不同免疫方式对雏鸡免疫器官发育、细胞增殖及凋亡相关基因表达的影响
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摘要
为研究不同免疫方式对雏鸡的应激以及对雏鸡免疫器官发育、细胞增殖、分化及凋亡相关基因表达的影响,揭示免疫应激对免疫器官发育抑制的机理,进行如下试验。试验设计
选取1日龄健康固始鸡公雏240只,随机分为A、B、C、D四个处理,每个处理4个重复,每个重复15只鸡。其中A组为对照组,抓捕后放回原笼;B组采用ND-IB弱毒苗进行滴鼻点眼免疫;C组和D组均采用ND-IB弱毒苗和灭活苗进行滴鼻点眼和皮下注射免疫,且D组从7日龄起在日粮中添加维生素E(100 IU/kg)。所有鸡只于10日龄早上8:00进行免疫。
试验一不同免疫方式对雏鸡应激敏感激素水平的影响
于免疫后的1d、3d、5d和7d从每个处理每重复取3只鸡,称活体重,心脏采血,分离血清,ELISA试剂盒检测血清应激敏感激素促肾上腺皮质激素(ACTH)和皮质酮(CORT)的浓度,结果表明:滴鼻点眼和皮下注射免疫均对雏鸡造成一定应激;应激从免疫后第3d开始得到缓解;日粮添加VE(100IU/kg)能够有效缓解应激。
试验二不同免疫方式对雏鸡体重与免疫器官发育的影响
于免疫后的1d、3d、5d和7d从每个处理每重复取3只鸡,称活体重,分离胸腺、脾脏和法氏囊并称重,计算免疫器官指数。结果表明:不同免疫方式对雏鸡的体重均无显著影响;皮下注射免疫导致雏鸡胸腺重量显著增加,添加VE(100IU/kg)后雏鸡的胸腺重略有降低;不同免疫方式对雏鸡脾脏和法氏囊发育均无显著影响。
试验三不同免疫方式对雏鸡免疫器官细胞增殖的影响
于免疫后的1d、3d、5d和7d从每个处理每重复取1只鸡,分离胸腺、脾脏和法氏囊,采用流式细胞术检测免疫器官细胞周期百分比,包括:分裂间期1(G1期)、DNA合成期(S期)和有丝分裂期(M期),计算细胞增殖指数。结果表明:皮下注射免疫导致雏鸡胸腺G1细胞比例在免疫后第3-5d时升高,抑制了胸腺细胞的增殖;添加VE(100IU/kg)后胸腺G1细胞比例降低,更多细胞进入DNA合成期;各组的脾脏细胞增殖指数在免疫后1-5d呈下降趋势。
试验四不同免疫方式对免疫器官细胞凋亡相关基因表达量的影响
于免疫后的1d、3d、5d和7d从每个处理每重复取1只鸡,分离胸腺、脾脏和法氏囊,提取总RNA,反转录出cDNA,用SYBR Green I做染料,以β-actin为参照基因,采用实时荧光定量PCR法检测抗凋亡基因Bcl-2、促凋亡基因Fas和FasL的相对表达量。结果表明:皮下注射免疫导致法氏囊Bcl-2的表达量显著降低,添加VE(100IU/kg)显著提高了FasL的表达量;胸腺和法氏囊内Bcl-2和FasL的表达量在免疫后第5d均显著提高,Fas和FasL的表达量也显著提高,共同参与细胞凋亡的调控。
The experiment was conducted to determine the stress of different immunizations on Gushi chicken and effects on the development of immune organs,cell cycle,cell proliferation and differentiation and expression level of apoptosis-related gene.
Experimental design
240 one-day-old healthy Gushi male chickens were randomly assigned to 4 groups,each group contained 4 pens and 15 chickens per pen.Group A was control group without any immune, group B was given intranasal-conjunctival immunization, group C and D were given both intranasal-conjunctival immunization and subcutaneous-injection immunization. Additionally, vitamin E (100IU/kg) was added into diet of group D from 7-day-old. Chicken were treated with different immunizations at 10 days.
