CN113413382A - 褪黑激素对子代獭兔毛囊细胞凋亡率影响的研究方法 - Google Patents
褪黑激素对子代獭兔毛囊细胞凋亡率影响的研究方法 Download PDFInfo
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Abstract
本发明提供褪黑激素对子代獭兔毛囊细胞凋亡率影响的研究方法,涉及褪黑激素应用獭兔养殖领域,本发明包括如下研究步骤:确定褪黑激素埋植剂量;将实验用兔分为激素埋植组以及不埋植激素的对照组,以在实验用兔产子后,采集子代皮肤组织样本;制作皮肤组织样本的切片;切片的综合处理,以及对细胞凋亡数据的测定,通过对母代埋植褪黑激素,可显著减少了子代毛囊细胞的凋亡率,并且毛囊细胞与毛囊密度有直接关系,即,降低毛囊细胞的凋亡率可提高獭兔的毛囊密度,而毛囊密度是评定皮张的主要依据,提高毛密度可以提高獭兔皮张的价格,以增加养殖户的经济收益。
Description
技术领域
本发明涉及褪黑激素应用獭兔养殖领域,尤其涉及褪黑激素对子代獭兔毛囊细胞凋亡率影响的研究方法。
背景技术
獭兔是世界著名的大型短毛兔品种,个性相当活泼好动,是一种典型的皮用型兔,生产方向是以皮为主,兼用其肉,因其毛皮酷似珍贵毛皮兽水獭,故称为獭兔。
獭兔养殖中,对于獭兔的皮毛管理是关于养殖户切身利益的重大事项,目前对于褪黑激素应用至獭兔养殖,来提高獭兔的毛发密度具有较高的正向作用,而现有褪黑激素的使用都是通过自身的埋植来提高毛囊密度,这样埋植主要有两个问题:
其一,自身埋植只能提高獭兔的次级毛囊密度,而对初级毛囊密度无影响,毛囊由初级毛囊和次级毛囊共同构成,因此如果要显著提高毛囊密度,需要从初级和次级毛囊两方面着手,胎儿是初级毛囊发生的主要时期,孕期埋植褪黑激素可以对胎儿的毛囊发生起作用,减少子代毛囊细胞的凋亡,提高獭兔的毛囊密度。
其二,自身埋植褪黑激素只能影响自身的毛囊密度,而在规模化养殖獭兔中,对单个体进行埋植,整体埋植数量大,成本高,且不排除獭兔个体因埋植褪黑激素不适宜,所导致的个体死亡问题。
遂,在獭兔养殖中,需求一种减少獭兔毛囊细胞的凋亡来提高獭兔毛囊密度的褪黑激素埋植方法。
发明内容
本发明的目的在于提供褪黑激素对子代獭兔毛囊细胞凋亡率影响的研究方法,以解决上述技术问题。
本发明为解决上述技术问题,采用以下技术方案来实现:
褪黑激素对子代獭兔毛囊细胞凋亡率影响的研究方法,其特征在于,包括如下研究步骤:
1)确定褪黑激素埋植剂量;
2)将实验用兔分为激素埋植组以及不埋植激素的对照组,以在实验用兔产子后,采集子代皮肤组织样本;
3)制作皮肤组织样本的切片;
4)切片的综合处理,以及对细胞凋亡数据的测定。
所述步骤2)中激素埋植组埋植褪黑激素缓释颗粒,不埋植激素的对照组埋植不含激素的硅胶颗粒;
所述步骤3)中切片的制作包括脱水、透明、包埋、切片、摊片和烤片;
所述步骤4)中切片的综合处理包括蛋白酶K修复、破膜、室温平衡、加反应液、DAPI复染细胞核、封片以及镜检拍照。
优选的,所述步骤1)中褪黑激素的埋植剂量为10mg。
优选的,所述步骤2)中实验用兔为若干母兔与同一只公兔精液进行人工授精,且在人工授精后诊断出受孕的孕兔作为实验用兔;
所述步骤2)中实验用兔产子后,自激素埋植组以及不埋植激素的对照组中选择数量相一致的窝仔兔,采用颈部移位法进行屠宰,屠宰后立即剥皮,于臀十字部位取2cm边长的方形皮肤块,用生理盐水冲洗后立即放入多聚甲醛溶液中固定。
优选的,所述步骤3)中皮肤组织样本的脱水为梯度脱水;
梯度脱水的具体步骤如下:
1)75%乙醇内脱水1小时;
2)85%乙醇内脱水1小时;
3)将95%乙醇分为两组,各组内脱水1小时;
