济宁青山羊皮肤毛囊体外培养和基因表达差异研究
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摘要
济宁青山羊是具有独特毛色和花形的羔皮用山羊品种,其主要产品是猾子皮、板皮和山羊绒。羔羊生后1-3天内屠宰后得到的猾子皮具有天然的色彩和花纹,毛色品质好,光润,皮板轻巧而美观。猾子皮所制的翻毛皮衣和帽领等在国际市场上极为畅销,享有盛誉。本研究主要采用组织学技术和差异显示反转录PCR(DDRT-PCR)等分子细胞生物学技术,对济宁青山羊的皮肤毛囊组织结构和基因表达差异进行了研究,研究结果有助于揭示动物毛囊器官的生长发育规律;并为揭示济宁青山羊猾子皮花纹品质的分子遗传机理奠定基础。
本文首先以组织学方法,研究了济宁青山羊猾子皮和成年羊皮肤毛囊的组织学结构。结果表明:猾子皮的毛囊与表皮呈45度倾斜,密度较稀。成年羊皮肤初级毛囊的毛球较大,毛囊较粗长,具有汗腺、皮脂腺、竖毛肌等附属结构,毛干有髓质;次级毛囊的毛球较小,皮脂腺不发达或缺如,毛干没有髓质。
比较了揉后小猾皮和大猾皮的品质和组织学结构特点。结果表明:小猾皮干皮重平均为88.75±11.33,比大猾皮(出生30-60日龄)的皮板(重159.83±10.72)轻薄;有花面积比例为67.84±2.97,明显高于大猾皮(60.00±7.39),且优等的波浪花所占比例较高;单位皮重面积产率(14.83±1.49)亦高于大猾皮(12.98±0.61)。但是,小猾皮的抗张强度、撕裂强度、崩裂强度均不及大猾皮。大猾皮与小猾皮皮厚的差异极显著(P<0.01);大猾皮各个部位皮厚的部位差差异不显著(P>0.05);而小猾皮各个部位皮厚的部位差明显大于大猾皮,且差异显著(P<0.05)。
济宁青山羊是生产猾子皮的一个专用品种,今后应加强该品种的选育和改良,增大羊的体型,提高猾皮总面积、波浪花的比例和皮板的物理机械指标,以便获得品质更好的猾子皮,进一步提高经济效益。
进一步采用差异显示反转录PCR技术,研究了济宁青山羊胎儿期、小猾皮和大猾皮皮肤组织的基因表达差异,结果表明:8种引物组合在3个年龄段的青山羊皮肤组织中共扩增出482条清晰可辩的条带,其中,在三个发育阶段的皮肤组织中未发现“有或无”的差异表达条带;但发现了99条表达“强或弱”的差异表达片段,占总条带数的20.54%。若进一步对这些差异表达条带进行回收、杂交验证和克隆测序,则有望得到与毛囊生长发育相关的差异表达基因。
采用三因子三水平的拉丁方正交试验设计(L9(34))方法,对比研究了无血清的Williams E培养基、无血清的DMEM培养基和添加10%FCS(胎牛血清)的DMEM培养基,及其在三种培养基中分别添加0、2ng/mL和10ng/mL三种不同浓度的血管内皮细胞生长因子(VEGF)和表皮细胞生长因子(EGF)时对成年羊毛囊体外培养的影响。将山羊的单根毛囊完整地从皮肤组织中分离出来,同时平行培养在9种培养基中,借助于倒置显微镜和目镜测微尺随时不断地观察毛囊生长的形态变化和测量毛根的长度。结果发现:山羊游离毛囊在9种培养基中均能很好地生长;但以无血清的培养基和在Williams E培养基的效果最好;分别和同时添加2%-10%的VEGF和EGF时可加快毛囊的生长;添加10%FCS和不添加细胞生长因子时毛囊也能生长,但生长速度较慢。
本研究结果为揭示青山羊毛囊发育规律奠定了基础;为进一步揭示济宁青山羊花纹品质的遗传机理奠定了基础。
Jining Gray Goat is the world-famous fur-bearing animal with its special color and patterns. Jining Gray goat has small size and delicious meat, and the main products of it are skins and cashmere. The lamb’s skins obtained 1-3 days after lamb was born have natural color and patterns, and also have the characters of good color, brilliancy and beauty. The products made by this kind of skin always have a good market all over the world. The methods of tissue slice and differential display reverse transcription polymerase chain reaction (DDRT-PCR) were used to study the developmental mechanism and structure about hair follicle of Jining Gray goat. The results were also helpful to indicate genetic mechanism of color quality of Jining Gray goat.
