FGF10和Spry4基因的克隆及其在绵羊皮肤中的表达

发布时间：2018-04-20 15:07

本文选题：中国美利奴羊 + 毛囊　；参考：《新疆农业大学》2015年硕士论文

【摘要】：本文以中国美利奴羊妊娠55、75、85、105和135天胎羊皮肤为研究对象。首先对FGF10和Spry4基因的CDS区进行克隆及真核表达;紧接着采用荧光定量PCR的方法,探究上述四个时期时FGF10和Spry4基因的转录规律;随后利用石蜡切片HE染色方法,观察胎羊体侧皮肤的毛囊在不同时期的发育情况;最后通过免疫组织化学对FGF10和Spry4两种蛋白在毛囊发育过程中的表达进行定位,从而探讨FGF10和Spry4在毛囊发育调控中的作用。通过上述研究方法,得到的结论如下:1.以美利奴羊皮肤组织的RNA为模板反转录获得cDNA,再利用PCR技术扩增获得Spry4和FGF10基因全长编码区,克隆到zero-PCR@TM-Blunt载体进行测序。随后将ToPo-Spry4和ToPo-FGF10分别亚克隆到pcDNA3.0真核表达载体,经脂质体转染293T细胞中表达,Western blot鉴定表达产物。进一步对Spry4及FGF10核酸和氨基酸序列进行分析。首次克隆获得绵羊Spry4基因编码序列(GenBank登录号:KP280032)。SDS-PAGE结果显示,Spry4重组表达的蛋白大小为36 kDa,FGF10重组表达的蛋白大小为24 kDa,均与预期蛋白大小一致。2.中国美利奴羊的毛囊发育过程中,FGF10和Spry4在五个时期的表达存着在较大的差异。FGF10基因从娠55天到75天时有所降低,当到达85天时表达量成倍增高从而到达峰值,从85天到105天时表达量迅速下降至与55天表达量相当。到135天的时候表达量稍有增长。Spry4基因的表达量在妊娠55天表时最高,随后保持下降趋势,直到85天时降至较低的水平,到105天时迅速上升,上升至几乎与55天时相等的表达量。到妊娠135天表达量降至最低。3.在75天时,初级毛囊原始体形成。FGF10和Spry4在表皮层和初级毛囊原体始处均有表达。85天初级毛囊继续向真皮方向生长,周围有次级毛囊原始体形成。此时FGF10主要表达于表皮层和次级毛囊原始体。而Spry4在表皮层、初级毛囊和次级毛囊原始体均有表达。当胚胎发育到妊娠105天时,FGF10的组化染色明显变浅,与定量呈现相同趋势。其主要在表皮层表达。与此同时,Spry4有着很广范的表达部位。在表皮层、初级和次级毛囊内均有表达。胚胎发育到135天时,初级毛囊和次级毛囊基本发育完全,FGF10和Spry4的表达部位一致,在表皮层和毛囊的内根鞘和外根鞘处有表达。在出生后8周时FGF10和Spry4的表达部位几乎与妊娠期第135天时相同。
[Abstract]:In this paper, the skin of Chinese Merino sheep was studied. Firstly, the CDS region of FGF10 and Spry4 gene was cloned and expressed in eukaryotic cells. Then, the transcriptional regularity of FGF10 and Spry4 genes in the four periods was studied by fluorescence quantitative PCR, and then paraffin sections were stained with HE staining. To observe the development of hair follicles in different stages of fetal sheep skin, and finally to investigate the role of FGF10 and Spry4 in the regulation of hair follicle development by immunohistochemical localization of the expression of FGF10 and Spry4 in hair follicle development. Through the above research method, the conclusion is as follows: 1. The full-length coding regions of Spry4 and FGF10 genes were amplified by reverse transcription of RNA from Merino sheep skin tissue and cloned into zero-PCR@TM-Blunt vector for sequencing. Then ToPo-Spry4 and ToPo-FGF10 were subcloned into pcDNA3.0 eukaryotic expression vector, and expressed in 293T cells by liposome. The expressed product was identified by Western blot. The nucleic acid and amino acid sequences of Spry4 and FGF10 were further analyzed. Sheep Spry4 gene coding sequence was cloned for the first time. The result of SDS-PAGE showed that the recombinant protein expressed in Spry4 was 36 kDa FGF10 with a size of 24 kDa, which was consistent with the expected protein size of 0.2. The expression of FGF10 and Spry4 in the hair follicles of Chinese Merino sheep decreased from 55 days to 75 days after gestation. The expression of FGF10 gene doubled and reached the peak at 85 days. From day 85 to day 105, the expression level decreased rapidly to the same level as that at day 55. The expression of Spry4 gene increased slightly at 135 days. The expression of Spry4 gene reached the highest level on day 55 of gestation, and then maintained a downward trend until it dropped to a lower level at day 85 and rose rapidly at day 105 to almost the same level as that at day 55. At 135 days of gestation, the expression level decreased to the lowest. 3. At 75 days, the primary hair follicle primordium formation. FGF10 and Spry4 were expressed in the epidermis and the primordia of the primary hair follicles. At day 85, the primary hair follicles continued to grow to the dermis, and the secondary hair follicle primordium formed around them. At this time, FGF10 was mainly expressed in epidermis and secondary hair follicle primordium. Spry4 was expressed in epidermis, primary hair follicle and secondary hair follicle primordium. When the embryo reached 105 days of gestation, the histochemical staining of FGF10 was obviously shallower, and showed the same trend as that of quantitative analysis. It is mainly expressed in the epidermis. At the same time, the Spry4 has a wide range of expression sites. In the epidermis, primary and secondary hair follicles are expressed. The expression of FGF10 and Spry4 in the primary hair follicle and the secondary hair follicle was the same at the 135th day of embryo development, and was expressed in the inner root sheath and the outer root sheath of the epidermis and hair follicle. The expression of FGF10 and Spry4 at 8 weeks after birth was almost the same as that on the 135th day of gestation.
【学位授予单位】：新疆农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S826

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