碱性成纤维细胞生长因子基因诱导表皮细胞去分化的实验研究
发布时间：2018-11-07 11:05
【摘要】： 目的1.构建碱性成纤维细胞生长因子基因真核表达载体pEGFP-C3-bFGF;2.将pEGFP-C3-bFGF转染至人表皮细胞,观察基因瞬时表达情况及表皮细胞的形态和表型改变,建立bFGF基因诱导表皮细胞去分化的体外实验模型;3.检测FGFR2在不同发育时期胎儿皮肤中的表达和分布,初步探索bFGF调控表皮细胞去分化的分子机制及FGFR2在皮肤发生、发育中的作用,为寻找调控表皮细胞去分化的内在驱动力提供实验基础,为深入研究皮肤的创伤修复与再生提供实验参考。方法1.参考分子克隆技术,采用PCR法从PBR322-bFGF质粒中扩增bFGF基因,同时在其两端引入HindⅢ和EcoRⅠ限制性内切酶酶切位点,将bFGF cDNA定向插入pEGFP-C3的多克隆位点中,构建真核表达质粒pEGFP-C3-bFGF,并对重组子进行DNA序列测定。2.采用脂质体(Lipofectamine~(TM) 2000)转染法,将pEGFP-C3-bFGF转染至人表皮细胞系HEKa细胞中,在荧光显微镜下观察基因瞬时表达情况及细胞形态。以同期培养的表皮细胞及pEGFP-C3转染的表皮细胞作为阴性对照,免疫细胞化学染色和免疫荧光标记法检测实验组及对照组细胞形态的改变和细胞中β_1整合素、CK19、CK14及CK10表达的差异。采用RT-PCR和Western-blot进一步检测pEGFP-C3-bFGF转染后表皮细胞表型的改变,建立bFGF基因诱导表皮细胞去分化的体外实验模型。3.对不同发育时期胎儿皮肤取材、固定、制成石蜡切片,S-P法检测FGFR2的表达及分布。结果1.构建了含有bFGF cDNA的真核表达载体pEGFP-C3-bFGF。2.荧光显微镜下观察pEGFP-C3-bFGF基因转染率为31.6%,转染阳性细胞体积小,呈圆形或圆梭形,核质比大。免疫细胞化学染色结果显示,实验组细胞中CK19、CK14表达增强,CK10表达减弱。免疫荧光染色结果显示,转染阳性细胞主要在细胞核、胞浆和胞膜中表达CK19和CK14,在胞膜上弱表达β_1整合素,而CK10表达为阴性。RT-PCR结果显示,pEGFP-C3-bFGF转染后表皮细胞中CK19、CK14 mRNA的转录水平明显上调,而CK10的mRNA表达明显下降。Western blot检测结果表明,pEGFP-C3-bFGF转染后表皮细胞中CK19、CK14的表达明显增强,而CK10的总体表达减少。pEGFP-C3-bFGF转染后表皮细胞发生去分化,重新获得干细胞的表达标志。3.胚胎发育期FGFR2在表皮细胞表达较弱,甚至呈阴性表达。表皮分层期时,表皮层开始增厚,并可见毛囊的初级原基;FGFR2在表皮层和毛囊初级原基内呈弱阳性表达。毛囊形成期时,毛囊的结构初步形成,体积细小,形态幼稚;FGFR2主要在毛母质细胞内表达,并且随着胎龄增加,表达范围逐渐扩大,表达量逐渐增多。毛囊发育成熟期后,毛囊的结构发育相对成熟,FGFR2则在表皮层、毛细血管内皮细胞及皮脂腺的导管部均有表达,尤其在毛母质细胞呈强阳性表达。结论1.将bF-GF基因插入pEGFP-C3的C末端,可以提高bFGF在真核细胞中的表达,而且不影响其目的蛋白的结构和功能。2.pEGFP-C3-bFGF能够转染至人表皮细胞并表达,在pEG-FP-C3-bFGF转染作用后,表皮细胞重新程序化,并获得其前体干细胞的某些潜在能力,表达表皮干细胞的一些未分化蛋白,成功建立bFGF基因诱导表皮细胞去分化的体外实验模型,为进一步阐述bFGF调控表皮细胞去分化的分子机制提供实验基础,为寻找调控表皮细胞去分化的内在驱动力提供实验参考。3.FGFR2在胎儿皮肤中的表达与分布与毛囊的发生发育密切相关。FGFR2的表达上调可能促进毛囊干细胞的增殖,并诱导其定向分化形成终末组织功能细胞。
[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.
【学位授予单位】：内蒙古医学院
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R329
本文编号：2316159
[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.
【学位授予单位】：内蒙古医学院
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R329
【引证文献】
相关期刊论文 前1条
1 江波;廖毅;童庭辉;;脂肪来源干细胞诱导表皮细胞化的研究现状与进展[J];中国组织工程研究与临床康复;2011年27期
，本文编号：2316159
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