【摘要】： 目的1.構(gòu)建堿性成纖維細(xì)胞生長因子基因真核表達(dá)載體pEGFP-C3-bFGF;2.將pEGFP-C3-bFGF轉(zhuǎn)染至人表皮細(xì)胞,觀察基因瞬時(shí)表達(dá)情況及表皮細(xì)胞的形態(tài)和表型改變,建立bFGF基因誘導(dǎo)表皮細(xì)胞去分化的體外實(shí)驗(yàn)?zāi)Ｐ?3.檢測FGFR2在不同發(fā)育時(shí)期胎兒皮膚中的表達(dá)和分布,初步探索bFGF調(diào)控表皮細(xì)胞去分化的分子機(jī)制及FGFR2在皮膚發(fā)生、發(fā)育中的作用,為尋找調(diào)控表皮細(xì)胞去分化的內(nèi)在驅(qū)動(dòng)力提供實(shí)驗(yàn)基礎(chǔ),為深入研究皮膚的創(chuàng)傷修復(fù)與再生提供實(shí)驗(yàn)參考。方法1.參考分子克隆技術(shù),采用PCR法從PBR322-bFGF質(zhì)粒中擴(kuò)增bFGF基因,同時(shí)在其兩端引入HindⅢ和EcoRⅠ限制性內(nèi)切酶酶切位點(diǎn),將bFGF cDNA定向插入pEGFP-C3的多克隆位點(diǎn)中,構(gòu)建真核表達(dá)質(zhì)粒pEGFP-C3-bFGF,并對(duì)重組子進(jìn)行DNA序列測定。2.采用脂質(zhì)體(Lipofectamine~(TM) 2000)轉(zhuǎn)染法,將pEGFP-C3-bFGF轉(zhuǎn)染至人表皮細(xì)胞系HEKa細(xì)胞中,在熒光顯微鏡下觀察基因瞬時(shí)表達(dá)情況及細(xì)胞形態(tài)。以同期培養(yǎng)的表皮細(xì)胞及pEGFP-C3轉(zhuǎn)染的表皮細(xì)胞作為陰性對(duì)照,免疫細(xì)胞化學(xué)染色和免疫熒光標(biāo)記法檢測實(shí)驗(yàn)組及對(duì)照組細(xì)胞形態(tài)的改變和細(xì)胞中β_1整合素、CK19、CK14及CK10表達(dá)的差異。采用RT-PCR和Western-blot進(jìn)一步檢測pEGFP-C3-bFGF轉(zhuǎn)染后表皮細(xì)胞表型的改變,建立bFGF基因誘導(dǎo)表皮細(xì)胞去分化的體外實(shí)驗(yàn)?zāi)Ｐ汀?.對(duì)不同發(fā)育時(shí)期胎兒皮膚取材、固定、制成石蠟切片,S-P法檢測FGFR2的表達(dá)及分布。結(jié)果1.構(gòu)建了含有bFGF cDNA的真核表達(dá)載體pEGFP-C3-bFGF。2.熒光顯微鏡下觀察pEGFP-C3-bFGF基因轉(zhuǎn)染率為31.6%,轉(zhuǎn)染陽性細(xì)胞體積小,呈圓形或圓梭形,核質(zhì)比大。免疫細(xì)胞化學(xué)染色結(jié)果顯示,實(shí)驗(yàn)組細(xì)胞中CK19、CK14表達(dá)增強(qiáng),CK10表達(dá)減弱。免疫熒光染色結(jié)果顯示,轉(zhuǎn)染陽性細(xì)胞主要在細(xì)胞核、胞漿和胞膜中表達(dá)CK19和CK14,在胞膜上弱表達(dá)β_1整合素,而CK10表達(dá)為陰性。RT-PCR結(jié)果顯示,pEGFP-C3-bFGF轉(zhuǎn)染后表皮細(xì)胞中CK19、CK14 mRNA的轉(zhuǎn)錄水平明顯上調(diào),而CK10的mRNA表達(dá)明顯下降。Western blot檢測結(jié)果表明,pEGFP-C3-bFGF轉(zhuǎn)染后表皮細(xì)胞中CK19、CK14的表達(dá)明顯增強(qiáng),而CK10的總體表達(dá)減少。pEGFP-C3-bFGF轉(zhuǎn)染后表皮細(xì)胞發(fā)生去分化,重新獲得干細(xì)胞的表達(dá)標(biāo)志。3.胚胎發(fā)育期FGFR2在表皮細(xì)胞表達(dá)較弱,甚至呈陰性表達(dá)。表皮分層期時(shí),表皮層開始增厚,并可見毛囊的初級(jí)原基;FGFR2在表皮層和毛囊初級(jí)原基內(nèi)呈弱陽性表達(dá)。毛囊形成期時(shí),毛囊的結(jié)構(gòu)初步形成,體積細(xì)小,形態(tài)幼稚;FGFR2主要在毛母質(zhì)細(xì)胞內(nèi)表達(dá),并且隨著胎齡增加,表達(dá)范圍逐漸擴(kuò)大,表達(dá)量逐漸增多。毛囊發(fā)育成熟期后,毛囊的結(jié)構(gòu)發(fā)育相對(duì)成熟,FGFR2則在表皮層、毛細(xì)血管內(nèi)皮細(xì)胞及皮脂腺的導(dǎo)管部均有表達(dá),尤其在毛母質(zhì)細(xì)胞呈強(qiáng)陽性表達(dá)。結(jié)論1.將bF-GF基因插入pEGFP-C3的C末端,可以提高bFGF在真核細(xì)胞中的表達(dá),而且不影響其目的蛋白的結(jié)構(gòu)和功能。2.pEGFP-C3-bFGF能夠轉(zhuǎn)染至人表皮細(xì)胞并表達(dá),在pEG-FP-C3-bFGF轉(zhuǎn)染作用后,表皮細(xì)胞重新程序化,并獲得其前體干細(xì)胞的某些潛在能力,表達(dá)表皮干細(xì)胞的一些未分化蛋白,成功建立bFGF基因誘導(dǎo)表皮細(xì)胞去分化的體外實(shí)驗(yàn)?zāi)Ｐ?為進(jìn)一步闡述bFGF調(diào)控表皮細(xì)胞去分化的分子機(jī)制提供實(shí)驗(yàn)基礎(chǔ),為尋找調(diào)控表皮細(xì)胞去分化的內(nèi)在驅(qū)動(dòng)力提供實(shí)驗(yàn)參考。3.FGFR2在胎兒皮膚中的表達(dá)與分布與毛囊的發(fā)生發(fā)育密切相關(guān)。FGFR2的表達(dá)上調(diào)可能促進(jìn)毛囊干細(xì)胞的增殖,并誘導(dǎo)其定向分化形成終末組織功能細(xì)胞。
