本文選題：牦牛 + 毛囊��； 參考：《甘肅農(nóng)業(yè)大學》2017年博士論文

【摘要】：試驗目的牦牛是高原地區(qū)寶貴的畜牧資源,可以為牧民提供乳、肉、毛絨等多種產(chǎn)品,是高原地區(qū)畜牧業(yè)經(jīng)濟的重要支柱。為了深入了解牦牛皮膚毛囊的發(fā)生發(fā)育規(guī)律及相關(guān)因子調(diào)控機制,本研究應用組織化學方法對牦牛皮膚毛囊組織結(jié)構(gòu)進行系統(tǒng)觀察,克隆了牦牛GSDMA基因,并選取HSP27、HSP70、HSP90及GSDMA四種參與調(diào)控毛囊發(fā)育的相關(guān)因子,采用熒光定量PCR、Western Blot及免疫組織化學技術(shù)探討毛發(fā)生長周期中上述因子在牦牛皮膚的表達模式,為進一步研究相關(guān)因子對牦牛毛發(fā)生長調(diào)控的作用機制奠定基礎。試驗方法1.采用組織化學方法研究毛發(fā)生長周期中牦牛皮膚毛囊的結(jié)構(gòu)特征。研究對象為1歲齡健康牦牛,體側(cè)近肩胛部取皮樣,分為生長期、退行期、休止期三組,平攤固定于硬紙板上,放入4%多聚甲醛溶液中固定。固定48h以上常規(guī)程序脫水,軟化,石蠟包埋,制備6μm橫縱連續(xù)切片。Sacpic法染色,用以觀察毛囊結(jié)構(gòu)。使用IPP6.0軟件測量次級毛囊的密度、活性、深度及毛囊寬度等數(shù)據(jù)。2.采用RT-PCR技術(shù)克隆牦牛GSDMA基因序列,并對序列進行相關(guān)生物信息學分析。采用熒光定量PCR技術(shù)研究GSDMA基因mRNA在牦牛主要器官組織中的表達情況。3.采用熒光定量PCR、Western blot及免疫組織化學技術(shù),從mRNA和蛋白質(zhì)兩個方面探討毛發(fā)生長周期中HSP27、HSP70、HSP90及GSDMA在牦牛皮膚的表達情況。結(jié)果1.皮膚組織經(jīng)Sacpic法染色后不同組織結(jié)構(gòu)區(qū)分清晰,易于觀察,并且突出了內(nèi)根鞘結(jié)構(gòu),可以作為判斷毛囊活性的依據(jù)。毛囊群結(jié)構(gòu)隨毛發(fā)生長周期變化而變化,休止期毛囊群結(jié)構(gòu)松散,毛干脫落,基本無內(nèi)根鞘結(jié)構(gòu);生長期毛囊群結(jié)構(gòu)逐漸清晰,初級毛囊、次級毛囊數(shù)量明顯增加,結(jié)締組織鞘和外根鞘逐漸增厚,內(nèi)根鞘結(jié)構(gòu)產(chǎn)生并逐漸完整;退行期,毛囊群開始變得不完整,毛囊個數(shù)減少,次級毛囊萎縮變小,內(nèi)根鞘向上移動變短。次級毛囊的大小、數(shù)量、活性及在皮膚內(nèi)的深度也隨著毛發(fā)生長周期變化而變化,生長期是次級毛囊發(fā)育最旺盛的時期。次級毛囊的發(fā)育并不是完全同步的。2.采用RT-PCR技術(shù)克隆牦牛GSDMA基因序列并進行相關(guān)生物信息學分析,結(jié)果如下:克隆得到牦牛GSDMA基因(GenBank登錄號:XM_1995113),其開放閱讀框最長為750bp,起始密碼子位于34bp處,終止密碼子位于783bp處,編碼249個氨基酸。其編碼蛋白為可溶性非跨膜蛋白。同源性分析和系統(tǒng)進化樹顯示,牦牛gsdma基因與瘤牛、野牦牛和家牛的進化水平較為相近,與豬、馬和人類進化水平較遠。采用熒光定量pcr技術(shù)研究gsdma基因mrna在牦牛主要器官組織中的表達情況,結(jié)果如下:gsdma基因mrna主要在牦牛皮膚及胃腸道中表達,卵巢與輸卵管次之,在心臟、肺臟與腎臟中表達量較低,其余組織中幾乎無表達。gsdma基因mrna在胃部的表達量由高到低依次為皺胃瓣胃網(wǎng)胃瘤胃。在小腸中,表達量由高到低依次為空腸十二指腸回腸。在大腸中,表達量由高到低依次為結(jié)腸盲腸直腸。采用熒光定量pcr、westernblot及免疫組織化學技術(shù)檢測毛發(fā)生長周期中g(shù)sdma在皮膚的表達情況,結(jié)果發(fā)現(xiàn):gsdma基因mrna和蛋白質(zhì)在牦牛皮膚的表達模式相同,表達量由高到低依次為生長期退行期休止期。各期gsdma基因mrna表達水平差異不顯著(p0.05)。各期gsdma蛋白質(zhì)表達量之間差異顯著(p0.05)。gsdma在整個毛發(fā)生長周期的各個階段均有表達。gsdma在表皮基底層持續(xù)表達,而在毛囊的表達位置因時期不同而發(fā)生變化。退行期時主要在內(nèi)根鞘及殘留的毛髓質(zhì)表達。休止期gsdma主要在次級毛芽表達,皮脂腺細胞也有表達。生長期時gsdma在外根鞘、毛髓質(zhì)及正在分化形成的內(nèi)根鞘表達,其表達強度隨內(nèi)根鞘的逐漸完整而增強。3.采用熒光定量pcr、westernblot及免疫組織化學技術(shù)檢測毛發(fā)生長周期中hsp27、hsp70、hsp90在皮膚的表達情況,結(jié)果發(fā)現(xiàn):這三種因子在牦牛皮膚的表達模式各異,hsp27的表達量由高到低依次為生長期退行期休止期;hsp70的表達量由高到低依次為休止期生長期退行期;hsp90的表達量由高到低依次為生長期休止期退行期。這三種因子的mrna和蛋白質(zhì)的表達模式相同,但差異顯著性各不相同。hsp27mrna生長期表達量與退行期和休止期表達量相比差異顯著(p0.05),退行期和休止期之間差異不顯著(p0.05)。而各期hsp27蛋白質(zhì)表達量之間差異均顯著(p0.05)。hsp70與hsp90各期mrna表達量之間差異不顯著(p0.05),但hsp70蛋白表達量各期之間差異顯著(p0.05),hsp90蛋白生長期表達量與退行期和休止期表達量相比差異顯著(p0.05),退行期和休止期之間差異不顯著(p0.05)。三種因子在整個毛發(fā)生長周期的各個階段均有表達。hsp27、hsp70及hsp90主要在皮膚表皮層和毛囊的外根鞘部位表達,也在皮脂腺和汗腺表達。結(jié)論1.牦牛皮膚毛囊群結(jié)構(gòu)隨毛發(fā)生長周期變化而變化,次級毛囊的大小、數(shù)量、活性及在皮膚內(nèi)的深度也隨著毛發(fā)生長周期變化而變化,生長期是次級毛囊發(fā)育最旺盛的時期。次級毛囊的發(fā)育并不是完全同步的。2.本試驗成功克隆出牦牛GSDMA基因(GenBank登錄號:XM_1995113),同源性分析和系統(tǒng)進化樹顯示,牦牛GSDMA基因與瘤牛、野牦牛和家牛的進化水平較為相近,與豬、馬和人類進化水平較遠,說明該基因在進化過程中既高度保守又具有種屬特異性。3.牦牛GSDMA基因mRNA主要在皮膚和胃腸道中表達,其余組織中幾乎無表達。在胃腸道的表達表現(xiàn)出了明顯的軸向性,這與其特異性的上皮表達譜有關(guān)。4.在毛發(fā)生長周期中,GSDMA表達量的變化提示其參與毛囊細胞凋亡與增殖的調(diào)控。GSDMA在皮膚表皮層的持續(xù)性表達推測與表皮干細胞的增殖分化有關(guān)。5.毛發(fā)生長周期中HSP27、HSP70、HSP90三種因子都參與牦牛皮膚毛囊細胞周期活動,還可能參與細胞凋亡調(diào)控。表達模式各異,表達水平也存在差異,說明每一種熱休克蛋白具體發(fā)揮的功能不同,其中HSP27占據(jù)主導地位。
