牦牛皮肤毛囊周期性结构变化规律及其相关调控因子的研究

发布时间：2018-06-06 20:27

本文选题：牦牛 + 毛囊　；参考：《甘肃农业大学》2017年博士论文

【摘要】：试验目的牦牛是高原地区宝贵的畜牧资源,可以为牧民提供乳、肉、毛绒等多种产品,是高原地区畜牧业经济的重要支柱。为了深入了解牦牛皮肤毛囊的发生发育规律及相关因子调控机制,本研究应用组织化学方法对牦牛皮肤毛囊组织结构进行系统观察,克隆了牦牛GSDMA基因,并选取HSP27、HSP70、HSP90及GSDMA四种参与调控毛囊发育的相关因子,采用荧光定量PCR、Western Blot及免疫组织化学技术探讨毛发生长周期中上述因子在牦牛皮肤的表达模式,为进一步研究相关因子对牦牛毛发生长调控的作用机制奠定基础。试验方法1.采用组织化学方法研究毛发生长周期中牦牛皮肤毛囊的结构特征。研究对象为1岁龄健康牦牛,体侧近肩胛部取皮样,分为生长期、退行期、休止期三组,平摊固定于硬纸板上,放入4%多聚甲醛溶液中固定。固定48h以上常规程序脱水,软化,石蜡包埋,制备6μm横纵连续切片。Sacpic法染色,用以观察毛囊结构。使用IPP6.0软件测量次级毛囊的密度、活性、深度及毛囊宽度等数据。2.采用RT-PCR技术克隆牦牛GSDMA基因序列,并对序列进行相关生物信息学分析。采用荧光定量PCR技术研究GSDMA基因mRNA在牦牛主要器官组织中的表达情况。3.采用荧光定量PCR、Western blot及免疫组织化学技术,从mRNA和蛋白质两个方面探讨毛发生长周期中HSP27、HSP70、HSP90及GSDMA在牦牛皮肤的表达情况。结果1.皮肤组织经Sacpic法染色后不同组织结构区分清晰,易于观察,并且突出了内根鞘结构,可以作为判断毛囊活性的依据。毛囊群结构随毛发生长周期变化而变化,休止期毛囊群结构松散,毛干脱落,基本无内根鞘结构;生长期毛囊群结构逐渐清晰,初级毛囊、次级毛囊数量明显增加,结缔组织鞘和外根鞘逐渐增厚,内根鞘结构产生并逐渐完整;退行期,毛囊群开始变得不完整,毛囊个数减少,次级毛囊萎缩变小,内根鞘向上移动变短。次级毛囊的大小、数量、活性及在皮肤内的深度也随着毛发生长周期变化而变化,生长期是次级毛囊发育最旺盛的时期。次级毛囊的发育并不是完全同步的。2.采用RT-PCR技术克隆牦牛GSDMA基因序列并进行相关生物信息学分析,结果如下:克隆得到牦牛GSDMA基因(GenBank登录号:XM_1995113),其开放阅读框最长为750bp,起始密码子位于34bp处,终止密码子位于783bp处,编码249个氨基酸。其编码蛋白为可溶性非跨膜蛋白。同源性分析和系统进化树显示,牦牛gsdma基因与瘤牛、野牦牛和家牛的进化水平较为相近,与猪、马和人类进化水平较远。采用荧光定量pcr技术研究gsdma基因mrna在牦牛主要器官组织中的表达情况,结果如下:gsdma基因mrna主要在牦牛皮肤及胃肠道中表达,卵巢与输卵管次之,在心脏、肺脏与肾脏中表达量较低,其余组织中几乎无表达。gsdma基因mrna在胃部的表达量由高到低依次为皱胃瓣胃网胃瘤胃。在小肠中,表达量由高到低依次为空肠十二指肠回肠。在大肠中,表达量由高到低依次为结肠盲肠直肠。采用荧光定量pcr、westernblot及免疫组织化学技术检测毛发生长周期中gsdma在皮肤的表达情况,结果发现:gsdma基因mrna和蛋白质在牦牛皮肤的表达模式相同,表达量由高到低依次为生长期退行期休止期。各期gsdma基因mrna表达水平差异不显著(p0.05)。各期gsdma蛋白质表达量之间差异显著(p0.05)。gsdma在整个毛发生长周期的各个阶段均有表达。gsdma在表皮基底层持续表达,而在毛囊的表达位置因时期不同而发生变化。退行期时主要在内根鞘及残留的毛髓质表达。休止期gsdma主要在次级毛芽表达,皮脂腺细胞也有表达。生长期时gsdma在外根鞘、毛髓质及正在分化形成的内根鞘表达,其表达强度随内根鞘的逐渐完整而增强。3.采用荧光定量pcr、westernblot及免疫组织化学技术检测毛发生长周期中hsp27、hsp70、hsp90在皮肤的表达情况,结果发现:这三种因子在牦牛皮肤的表达模式各异,hsp27的表达量由高到低依次为生长期退行期休止期;hsp70的表达量由高到低依次为休止期生长期退行期;hsp90的表达量由高到低依次为生长期休止期退行期。这三种因子的mrna和蛋白质的表达模式相同,但差异显著性各不相同。hsp27mrna生长期表达量与退行期和休止期表达量相比差异显著(p0.05),退行期和休止期之间差异不显著(p0.05)。而各期hsp27蛋白质表达量之间差异均显著(p0.05)。hsp70与hsp90各期mrna表达量之间差异不显著(p0.05),但hsp70蛋白表达量各期之间差异显著(p0.05),hsp90蛋白生长期表达量与退行期和休止期表达量相比差异显著(p0.05),退行期和休止期之间差异不显著(p0.05)。三种因子在整个毛发生长周期的各个阶段均有表达。hsp27、hsp70及hsp90主要在皮肤表皮层和毛囊的外根鞘部位表达,也在皮脂腺和汗腺表达。结论1.牦牛皮肤毛囊群结构随毛发生长周期变化而变化,次级毛囊的大小、数量、活性及在皮肤内的深度也随着毛发生长周期变化而变化,生长期是次级毛囊发育最旺盛的时期。次级毛囊的发育并不是完全同步的。2.本试验成功克隆出牦牛GSDMA基因(GenBank登录号:XM_1995113),同源性分析和系统进化树显示,牦牛GSDMA基因与瘤牛、野牦牛和家牛的进化水平较为相近,与猪、马和人类进化水平较远,说明该基因在进化过程中既高度保守又具有种属特异性。3.牦牛GSDMA基因mRNA主要在皮肤和胃肠道中表达,其余组织中几乎无表达。在胃肠道的表达表现出了明显的轴向性,这与其特异性的上皮表达谱有关。4.在毛发生长周期中,GSDMA表达量的变化提示其参与毛囊细胞凋亡与增殖的调控。GSDMA在皮肤表皮层的持续性表达推测与表皮干细胞的增殖分化有关。5.毛发生长周期中HSP27、HSP70、HSP90三种因子都参与牦牛皮肤毛囊细胞周期活动,还可能参与细胞凋亡调控。表达模式各异,表达水平也存在差异,说明每一种热休克蛋白具体发挥的功能不同,其中HSP27占据主导地位。
[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.
【学位授予单位】：甘肃农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S823.85

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