[Abstract]:Purpose 1. and constructing a basic fibroblast growth factor gene true nuclear expression vector pEGFP-C3-bFGF; pEGFP-C3-bFGF was transfected into human epidermal cells, the transient expression of the gene and the morphological and phenotypic changes of the epidermal cells were observed, and the in vitro experimental model of the development of the bFGF gene to induce the dedifferentiation of the epidermal cells was established. the expression and distribution of FGFR2 in the skin of the fetus during different development periods are detected, the molecular mechanism of the bFGF to regulate the dedifferentiation of the epidermal cells and the role of the FGFR2 in the generation and development of the skin are preliminarily explored, and an experimental basis is provided for finding the intrinsic driving force for regulating the dedifferentiation of the epidermal cells, provides an experimental reference for deep-depth study of wound healing and regeneration of the skin. Method 1. In this paper, the expression of bFGF gene from PBR322-bFGF plasmid was amplified by PCR, and the restriction site of HindIII and EcoR I was introduced at both ends, and the bFGF cDNA was directionally inserted into the multiple cloning site of pEGFP-C3 to construct the true nuclear expression plasmid pEGFP-C3-bFGF, and the DNA sequence of the recombinant was determined. pEGFP-C3-bFGF was transfected into the human epidermal cell line HEKa cell by lipofectamine-(TM) 2000) transfection method, and the transient expression of the gene and the cell morphology were observed under the fluorescence microscope. The changes of cell morphology and the expression of CD1 integrin, CK19, CK14 and CK10 in the experimental group and the control group were detected with the epidermal cells cultured in the same period and the epidermal cells transfected with pEGFP-C3 as the negative control, the immunocytochemical staining and the immunofluorescence labeling method. The expression of pEGFP-C3-bFGF after transfection of pEGFP-C3-bFGF was further detected by RT-PCR and Western-blot. The expression and distribution of FGFR2 were detected by S-P method. Results 1. a true nuclear expression vector pEGFP-C3-bFGF containing bFGF cDNA was constructed. The transfection rate of pEGFP-C3-bFGF was 31.6% under a fluorescence microscope, and the transfected cells were small, circular or circular, and the nuclear mass ratio was large. The results showed that the expression of CK19 and CK14 in the cells of the experimental group was enhanced and the expression of CK10 decreased. Immunofluorescence staining showed that the transfected cells expressed CK19 and CK14 mainly in the nucleus, the cytoplasm and the cell membrane, and the expression of the CD1 integrin was weakly expressed on the cell membrane, while the CK10 expression was negative. The results of