4)将100%乙醇分为两组,各组内脱水45分钟;
5)苯乙醇内脱水45分钟,其中苯乙醇溶液为体积比为1:1的二甲苯和乙醇;
所述步骤3)中皮肤组织样本的透明由二甲苯作用,步骤3)中皮肤组织样本的包埋由石蜡固定;
透明和包埋的具体步骤如下:
1)将二甲苯分为两份,皮肤组织样本置于每份二甲苯内7分钟;
2)其后将皮肤组织样本置于苯蜡中1小时;
3)将石蜡分为两份,皮肤组织样本置于每份石蜡内1小时;
所述步骤3)中皮肤组织样本的切片步骤如下:
石蜡包埋48小时后切片,将蜡块放在切片机上,先按住调平按钮调节刀片和蜡块的间隔以及视点,试切,切片机档位先调到10微米,当切到组织时调至6微米;
所述步骤3)中皮肤组织样本的摊片和烤片步骤如下:
1)切下的组织轻轻放入摊片机中,待皮肤组织彻底展平后,用载玻片将组织捞出;
2)去除剩余的水后放置于烘片机上,进行烘干处理。
优选的,所述步骤4)中蛋白酶K修复的操作步骤下:
1)切片完全甩干前,用组化笔在组织周围画圈,在圈内滴加蛋白酶K工作液覆盖组织,37℃温箱孵育22分钟;
2)将玻片置于pH为7.4的PBS中后在脱色摇床上晃动洗涤3次,每次5分钟;
所述蛋白酶K工作液浓度为原液:PBS=1:9。
优选的,所述步骤4)中破膜的操作步骤如下:
1)切片完全甩干前,在圈内滴加破膜工作液覆盖组织,常温下孵育20分钟;
2)将玻片置于PBS中后脱色摇床上晃动洗涤3次,每次5分钟;
所述破膜液为0.1%Triton,即Triton原液:PBS=1:1000的混合溶;
所述室温平衡是将未完全甩干的切片,在圈内滴加缓冲液覆盖组织,缓冲液常温孵育10分钟。
优选的,所述步骤4)中加反应液的操作步骤如下:
1)按片子数量和组织大小取Tunel试剂盒内适量末端转移酶、核酸标记荧光染料、缓冲液,按1:5:50比例混合,加到圈内覆盖组织,
2)切片平放于湿盒内,37℃恒温箱孵育2小时,湿盒内加少量水保持湿度;
所述步骤4)中DAPI复染细胞核的操作步骤如下:
1)将切片用pH为7.4的PBS洗涤三次,每次5分钟;
2)去除PBS后在圈内滴加DAPI染液,避光室温孵育10分钟。
优选的,所述步骤4)中封片的操作步骤如下:
1)玻片置于pH为7.4的PBS中在脱色摇床上晃动洗涤3次,每次5分钟;
2)切片未甩干前,用抗荧光淬灭封片剂封片。
优选的,所述镜检拍照中切片于荧光显微镜下观察并采集图像,其中DAPI紫外激发波长330-380纳米,发射波长420纳米,发蓝光;FITC激发波长465-495纳米,发射波长515-555纳米,发绿光。
优选的,所述细胞凋亡数据的测定步骤如下:
1)DAPI染出来的细胞核在紫外的激发下为蓝色,试剂盒为FITC荧光素标记,阳性凋亡细胞核为绿色;
2)每张片子选取5个完整视野,对蓝色细胞核和荧光绿色凋亡的细胞核分别计数,每组做5个生物学重复。
哺乳动物的毛囊发生是在胚胎时期,毛囊的发育与胚胎表皮分层有着密切的联系,在胎儿时期毛囊发生经历了毛基板的形成,毛囊器官的形成和细胞的分化三个阶段,表皮上皮和间质真皮的相互作用形成了毛囊,出生后的各种营养和外界条件等干预和处理对次级毛囊的周期可以起到改善作用,但对于初级毛囊效果甚微,由于毛囊是固定在皮下组织中的器官,在胎儿时期毛囊将固定并伴随终身,因此,从胎儿时期着手能够从根本上提高毛皮动物毛囊密度。
本发明的有益效果是:
本发明中通过对母代埋植褪黑激素,可显著减少了子代毛囊细胞的凋亡率,并且毛囊细胞与毛囊密度有直接关系,即,降低毛囊细胞的凋亡率可提高獭兔的毛囊密度,而毛囊密度是评定皮张的主要依据,提高毛密度可以提高獭兔皮张的价格,以增加养殖户的经济收益。
附图说明
图1为本发明褪黑激素对子代獭兔毛囊细胞凋亡率影响的研究方法的工艺图示意图;