[Abstract]:Yak is a valuable animal resource in plateau area. It can provide milk, meat and wool for herdsmen. It is an important pillar of animal husbandry in plateau area. In order to understand the development law of yak skin follicle and the regulation mechanism of related factors, this research applies histochemical method to the yak skin follicle tissue. The structure was systematically observed, the yak GSDMA gene was cloned, and four kinds of related factors involved in the development of hair follicle were selected as HSP27, HSP70, HSP90 and GSDMA. The expression pattern of the above factors in yak skin was investigated by fluorescence quantitative PCR, Western Blot and immunohistochemical technique, so as to further study the relative factors to yak. The experimental method 1. studied the structure characteristics of yak skin follicle in hair growth cycle by histochemical method. The study object was 1 years old healthy yak, the body side near the scapula, divided into three groups, the growth period, the degenerative period and the rest period, which were fixed on the cardboard and placed in 4% Polya. It was fixed in the aldehyde solution. The routine procedures above 48h were dehydrated, softened and paraffin embedded, and the 6 m transverse section was prepared by.Sacpic staining to observe the hair follicle structure. The IPP6.0 software was used to measure the density, activity, depth and the width of the follicle by the IPP6.0 software. The sequence of the GSDMA gene was cloned by RT-PCR technology, and the sequence was carried out. Biological informatics analysis. The expression of GSDMA gene mRNA in the main organs of yak was studied by fluorescence quantitative PCR technique.3. using fluorescence quantitative PCR, Western blot and immunohistochemical technique. The expression of HSP27, HSP70, HSP90 and GSDMA in the yak skin was investigated from two aspects of mRNA and protein. Results 1. the tissue structure of 1. skin tissues is distinct, easy to observe, and easy to observe. The structure of inner root sheath is easy to be observed. The structure of inner root sheath can be used as a basis for judging the activity of hair follicle. The structure of hair follicle group changes with the growth cycle of hair. The structure of hair follicle in the repose period is loose, the hair shedding, basically no internal root sheath structure, and the growth period of hair follicle group. The number of primary follicles and secondary follicles increased obviously, the sheath and outer root sheath of connective tissue and outer root sheath gradually thickened, and the inner root sheath formed and completed gradually; the follicle group began to become incomplete, the number of hair follicles decreased, the secondary follicle atrophied and the inner root sheath moved up to a shorter time. The size, quantity, activity of secondary hair follicle and the skin were in the skin. The internal depth also changes with the growth cycle of hair. The growth period is the most vigorous period of secondary hair follicle development. The development of secondary hair follicle is not completely synchronous.2. using RT-PCR technology to clone yak GSDMA gene sequence and carry out related bioinformatics analysis. The results are as follows: the GSDMA gene of Yak (GenBank login number: XM_) was cloned. 