The structure of the adult goat hair follicle was studied by the method of histology. The results show that, the density of the hair follicle of the lamb skin is rather thin and there is a 45°of anguli between it and epiderm. The characteristics of the primary hair follicle of elder goat’s skin are big hair bulb, long and pachy hair follicle, and there are accessory structures such as sweat glands, sebaceous glands, arrector muscle, and there is medulla in hair shaft; The characteristics of secondary hair follicle are small hair bulb, undeveloped or absent sebaceous glands, and there is no medulla in the hair shaft.
Furthermore, we compared the characteristics of structure between kid lamb skin and elder lamb (born within 30-60 days) skin. The results indicated that: the mean weight of dry skin of kid lamb skin is 88.75±11.33,it is lighter and thinner more than that of elder lamb skin (159.83±10.72); the ratio of area with patterns is 67.84±2.97, it is significantly higher than that of elder lamb skin (60.00±7.39), moreover it has a high proportion in area with super wave patterns; and meanwhile the area within a unit of skin weight (14.83±1.49) is higher than that of elder lamb skin (12.98±0.61). However, compared to elder lamb’skin the kid lamb’skin shows inferiority in tensile strength, tearing strength and cracking strength. The thickness of elder lamb’skin is significantly higher (P<0.01) comparing to that of kid lamb skin; there is no significant difference in thickness between the different parts of elder lamb skin (P>0.05); while it shows a significant result in little lamb’skin (P<0.05).
Jining Gray goat is a special breed for lamb’skin. Thus some improvement and selection work in body type, the area of lamb’skin, wave patterns and physical traits is recommended to do for this breed so as to obtain superior lamb’skin and makes big economic returns.
482 cDNA fragments were isolated used 8 kinds of primers’combination in the skin tissues of Jining Gray goats among three age stages with the DDRT-PCR method in order to study the differential expressed genes of skin tissues among kid lamb(born whin 1-3 days)skin, elder lamb skin(born whin 30-60 days)and adult goat. These results showed that the patterns of expression are similar among the little lamb’skin, elder lamb’skin and adult goat skins. 99 bands, which accounted for 11.44%, were different between lamb’skin and adult skin. Furthermore, isolation, hybridization and sequencing of these differential expressied bands would help to attain the differential expression genes involved in the development of goat skin hair follicle.
For obtaining a succinct and effective method of in vitro culture, the effect on adult goat’s hair follicles was tested by orthogonal latin square (L9(34)) method under different circumstances. We completely separated the single hair follicle from skin tissue and cultured in 9 kinds of medium at the same time. Used the inverted microscope and eyepiece micrometer, we recorded hair follicles’shape change and length. The results indicated that the hair follicles grew well in all 9 kinds of medium in vitro. However, the mediums without serum and that with Wlilliams E were the best two choices. The growth of hair follicle was promoted by 2%-10% VEGF and/or EGF, and inhibited by 10% FCS. Furthermore, it grew slowly without growth factors.
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 Gray goat’s pattern quality.
本文首先以组织学方法,研究了济宁青山羊猾子皮和成年羊皮肤毛囊的组织学结构。结果表明:猾子皮的毛囊与表皮呈45度倾斜,密度较稀。成年羊皮肤初级毛囊的毛球较大,毛囊较粗长,具有汗腺、皮脂腺、竖毛肌等附属结构,毛干有髓质;次级毛囊的毛球较小,皮脂腺不发达或缺如,毛干没有髓质。
比较了揉后小猾皮和大猾皮的品质和组织学结构特点。结果表明:小猾皮干皮重平均为88.75±11.33,比大猾皮(出生30-60日龄)的皮板(重159.83±10.72)轻薄;有花面积比例为67.84±2.97,明显高于大猾皮(60.00±7.39),且优等的波浪花所占比例较高;单位皮重面积产率(14.83±1.49)亦高于大猾皮(12.98±0.61)。但是,小猾皮的抗张强度、撕裂强度、崩裂强度均不及大猾皮。大猾皮与小猾皮皮厚的差异极显著(P<0.01);大猾皮各个部位皮厚的部位差差异不显著(P>0.05);而小猾皮各个部位皮厚的部位差明显大于大猾皮,且差异显著(P<0.05)。
济宁青山羊是生产猾子皮的一个专用品种,今后应加强该品种的选育和改良,增大羊的体型,提高猾皮总面积、波浪花的比例和皮板的物理机械指标,以便获得品质更好的猾子皮,进一步提高经济效益。
进一步采用差异显示反转录PCR技术,研究了济宁青山羊胎儿期、小猾皮和大猾皮皮肤组织的基因表达差异,结果表明:8种引物组合在3个年龄段的青山羊皮肤组织中共扩增出482条清晰可辩的条带,其中,在三个发育阶段的皮肤组织中未发现“有或无”的差异表达条带;但发现了99条表达“强或弱”的差异表达片段,占总条带数的20.54%。若进一步对这些差异表达条带进行回收、杂交验证和克隆测序,则有望得到与毛囊生长发育相关的差异表达基因。
采用三因子三水平的拉丁方正交试验设计(L9(34))方法,对比研究了无血清的Williams E培养基、无血清的DMEM培养基和添加10%FCS(胎牛血清)的DMEM培养基,及其在三种培养基中分别添加0、2ng/mL和10ng/mL三种不同浓度的血管内皮细胞生长因子(VEGF)和表皮细胞生长因子(EGF)时对成年羊毛囊体外培养的影响。将山羊的单根毛囊完整地从皮肤组织中分离出来,同时平行培养在9种培养基中,借助于倒置显微镜和目镜测微尺随时不断地观察毛囊生长的形态变化和测量毛根的长度。结果发现:山羊游离毛囊在9种培养基中均能很好地生长;但以无血清的培养基和在Williams E培养基的效果最好;分别和同时添加2%-10%的VEGF和EGF时可加快毛囊的生长;添加10%FCS和不添加细胞生长因子时毛囊也能生长,但生长速度较慢。