图2为本发明中两组初生仔兔皮肤样本于5倍显微镜下的TUNEL染色情况示意图;
图3为本发明中两组初生仔兔皮肤样本于10倍显微镜下的TUNEL染色情况示意图;
图4为本发明中两组初生仔兔皮肤样本于20倍显微镜下的TUNEL染色情况示意图;
图5为本发明中两组初生仔兔皮肤样本于40倍显微镜下的TUNEL染色情况示意图。
具体实施方式
基于实施方式中的实施例,本领域技术人员在没有做出创造性劳动的前提下所获得其它实施例,都属于本发明的保护范围。
下面基于图示阐述本发明的具体实施例。
实施例1
褪黑激素对子代獭兔毛囊细胞凋亡率影响的研究方法,包括如下研究步骤:
1)种母兔受孕:选择90只种母兔,为了减少遗传噪音,用同一只公兔精液进行人工授精,配种后10天妊娠诊断出79只孕兔作为试验用兔;
2)埋植颗粒:将孕兔分为两组,分别埋植10mg褪黑激素缓释颗粒(褪黑激素组,n=40)和不含激素的硅胶颗粒(对照组,n=39),其中獭兔的妊娠期共30天,妊娠鉴定最早是在胎龄10天,獭兔胎儿毛囊的发育始于胎龄19天,选择在胎龄10天时埋植,早于胚胎发育时间,让褪黑激素尽早的进入母体循环来影响胎儿,并且,埋植方式为后颈部皮肤埋植,其主要原因是獭兔后颈部皮肤皮下脂肪最少,埋植后褪黑激素缓释效果好,其次獭兔皮张中颈部皮较薄,埋植时相对容易、方便;第三个原因是獭兔平时活动不会触碰到后颈部,减少了将褪黑激素颗粒损害的风险;
3)采集样本:母兔生产当天,每组各选7只不同窝仔兔屠宰采用颈部移位法进行屠宰,屠宰后立即剥皮,在臀十字部位取2cm边长的方形皮肤块,用生理盐水冲洗后立即放入多聚甲醛溶液中固定,其中选择了仔兔出生当天取皮,原因是出生当天取皮可以减少环境、营养、饲养条件等对仔獭兔毛囊发育的干扰,试验结果就可以反应是由于埋植褪黑激素引起的毛囊细胞调亡的差异;
4)皮肤组织样本制作切片;
5)蛋白酶K修复:切片完全甩干前,用组化笔在组织周围画圈,在圈内滴加蛋白酶K工作液覆盖组织,37℃温箱孵育22分钟,将玻片置于pH为7.4的PBS中后在脱色摇床上晃动洗涤3次,每次5分钟(蛋白酶K工作液浓度为原液:PBS=1:9);
6)破膜:切片完全甩干前,在圈内滴加破膜工作液覆盖组织,常温下孵育20分钟,将玻片置于PBS中后脱色摇床上晃动洗涤3次,每次5分钟,其中破膜液为0.1%Triton,即Triton原液:PBS=1:1000的混合溶液;
7)室温平衡:将未完全甩干的切片,在圈内滴加缓冲液覆盖组织,缓冲液常温孵育10分钟;
8)加反应液:按片子数量和组织大小取Tunel试剂盒内适量末端转移酶、核酸标记荧光染料、缓冲液,按1:5:50比例混合,加到圈内覆盖组织,切片平放于湿盒内,37℃恒温箱孵育2小时,湿盒内加少量水保持湿度;
9)DAPI复染细胞核:将切片用pH为7.4的PBS洗涤三次,每次5分钟,去除PBS后在圈内滴加DAPI染液,避光室温孵育10分钟;
10)封片:玻片置于pH为7.4的PBS中在脱色摇床上晃动洗涤3次,每次5分钟,切片未甩干前,用抗荧光淬灭封片剂封片;
11)镜检拍照:镜检拍照中切片于荧光显微镜下观察并采集图像,其中DAPI紫外激发波长330-380纳米,发射波长420纳米,发蓝光;FITC激发波长465-495纳米,发射波长515-555纳米,发绿光;
12)细胞调亡数据的测定:DAPI染出来的细胞核在紫外的激发下为蓝色,试剂盒为FITC荧光素标记,阳性凋亡细胞核为绿色,每张片子选取5个完整视野,对蓝色细胞核和荧光绿色凋亡的细胞核分别计数,每组做5个生物学重复。
实施例2