1995113) the opening reading frame is the longest 750bp, the beginning codon is located at 34bp, the codon is located at 783bp and encodes 249 amino acids. Its encoding protein is soluble non transmembrane protein. Homology analysis and phylogenetic tree show that the evolution level of yak gsdma gene is similar to that of cattle, wild yak and domestic cattle. The expression of gsdma gene mRNA in the main organs of yak is studied by fluorescence quantitative PCR. The results are as follows: the gsdma gene mRNA is mainly expressed in the yak skin and the gastrointestinal tract, the ovary and the oviduct are second, the expression of the gsdma is low in the heart, the lungs and the kidneys, and the.Gsdma gene mRNA is almost no expression in the other tissues. In the small intestine, the expression amount from high to low is in the order of the jejunoduodenum ileum in the small intestine. In the large intestine, the expression amount from high to low is the colon cecum rectum. Fluorescence quantitative PCR, Westernblot and immunohistochemical technique are used to detect gsdma in the hair growth cycle. The results showed that the expression pattern of gsdma gene mRNA and protein in yak skin was the same, and the expression amount from high to low was in the period of degenerative period of growth period. The difference of mRNA expression level of gsdma gene in each stage was not significant (P0.05). The difference of gsdma protein expression in each phase (P0.05).Gsdma was in the whole hair growth cycle. The expression of.Gsdma was expressed in the basal layer of the epidermis at all stages, and the expression position of the follicle changed in different periods. The degenerative period was mainly expressed in the inner root sheath and the residual medulla. The gsdma was mainly expressed in the secondary hair buds, and the sebaceous gland cells were also expressed. In the long term, the gsdma was in the outer root sheath, the medulla and being divided. The expression of the formed inner root sheath increased with the gradual integrity of the internal root sheath and enhanced.3. using fluorescence quantitative PCR. Westernblot and immunohistochemistry were used to detect the expression of HSP27, HSP70 and HSP90 in the hair growth cycle. The results showed that the expression patterns of these three factors were different in yak skin, and the expression of HSP27 was high. The low expression of HSP70 from high to low is the degenerative period of the growth period, and the expression of Hsp90 from high to low is the degenerative period of the growth period. The