【作者】 董彬；

【导师】 王慧；

【作者基本信息】 山东农业大学 ， 动物遗传育种与繁殖， 2010， 硕士

【摘要】 为揭示济宁青山羊皮肤毛囊的发育性变化规律及其候选基因的差异表达对皮肤毛囊生长和发育的影响,本研究采用组织学和实时荧光定量PCR等分子细胞生物学技术,对青山羊不同发育阶段皮肤毛囊的组织结构及其发育性变化规律和皮肤组织中褪黑素受体1A(MTR-1A)、类胰岛素生长因子1(IGF-1)和酪氨酸酶(TYR)基因的mRNA差异表达量进行了比较研究。主要研究结果如下:一、采用组织学与显微观测技术,比较研究了济宁青山羊胎儿羊(妊娠80-85d)、小猾皮(出生后3d)、大猾皮(出生后30d)和1龄成年羊皮肤毛囊的组织学结构,以及成年羊皮肤不同部位的组织学差异,结果表明:(1)胎儿羊(妊娠80-85d)胚胎性表皮分化为三层并增厚,真皮分化为两层,交织分布着成纤维细胞。在表皮区可以发现次级毛囊毛基的发生。(2)小猾皮毛囊层中的胶原纤维平行纵横交错,与网状层相比排列较为紧密;而网状层中的胶原纤维呈水平走向,为平行交织状排列。毛囊层内的弹性纤维细而弯曲,相互交织,纵横排列。网状层内无弹性纤维的分布。小猾皮的毛囊组织间隙较大,密度较稀,初级毛囊发达,次级毛囊较少。小猾皮每个毛囊群仅由2-3个初级毛囊和1-2个次级毛囊组成。(3)大猾皮毛囊层和网状层明显增厚,有粗大的胶原纤维束分布其中。大猾皮的毛囊密度和毛囊群直径极显著地大于小猾皮。次级毛囊密集排列在初级毛囊的周围。大猾皮毛囊群由1-5个初级毛囊和若干个次级毛囊组成,其中3毛囊群最多,发达的毛囊群使大猾皮表面粗糙。(4)成年羊皮肤毛囊层的厚度超过网状层,毛囊层内含有较多的结缔组织、胶原纤维和弹性纤维,形成致密的弹性纤维网。网状层中的胶原纤维粗大,胶原纤维呈平行疏松排列。成年羊皮肤初级毛囊较粗长,具有皮脂腺等附属结构,毛干有髓质;次级毛囊没有髓质且毛球较小,皮脂腺不发达或缺如。成年羊毛囊群和大猾皮相似,3毛囊群最多,占81.32%。(5)成年羊颈、背、腹和臀四个部位的皮肤厚度、表皮厚、真皮各层厚均以颈部最厚,背部次之,腹部最薄,颈部与背部、腹部差异极显著,背部与臀部差异不显著;四个部位的羊毛密度,以背部最密,腹部最稀(P>0.05)。羊毛细度在各个部位的差异不显著。毛囊群直径,以腹部最粗,背部次之,臀部最细(P<0.01)。初级毛囊和次级毛囊的深度,均以颈部最深,背部次之,臀部最浅(P<0.01)。二、采用实时荧光定量PCR技术,研究了MTR-1A、IGF-1和TYR基因在济宁青山羊胎儿羊、小猾皮、大猾皮和1龄成年羊背部皮肤组织中mRNA的差异表达量,结果表明:(1)MTR-1A在大猾皮的表达量极显著地高于其他三个时期(P<0.01),为胎儿期的19.5倍,为小猾皮的14.98倍,为1龄成年羊的8.87倍;IGF-1在大猾皮的表达量极显著地高于其他三个时期(P<0.01),为胎儿期的39.26倍,为小猾皮的24.61倍,为1龄成年羊的19.52倍;表明,MT和IGF-1参与调控次级毛囊的分化与发育。在大猾皮时期,MTR-1A和IGF-1的高表达说明,在此时期大量初级毛囊的毛纤维生长和次级毛囊毛基的发育启动了次级毛囊的毛纤维的发生和生长。(2)MTR-1A在1龄成年羊皮肤颈部和背部的表达量与臀部的表达量差异显著(P<0.05);IGF1在1龄成年羊皮肤颈部、背部和臀部的表达量与腹部的表达量差异显著(P<0.05)。成年羊皮肤组织中MTR-1A和IGF-1的差异表达以及毛囊群分布提示MT对初级毛囊的作用大于IGF-1。(3)MTR-1A和IGF-1的表达量与成年羊的羊毛细度存在一定的相关性(P>0.05),说明MT和IGF-1是影响羊毛细度的重要候选基因。(4)TYR在小猾皮和成年羊皮肤组织的表达量极显著地高于胎儿期和大猾皮阶段(P<0.01),其在小猾皮和成年羊背部皮肤中的表达量分别为胎儿期的14.4倍和10.89倍。TYR在小猾皮中的高表达说明,胎儿出生后,由于光照等环境因素的影响,位于毛囊乳头顶部的黑素细胞活化,皮肤组织中有大量黑素颗粒生成,在毛干堆积影响毛纤维的颜色。TYR在成年羊腹部皮肤的低表达与青山羊腹部羊毛多为白色有关。以上研究结果为揭示青山羊皮肤毛囊发育的分子遗传机理以及进一步揭示济宁青山羊毛色花纹品质形成的遗传机理奠定了基础。更多还原