RT-PCR showed that the transcription level of CK19 and CK14 mRNA was up-regulated after pEGFP-C3-bFGF was transfected with pEGFP-C3-bFGF, while the expression of CK10 mRNA was significantly decreased. The results of Western blot showed that the expression of CK19 and CK14 in the epidermal cells after transfection with pEGFP-C3-bFGF was significantly enhanced, while the overall expression of CK10 was reduced. After the transfection of pEGFP-C3-bFGF, the epidermal cells were dedifferentiated and the expression of the stem cells was re-obtained. The expression of FGFR2 in the embryonic development stage was weak and even negative in the epidermal cells. In the skin layer, the epidermis of the hair follicle is thickened and the primary primordium of the hair follicle is visible; and the FGFR2 is weakly positive in the primary primordia of the epidermis and the hair follicle. When the hair follicle is in the form of hair follicle, the structure of the hair follicle is primarily formed, the volume is small, the shape is childish, the FGFR2 is mainly expressed in the maternally-like cells, and the expression range is gradually expanded with the increase of the gestational age, and the expression amount is gradually increased. After the development of the hair follicle, the structure of the hair follicle is relatively mature, and the FGFR2 is expressed in the surface layer, the capillary endothelial cell and the catheter part of the sebaceous gland, in particular, the hair follicle has strong positive expression. Conclusion 1. the bF-GF gene is inserted into the C-terminal of pEGFP-C3, so that the expression of bFGF in the eukaryotic cell can be improved, and the structure and the function of the target protein thereof are not influenced. and obtaining some potential capability of the precursor stem cells, expressing some undifferentiated proteins of the epidermal stem cells, successfully establishing an in vitro experimental model of the bFGF gene to induce the dedifferentiation of the epidermal cells, and providing an experimental basis for further elucidating the molecular mechanism of the bFGF to regulate the dedifferentiation of the epidermal cells, The expression and distribution of FGFR2 in the skin of the fetus are closely related to the development of the hair follicle. The up-regulation of FGFR2 may promote the proliferation of hair follicle stem cells and induce its directional differentiation to form terminal tissue functional cells.
【學(xué)位授予單位】：內(nèi)蒙古醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類號(hào)】：R329

【引證文獻(xiàn)】

相關(guān)期刊論文 前1條

1 江波;廖毅;童庭輝;;脂肪來源干細(xì)胞誘導(dǎo)表皮細(xì)胞化的研究現(xiàn)狀與進(jìn)展[J];中國組織工程研究與臨床康復(fù);2011年27期

，

本文編號(hào)：2316159