expression pattern of the three factors is the same as the expression pattern of the protein, but the difference is not the same for the expression of the.Hsp27mrna growth period and the degenerative period. There was no significant difference in the period and repose period (P0.05), and there was no significant difference between the degenerative period and the rest period (P0.05), but the difference in the expression of HSP27 protein at all stages was significant (P0.05) and there was no significant difference between the mRNA expressions of.Hsp70 and HSP90 at each stage (P0.05), but the difference between the Hsp70 egg white expression periods was significant (P0.05) and the Hsp90 protein growth period. There was significant difference in expression amount compared with the expression of degenerative and resting periods (P0.05). There was no significant difference between the degenerative and repose periods (P0.05). The three factors expressed.Hsp27 at all stages of the whole hair growth cycle, and HSP70 and HSP90 were expressed mainly in the epidermis of the skin and the outer root sheath of the hair follicle, but also in the sebaceous glands and sweat glands. 1. yak skin follicle structure changes with the growth cycle of hair. The size, quantity, activity and depth of secondary hair follicle change with the growth cycle of hair. The growth period is the most vigorous period of secondary hair follicle development. The development of secondary hair follicle is not completely synchronous.2. experiment successfully cloned the yak GSDM The A gene (GenBank login number: XM_1995113), homology analysis and phylogenetic tree show that the evolution level of the yak GSDMA gene is similar to the cattle, wild yak and home cattle, which is far from the pig, horse and human evolution. It shows that the gene is highly conservative and the specific.3. yak GSDMA gene mRNA is mainly in the skin during the evolution process. The expression in the other tissues was almost nonexpressive in the gastrointestinal tract. The expression in the gastrointestinal tract was obviously axial, which was related to the specific epithelial expression profiles related to the changes in the expression of.4. in the hair growth cycle. The changes in the expression of GSDMA suggest that the.GSDMA is involved in the apoptosis and proliferation of the hair follicle cells in the continuous expression of the skin layer. With the proliferation and differentiation of epidermal stem cells, the three factors of HSP27, HSP70, and HSP90 in the growth cycle of.5. hair are involved in the cycle activities of the yak hair follicle cells, and may also participate in the regulation of cell apoptosis. The expression patterns are different and the expression levels are different, indicating that each kind of heat shock protein plays different functions, in which HSP27 occupies the main body. Guide position.
【學位授予單位】：甘肅農(nóng)業(yè)大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：S823.85

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