本研究结果为揭示青山羊毛囊发育规律奠定了基础;为进一步揭示济宁青山羊花纹品质的遗传机理奠定了基础。
Jining Gray Goat is the world-famous fur-bearing animal with its special color and patterns. Jining Gray goat has small size and delicious meat, and the main products of it are skins and cashmere. The lamb’s skins obtained 1-3 days after lamb was born have natural color and patterns, and also have the characters of good color, brilliancy and beauty. The products made by this kind of skin always have a good market all over the world. The methods of tissue slice and differential display reverse transcription polymerase chain reaction (DDRT-PCR) were used to study the developmental mechanism and structure about hair follicle of Jining Gray goat. The results were also helpful to indicate genetic mechanism of color quality of Jining Gray goat.
The structure of the adult goat hair follicle was studied by the method of histology. The results show that, the density of the hair follicle of the lamb skin is rather thin and there is a 45°of anguli between it and epiderm. The characteristics of the primary hair follicle of elder goat’s skin are big hair bulb, long and pachy hair follicle, and there are accessory structures such as sweat glands, sebaceous glands, arrector muscle, and there is medulla in hair shaft; The characteristics of secondary hair follicle are small hair bulb, undeveloped or absent sebaceous glands, and there is no medulla in the hair shaft.
Furthermore, we compared the characteristics of structure between kid lamb skin and elder lamb (born within 30-60 days) skin. The results indicated that: the mean weight of dry skin of kid lamb skin is 88.75±11.33,it is lighter and thinner more than that of elder lamb skin (159.83±10.72); the ratio of area with patterns is 67.84±2.97, it is significantly higher than that of elder lamb skin (60.00±7.39), moreover it has a high proportion in area with super wave patterns; and meanwhile the area within a unit of skin weight (14.83±1.49) is higher than that of elder lamb skin (12.98±0.61). However, compared to elder lamb’skin the kid lamb’skin shows inferiority in tensile strength, tearing strength and cracking strength. The thickness of elder lamb’skin is significantly higher (P<0.01) comparing to that of kid lamb skin; there is no significant difference in thickness between the different parts of elder lamb skin (P>0.05); while it shows a significant result in little lamb’skin (P<0.05).
Jining Gray goat is a special breed for lamb’skin. Thus some improvement and selection work in body type, the area of lamb’skin, wave patterns and physical traits is recommended to do for this breed so as to obtain superior lamb’skin and makes big economic returns.
482 cDNA fragments were isolated used 8 kinds of primers’combination in the skin tissues of Jining Gray goats among three age stages with the DDRT-PCR method in order to study the differential expressed genes of skin tissues among kid lamb(born whin 1-3 days)skin, elder lamb skin(born whin 30-60 days)and adult goat. These results showed that the patterns of expression are similar among the little lamb’skin, elder lamb’skin and adult goat skins. 99 bands, which accounted for 11.44%, were different between lamb’skin and adult skin. Furthermore, isolation, hybridization and sequencing of these differential expressied bands would help to attain the differential expression genes involved in the development of goat skin hair follicle.
For obtaining a succinct and effective method of in vitro culture, the effect on adult goat’s hair follicles was tested by orthogonal latin square (L9(34)) method under different circumstances. We completely separated the single hair follicle from skin tissue and cultured in 9 kinds of medium at the same time. Used the inverted microscope and eyepiece micrometer, we recorded hair follicles’shape change and length. The results indicated that the hair follicles grew well in all 9 kinds of medium in vitro. However, the mediums without serum and that with Wlilliams E were the best two choices. The growth of hair follicle was promoted by 2%-10% VEGF and/or EGF, and inhibited by 10% FCS. Furthermore, it grew slowly without growth factors.
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 Gray goat’s pattern quality.
引文
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