本发明对孕期獭兔开展试验,将孕兔分为两组,分别埋植10mg褪黑激素缓释颗粒(褪黑激素组)和不含激素的硅胶颗粒(对照组),母兔生产当天每组各选不同窝仔兔屠宰,对仔兔的毛囊细胞凋亡情况进行测定,通过转录组学测序技术分析两组仔兔皮肤差异表达基因,富集通路,筛选并鉴定出影响子代毛囊发育和毛囊细胞凋亡的关键基因,利用采用TUNEL法对仔兔毛囊细胞调亡情况进行检测。
通过TUNEL法对仔兔毛囊细胞调亡情况进行检测的图片数据如图2-5,如图2-5数据所测算的表格数据如表一:
表一 TUNEL法检测两组仔兔毛囊细胞凋亡率
其中数据统计方法:DAPI染出来的细胞核在紫外的激发下为蓝色,试剂盒为FITC荧光素标记,阳性凋亡细胞核为绿色,每张片子选取5个完整视野,在DAPI模式下对每个视野中蓝色细胞核进行计数并记录,记为DA,在FITC模式下对荧光绿色凋亡的细胞核进行计数并记录,记为FI,细胞凋亡率的计算算式为凋亡率(%)=FI/DA*100%。
数据分析方法是所有数据采用统计分析软件进行方差分析,数据采用单因素方差分析,以组别作为固定因子,凋亡率作为因变量,并进行方差齐性检验后计算平均值和标准差,平均值的显著性差异采用邓肯检验,显著性差异定义为p<0.05,最终数据以平均值±标准差表示。
由图2-5可知,与CR组比较,MT组DAPI染色细胞与凋亡细胞重合少,凋亡阳性细胞率低,由表一可知,MT组毛囊细胞凋亡率极显著低于CR组(p<0.01),这说明母体埋植MT减少了子代獭兔毛囊细胞的调亡。
以上显示和描述了本发明的基本原理、主要特征和本发明的优点。本行业的技术人员应该了解,本发明不受上述实施例的限制,上述实施例和说明书中描述的仅为本发明的优选例,并不用来限制本发明,在不脱离本发明精神和范围的前提下,本发明还会有各种变化和改进,这些变化和改进都落入要求保护的本发明范围内。本发明要求保护范围由所附的权利要求书及其等效物界定。
Claims (10)
1.褪黑激素对子代獭兔毛囊细胞凋亡率影响的研究方法,其特征在于,包括如下研究步骤:
1)确定褪黑激素埋植剂量;
2)将实验用兔分为激素埋植组以及不埋植激素的对照组,以在实验用兔产子后,采集子代皮肤组织样本;
3)制作皮肤组织样本的切片;
4)切片的综合处理,以及对细胞凋亡数据的测定。
所述步骤2)中激素埋植组埋植褪黑激素缓释颗粒,不埋植激素的对照组埋植不含激素的硅胶颗粒;
所述步骤3)中切片的制作包括脱水、透明、包埋、切片、摊片和烤片;
所述步骤4)中切片的综合处理包括蛋白酶K修复、破膜、室温平衡、加反应液、DAPI复染细胞核、封片以及镜检拍照。
2.根据权利要求1所述的褪黑激素对子代獭兔毛囊细胞凋亡率影响的研究方法,其特征在于:所述步骤1)中褪黑激素的埋植剂量为10mg。
3.根据权利要求1所述的褪黑激素对子代獭兔毛囊细胞凋亡率影响的研究方法,其特征在于:
所述步骤2)中实验用兔为若干母兔与同一只公兔精液进行人工授精,且在人工授精后诊断出受孕的孕兔作为实验用兔;
所述步骤2)中实验用兔产子后,自激素埋植组以及不埋植激素的对照组中选择数量相一致的窝仔兔,采用颈部移位法进行屠宰,屠宰后立即剥皮,于臀十字部位取2cm边长的方形皮肤块,用生理盐水冲洗后立即放入多聚甲醛溶液中固定。
4.根据权利要求1所述的褪黑激素对子代獭兔毛囊细胞凋亡率影响的研究方法,其特征在于:所述步骤3)中皮肤组织样本的脱水为梯度脱水;
梯度脱水的具体步骤如下:
1)75%乙醇内脱水1小时;
2)85%乙醇内脱水1小时;
3)将95%乙醇分为两组,各组内脱水1小时;
4)将100%乙醇分为两组,各组内脱水45分钟;
5)苯乙醇内脱水45分钟,其中苯乙醇溶液为体积比为1:1的二甲苯和乙醇;
所述步骤3)中皮肤组织样本的透明由二甲苯作用,步骤3)中皮肤组织样本的包埋由石蜡固定;
透明和包埋的具体步骤如下:
1)将二甲苯分为两份,皮肤组织样本置于每份二甲苯内7分钟;