Trail 1 Effects of different immunizations on stress-related hermone level in chicken
Three chickens of each pen were randomly caught and blood samples were collected after 1,3,5,and 7d of immuned, serum concentration of CORT and ACTH were measured.The results suggested that:1day after immunization, serum concentration of CORT in group D was highest and significantly higher than group A and B(p<0.05);during 3 to 7days after immunization,CORT concentration of all group decreased to a similar level,while group D kept in a lower level;ACTH concentration of all groups had no significant difference(p>0.05).It indicated that subcutaneous-injection immunization caused severe stress on chicken ,and it was down-regulated by VE added in diet.
Trail 2 Effects of different immunizations on body weights and the development of immune organs in chicken
Three chickens of each pen were randomly caught and weighted before killed,immune organs were collected and weighted,organ index were calculated. The results suggested that:body weights had no significant difference within each group(p>0.05);Thymus index of group C was significantly higher than other groups at 3,5 and 7 days after immunization(p<0.05),while spleen index and bursa index had no significant difference in all treatments.These results indicated that: different immunizations had no significant effect on body weights, subcutaneous-injection immunization caused higher development of thymus,while had no effects on spleen and brusa of chicken.
Trail 3 Effects of different immunizations on cell proliferation of immune organs in chicken
One chickens of each pen were randomly caught and weighted before killed,immune organs were collected for cell cycle assay by FCM. The results suggested that:it had significant effect on S phase and G2/M phase by different immunizations, cell proliferation indexes of thymus and brusa were down-regulated,while cell proliferation indexes of spleen had no effect.
Trail 4 Effects of different immunizations on relative expression level of apoptosis-related gene of immune organs in chicken
One chickens of each pen were randomly caught and weighted before killed,immune organs were collected,total RNA were detection and reverse transcription into cDNA,SYBR GreenⅠreal-time PCR were used to test expression level of Bcl-2,Fas,andFasL.The results showed that expression of Bcl-2 and FasL in thymus were higher by subcutaneous-injection immunization; Bcl-2 gene was lower by different immunization,while expression of Fas was higher in spleen; Relative expression level of Bcl-2 was higher in bursa at 5d after immunization,and relative expression level of Fas and FasL were also higher in bursa at 3-5d after immunization.
选取1日龄健康固始鸡公雏240只,随机分为A、B、C、D四个处理,每个处理4个重复,每个重复15只鸡。其中A组为对照组,抓捕后放回原笼;B组采用ND-IB弱毒苗进行滴鼻点眼免疫;C组和D组均采用ND-IB弱毒苗和灭活苗进行滴鼻点眼和皮下注射免疫,且D组从7日龄起在日粮中添加维生素E(100 IU/kg)。所有鸡只于10日龄早上8:00进行免疫。
试验一不同免疫方式对雏鸡应激敏感激素水平的影响
于免疫后的1d、3d、5d和7d从每个处理每重复取3只鸡,称活体重,心脏采血,分离血清,ELISA试剂盒检测血清应激敏感激素促肾上腺皮质激素(ACTH)和皮质酮(CORT)的浓度,结果表明:滴鼻点眼和皮下注射免疫均对雏鸡造成一定应激;应激从免疫后第3d开始得到缓解;日粮添加VE(100IU/kg)能够有效缓解应激。
试验二不同免疫方式对雏鸡体重与免疫器官发育的影响
于免疫后的1d、3d、5d和7d从每个处理每重复取3只鸡,称活体重,分离胸腺、脾脏和法氏囊并称重,计算免疫器官指数。结果表明:不同免疫方式对雏鸡的体重均无显著影响;皮下注射免疫导致雏鸡胸腺重量显著增加,添加VE(100IU/kg)后雏鸡的胸腺重略有降低;不同免疫方式对雏鸡脾脏和法氏囊发育均无显著影响。
试验三不同免疫方式对雏鸡免疫器官细胞增殖的影响
于免疫后的1d、3d、5d和7d从每个处理每重复取1只鸡,分离胸腺、脾脏和法氏囊,采用流式细胞术检测免疫器官细胞周期百分比,包括:分裂间期1(G1期)、DNA合成期(S期)和有丝分裂期(M期),计算细胞增殖指数。结果表明:皮下注射免疫导致雏鸡胸腺G1细胞比例在免疫后第3-5d时升高,抑制了胸腺细胞的增殖;添加VE(100IU/kg)后胸腺G1细胞比例降低,更多细胞进入DNA合成期;各组的脾脏细胞增殖指数在免疫后1-5d呈下降趋势。
试验四不同免疫方式对免疫器官细胞凋亡相关基因表达量的影响
