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  本文關(guān)鍵詞：小鼠胚胎干細胞體外建系及電轉(zhuǎn)染K14-EGFP基因的實驗研究　出處：《福建醫(yī)科大學(xué)》2008年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 胚胎干細胞 皮膚細胞 誘導(dǎo)分化 電轉(zhuǎn)染 角蛋白14 實驗

【摘要】： 背景:目前,皮膚缺損是臨床上經(jīng)常遇到的問題,應(yīng)用組織工程方法構(gòu)建人工皮膚并應(yīng)用于臨床是研究的熱點。在組織工程化皮膚的研究中,需要能快速大量擴增、長期體外傳代培養(yǎng)、細胞功能旺盛的種子細胞。由于胚胎干細胞(Embryonic stem cells,ESC)有很高的再生及多向分化的潛能,以胚胎干細胞誘導(dǎo)分化而來的皮膚細胞為種子細胞構(gòu)建組織工程化皮膚是一種很好的研究方法,而目前,國內(nèi)外學(xué)者對胚胎干細胞誘導(dǎo)分化為皮膚細胞的深入研究并不多。 目的:本實驗旨在研究和建立帶有角蛋白10-增強型綠色熒光蛋白(Keratin14- Enhanced Green Fluorescence Protein, K14-EGFP)基因的小鼠胚胎干細胞系并進行鑒定,為組織工程化皮膚的基礎(chǔ)研究及臨床應(yīng)用打下堅實的基礎(chǔ)。 方法:首先體外建系129/S1小鼠的胚胎干細胞,并對其進行形態(tài)學(xué)、堿性磷酸酶染色(Alkaline Phosphatase,AKP)、階段特異性胚胎表面抗原-1(Stage-specific embryonic surface antigen 1,SSEA-1)免疫組化染色、體內(nèi)及體外分化實驗等的鑒定分析,證明其胚胎干細胞的特性,再對建系成功的胚胎干細胞進行K14-EGFP基因電轉(zhuǎn)染,并對轉(zhuǎn)染成功的胚胎干細胞進行鑒定包括體內(nèi)外向分化能力鑒定。 結(jié)果: 一.成功建立兩株129/S1小鼠的胚胎干細胞系,并進行了形態(tài)學(xué)、堿性磷酸酶染色、階段特異性表面抗原-1、體內(nèi)及體外分化實驗等的指標鑒定。 二.對建系成功的胚胎干細胞進行K14-EGFP(抗G418)電轉(zhuǎn)染,經(jīng)G418篩選的陽性克隆進行體外擴增,獲得大量帶有K14-EGFP基因片段的小鼠胚胎干細胞。 結(jié)論: 一.利用生長3.5天的小鼠囊胚分離培養(yǎng)胚胎干細胞,并且在建系培養(yǎng)過程中需應(yīng)用MEF制備的飼養(yǎng)層和白血病抑制因子來促進胚胎干細胞的增殖和抑制其分化; 二.胚胎干細胞的電轉(zhuǎn)染效率很低,但是轉(zhuǎn)染成功的細胞經(jīng)體外擴增可獲得足夠數(shù)量的轉(zhuǎn)染陽性細胞; 三.轉(zhuǎn)染K14-EGFP片段基因的胚胎干細胞經(jīng)誘導(dǎo)有皮膚細胞表面標志物角蛋白14表達時就會有綠色熒光蛋白的表達。
[Abstract]:Background: at present, skin defect is a common problem in clinic. The application of tissue engineering to artificial skin is a hot topic in the research of tissue engineered skin. It needs seed cells that can be rapidly expanded, subcultured in vitro for a long period of time, and have high cell function. Because of embryonic stem cells, Embryonic stem cells. ESCC has a high potential for regeneration and multi-differentiation. It is a good method to construct tissue-engineered skin with skin cells derived from embryonic stem cells as seed cells. There are few researches on the induction and differentiation of embryonic stem cells into skin cells at home and abroad. Objective: to study and establish a new type of green fluorescent protein (GFP) with keratin 10 (K10)-enhanced green fluorescent protein (GFP). Keratin14-Enhanced Green Fluorescence Protein. The mouse embryonic stem cell lines of K14-EGFP gene were identified, which laid a solid foundation for the basic research and clinical application of tissue engineering skin. Methods: the embryonic stem cells of 129 / S1 mice were first established in vitro, and were morphologically stained with alkaline phosphatase (Alkaline Phosphatase). Stage-specific embryonic surface antigen 1. SSEA-1) immunohistochemical staining, in vivo and in vitro differentiation experiments, to prove the characteristics of its embryonic stem cells, and then successfully constructed embryonic stem cells for K14-EGFP gene electrotransfection. The successful transfection of embryonic stem cells was identified, including the ability of differentiation in vivo. Results: 1. Two embryonic stem cell lines of 129 / S1 mouse were successfully established and identified by morphology, alkaline phosphatase staining, phase specific surface antigen -1 and differentiation test in vitro and in vivo. 2. The successful embryonic stem cells were transfected with K14-EGFP (anti-G418), and the positive clones screened by G418 were amplified in vitro. A large number of mouse embryonic stem cells with K14-EGFP gene fragment were obtained. Conclusion: 1. Embryonic stem cells were isolated and cultured from mouse blastocysts for 3.5 days. The feeder layer and leukemia inhibitor prepared by MEF should be used to promote the proliferation and inhibit the differentiation of embryonic stem cells. 2. The efficiency of electrotransfection of embryonic stem cells is very low, but enough positive cells can be obtained by amplification of transfected cells in vitro. 3. The expression of green fluorescent protein was found in the embryonic stem cells transfected with K14-EGFP gene when it was induced to express keratin 14 on the skin cell surface.
【學(xué)位授予單位】：福建醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2008
【分類號】：R329.21

【引證文獻】

相關(guān)博士學(xué)位論文 前1條

1 梁洋;蜘蛛拖絲蛋白基因Spidroin1嵌合體小鼠的研究[D];東北林業(yè)大學(xué);2011年

相關(guān)碩士學(xué)位論文 前1條

1 呂一帆;胚胎干細胞在內(nèi)膜損傷小鼠宮腔內(nèi)移植的實驗研究[D];福建醫(yī)科大學(xué);2010年

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本文編號：1432267