2)其后将皮肤组织样本置于苯蜡中1小时;
3)将石蜡分为两份,皮肤组织样本置于每份石蜡内1小时;
所述步骤3)中皮肤组织样本的切片步骤如下:
石蜡包埋48小时后切片,将蜡块放在切片机上,先按住调平按钮调节刀片和蜡块的间隔以及视点,试切,切片机档位先调到10微米,当切到组织时调至6微米;
所述步骤3)中皮肤组织样本的摊片和烤片步骤如下:
1)切下的组织轻轻放入摊片机中,待皮肤组织彻底展平后,用载玻片将组织捞出;
2)去除剩余的水后放置于烘片机上,进行烘干处理。
5.根据权利要求1所述的褪黑激素对子代獭兔毛囊细胞凋亡率影响的研究方法,其特征在于:
所述步骤4)中蛋白酶K修复的操作步骤下:
1)切片完全甩干前,用组化笔在组织周围画圈,在圈内滴加蛋白酶K工作液覆盖组织,37℃温箱孵育22分钟;
2)将玻片置于pH为7.4的PBS中后在脱色摇床上晃动洗涤3次,每次5分钟;
所述蛋白酶K工作液浓度为原液:PBS=1:9。
6.根据权利要求1所述的褪黑激素对子代獭兔毛囊细胞凋亡率影响的研究方法,其特征在于:
所述步骤4)中破膜的操作步骤如下:
1)切片完全甩干前,在圈内滴加破膜工作液覆盖组织,常温下孵育20分钟;
2)将玻片置于PBS中后脱色摇床上晃动洗涤3次,每次5分钟;
所述破膜液为0.1%Triton,即Triton原液:PBS=1:1000的混合溶液;
所述室温平衡是将未完全甩干的切片,在圈内滴加缓冲液覆盖组织,缓冲液常温孵育10分钟。
7.根据权利要求1所述的褪黑激素对子代獭兔毛囊细胞凋亡率影响的研究方法,其特征在于:
所述步骤4)中加反应液的操作步骤如下:
1)按片子数量和组织大小取Tunel试剂盒内适量末端转移酶、核酸标记荧光染料、缓冲液,按1:5:50比例混合,加到圈内覆盖组织;
2)切片平放于湿盒内,37℃恒温箱孵育2小时,湿盒内加少量水保持湿度;
所述步骤4)中DAPI复染细胞核的操作步骤如下:
1)将切片用pH为7.4的PBS洗涤三次,每次5分钟;
2)去除PBS后在圈内滴加DAPI染液,避光室温孵育10分钟。
8.根据权利要求1所述的褪黑激素对子代獭兔毛囊细胞凋亡率影响的研究方法,其特征在于:
所述步骤4)中封片的操作步骤如下:
1)玻片置于pH为7.4的PBS中在脱色摇床上晃动洗涤3次,每次5分钟;
2)切片未甩干前,用抗荧光淬灭封片剂封片。
9.根据权利要求1所述的褪黑激素对子代獭兔毛囊细胞凋亡率影响的研究方法,其特征在于:所述镜检拍照中切片于荧光显微镜下观察并采集图像,其中DAPI紫外激发波长330-380纳米,发射波长420纳米,发蓝光;FITC激发波长465-495纳米,发射波长515-555纳米,发绿光。
10.根据权利要求1所述的褪黑激素对子代獭兔毛囊细胞凋亡率影响的研究方法,其特征在于:
所述细胞凋亡数据的测定步骤如下:
1)DAPI染出来的细胞核在紫外的激发下为蓝色,试剂盒为FITC荧光素标记,阳性凋亡细胞核为绿色;
2)每张片子选取5个完整视野,对蓝色细胞核和荧光绿色凋亡的细胞核分别计数,每组做5个生物学重复。
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封洋;姜天团;滚双宝;孙蕴文;张英杰;史浩;康景;段晓青;: "皮下埋植褪黑激素对獭兔毛囊发育和毛皮质量的影响", 家畜生态学报, vol. 40, no. 04, pages 52 - 56 * |
康景;封洋;: "母体埋植褪黑激素对子一代獭兔内脏组织中RORα基因表达的影响", 中国畜牧杂志, vol. 56, no. 01, pages 86 - 94 * |
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