尾 1 expression in epidermal cells using integrin 尾 1 promoter luciferase reporter gene system as a model of HaCaT cells. It lays a foundation for further study on the mechanism of proliferation and differentiation of epidermal stem cells. Objective 1. To down-regulate HaCaT integrin 尾 1 and observe its proliferation. 2. To study the activity of integrin 尾 1 promoter in HaCaT, and to provide a theoretical basis for studying the mechanism of stem cell differentiation and proliferation.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2006
【分類(lèi)號(hào)】：R329

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