發(fā)布時間：2018-04-20 15:07

  本文選題：中國美利奴羊 + 毛囊��； 參考：《新疆農(nóng)業(yè)大學(xué)》2015年碩士論文

【摘要】：本文以中國美利奴羊妊娠55、75、85、105和135天胎羊皮膚為研究對象。首先對FGF10和Spry4基因的CDS區(qū)進(jìn)行克隆及真核表達(dá);緊接著采用熒光定量PCR的方法,探究上述四個時期時FGF10和Spry4基因的轉(zhuǎn)錄規(guī)律;隨后利用石蠟切片HE染色方法,觀察胎羊體側(cè)皮膚的毛囊在不同時期的發(fā)育情況;最后通過免疫組織化學(xué)對FGF10和Spry4兩種蛋白在毛囊發(fā)育過程中的表達(dá)進(jìn)行定位,從而探討FGF10和Spry4在毛囊發(fā)育調(diào)控中的作用。通過上述研究方法,得到的結(jié)論如下:1.以美利奴羊皮膚組織的RNA為模板反轉(zhuǎn)錄獲得cDNA,再利用PCR技術(shù)擴(kuò)增獲得Spry4和FGF10基因全長編碼區(qū),克隆到zero-PCR@TM-Blunt載體進(jìn)行測序。隨后將ToPo-Spry4和ToPo-FGF10分別亞克隆到pcDNA3.0真核表達(dá)載體,經(jīng)脂質(zhì)體轉(zhuǎn)染293T細(xì)胞中表達(dá),Western blot鑒定表達(dá)產(chǎn)物。進(jìn)一步對Spry4及FGF10核酸和氨基酸序列進(jìn)行分析。首次克隆獲得綿羊Spry4基因編碼序列(GenBank登錄號:KP280032)。SDS-PAGE結(jié)果顯示,Spry4重組表達(dá)的蛋白大小為36 kDa,FGF10重組表達(dá)的蛋白大小為24 kDa,均與預(yù)期蛋白大小一致。2.中國美利奴羊的毛囊發(fā)育過程中,FGF10和Spry4在五個時期的表達(dá)存著在較大的差異。FGF10基因從娠55天到75天時有所降低,當(dāng)?shù)竭_(dá)85天時表達(dá)量成倍增高從而到達(dá)峰值,從85天到105天時表達(dá)量迅速下降至與55天表達(dá)量相當(dāng)。到135天的時候表達(dá)量稍有增長。Spry4基因的表達(dá)量在妊娠55天表時最高,隨后保持下降趨勢,直到85天時降至較低的水平,到105天時迅速上升,上升至幾乎與55天時相等的表達(dá)量。到妊娠135天表達(dá)量降至最低。3.在75天時,初級毛囊原始體形成。FGF10和Spry4在表皮層和初級毛囊原體始處均有表達(dá)。85天初級毛囊繼續(xù)向真皮方向生長,周圍有次級毛囊原始體形成。此時FGF10主要表達(dá)于表皮層和次級毛囊原始體。而Spry4在表皮層、初級毛囊和次級毛囊原始體均有表達(dá)。當(dāng)胚胎發(fā)育到妊娠105天時,FGF10的組化染色明顯變淺,與定量呈現(xiàn)相同趨勢。其主要在表皮層表達(dá)。與此同時,Spry4有著很廣范的表達(dá)部位。在表皮層、初級和次級毛囊內(nèi)均有表達(dá)。胚胎發(fā)育到135天時,初級毛囊和次級毛囊基本發(fā)育完全,FGF10和Spry4的表達(dá)部位一致,在表皮層和毛囊的內(nèi)根鞘和外根鞘處有表達(dá)。在出生后8周時FGF10和Spry4的表達(dá)部位幾乎與妊娠期第135天時相同。
[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.
【學(xué)位授予單位】：新疆農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S826

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