于免疫后的1d、3d、5d和7d从每个处理每重复取1只鸡,分离胸腺、脾脏和法氏囊,提取总RNA,反转录出cDNA,用SYBR Green I做染料,以β-actin为参照基因,采用实时荧光定量PCR法检测抗凋亡基因Bcl-2、促凋亡基因Fas和FasL的相对表达量。结果表明:皮下注射免疫导致法氏囊Bcl-2的表达量显著降低,添加VE(100IU/kg)显著提高了FasL的表达量;胸腺和法氏囊内Bcl-2和FasL的表达量在免疫后第5d均显著提高,Fas和FasL的表达量也显著提高,共同参与细胞凋亡的调控。
The experiment was conducted to determine the stress of different immunizations on Gushi chicken and effects on the development of immune organs,cell cycle,cell proliferation and differentiation and expression level of apoptosis-related gene.
Experimental design
240 one-day-old healthy Gushi male chickens were randomly assigned to 4 groups,each group contained 4 pens and 15 chickens per pen.Group A was control group without any immune, group B was given intranasal-conjunctival immunization, group C and D were given both intranasal-conjunctival immunization and subcutaneous-injection immunization. Additionally, vitamin E (100IU/kg) was added into diet of group D from 7-day-old. Chicken were treated with different immunizations at 10 days.
Trail 1 Effects of different immunizations on stress-related hermone level in chicken
Three chickens of each pen were randomly caught and blood samples were collected after 1,3,5,and 7d of immuned, serum concentration of CORT and ACTH were measured.The results suggested that:1day after immunization, serum concentration of CORT in group D was highest and significantly higher than group A and B(p<0.05);during 3 to 7days after immunization,CORT concentration of all group decreased to a similar level,while group D kept in a lower level;ACTH concentration of all groups had no significant difference(p>0.05).It indicated that subcutaneous-injection immunization caused severe stress on chicken ,and it was down-regulated by VE added in diet.
Trail 2 Effects of different immunizations on body weights and the development of immune organs in chicken
Three chickens of each pen were randomly caught and weighted before killed,immune organs were collected and weighted,organ index were calculated. The results suggested that:body weights had no significant difference within each group(p>0.05);Thymus index of group C was significantly higher than other groups at 3,5 and 7 days after immunization(p<0.05),while spleen index and bursa index had no significant difference in all treatments.These results indicated that: different immunizations had no significant effect on body weights, subcutaneous-injection immunization caused higher development of thymus,while had no effects on spleen and brusa of chicken.
Trail 3 Effects of different immunizations on cell proliferation of immune organs in chicken
One chickens of each pen were randomly caught and weighted before killed,immune organs were collected for cell cycle assay by FCM. The results suggested that:it had significant effect on S phase and G2/M phase by different immunizations, cell proliferation indexes of thymus and brusa were down-regulated,while cell proliferation indexes of spleen had no effect.
Trail 4 Effects of different immunizations on relative expression level of apoptosis-related gene of immune organs in chicken
One chickens of each pen were randomly caught and weighted before killed,immune organs were collected,total RNA were detection and reverse transcription into cDNA,SYBR GreenⅠreal-time PCR were used to test expression level of Bcl-2,Fas,andFasL.The results showed that expression of Bcl-2 and FasL in thymus were higher by subcutaneous-injection immunization; Bcl-2 gene was lower by different immunization,while expression of Fas was higher in spleen; Relative expression level of Bcl-2 was higher in bursa at 5d after immunization,and relative expression level of Fas and FasL were also higher in bursa at 3-5d after immunization.
引文
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