【Abstract】 To reveal the developmental changes of hair follicle and cytokines effected on the differentiation of hair follicle in Jining Gray goat skins. The tissue slice methods were used to study the skin and hair follicle structures at different stages; and real-time quantitative PCR technique was applied to detect the MTR-1A, IGF-1 and TYR mRNA expressions of skins. The main results are as follows:The histology methods and micro-observation approach were used to compare the characteristics of skin and hair follicle structures of Jining Gray goats within 85-days fetus, 3-days-old, 1-month-old, and one-year-old.(1) 85d fetal skin epidermis differentiated into three layers and thickening, dermis divided into upper and lower levels, interlacing with fibroblasts. Secondary follicle placode can be found in the cuticle. All placodes of primary follicle in fetus had formed before birth, placodes of secondary follicle were decreased with the birth to 1 month and developed into wool fiber.(2) In kid-skins, collagen fibers in follicle layer were interlaced level while in the plexiform layer parallel intertwined. Elastic fibers in follicle layer were thin and curved interwoven arrangement while reticular layer of inelastic fibers distributed. The numbers of primary follicles were larger than secondary follicles. The density of hair follicle was smaller. Hair folliculus group was made up from 2-3 primary follicle and 1-2 secondary follicle.(3) In lamb-skins, follicle and reticular layer were thickening with collagen fibers. Hair folliculus group was made up from 1-5 primary follicles and several secondary follicles and the most of follicle groups were three.(4) In adult-skins, the thickness follicle layer contained more connective tissue, collagen and elastic fibers was larger than reticular layer’s. Adult medullary primary follicle was large with sebaceous glands and other accessory structures around distributed; secondary follicle without medulla and hair bulb was small. Hair folliculus group was same with lamb.(5) In different parts of adult-skins, epidermis, derma, follicle layer and reticular layer thickness of cervical skin were larger than back’s, abdomen’s were the smallest, there was extremely significant difference between cervical, back and abdomen; wool density of adult back skins was larger than other positions (P>0.05); different parts of wool fineness were no significant difference; abdomen follicle groups diameter was larger than back’s, buttocks’was the smallest (P<0.01); cervical primary and secondary follicles depth were deeper than back’s, buttocks’was the smallest (P>0.01).Real-time quantitative PCR was used to study MTR-1A, IGF-1 and TYR mRNA expressions in skins of Jining Gray goats within 85-days fetus, 3-days-old, 1-month-old, and one-year-old.(1) MTR-1A and IGF-1 in lamb-skins were extremely larger than other developmental phases, 19.5 and 39.26 times than fetus. MTR-1A and IGF-1 largely mRNA expressions in lamb-skins indicated that they played a role in promoting the development of secondary follicle.(2) MTR-1A expressed in one year old adult cervical and back skins were different with buttocks (P<0.05).IGF-1 expressed in cervical, back and buttocks skin were larger than abdomen (P<0.05). MTR-1A and IGF-1 differential expression and distribution of hair follicle group indicated MT play an important role in primary hair follicle than IGF-1.(3) MTR-1A and IGF-1 differential expression related with wool fineness (P>0.05), they are important candidate genes effecting wool fineness.(4) TYR mRNA expressed in kid and adult-skins were significant larger than lamb-skins’, because of lighting the main activity of the skin was the occurrence of melanin granules after birth, melanocyte at the top of the dermal papilla generated melanin granules effecting the colour of wool fiber.The results lay the foundation to reveal the developmental pattern of Jining Gray goat’s hair follicle. Furthermore, these results also reveal the genetic mechanism of Jining Gray goat’s pattern quality.更多还原