生长抑素主动免疫对生长湖羊生长及生长激素基因表达影响的研究
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摘要
动物的生长和发育受多种因素的调节和控制，包括遗传，营养，环境和内分泌等。在内分泌系统中，尽管任何一种内分泌激素都不能单独地调控动物全部生长和发育过程，不过生长激素在其中仍占据着中心调节作用的位置。已知，巨人症患者生长激素水平极度的升高，而侏儒症患者的生长激素水平则显著地降低，同时，注射外源性生长激素可明显地提高动物的生长速度，改善了动物的胴体组成。因此，采用一系列技术手段提高或促进动物垂体生长激素的合成和分泌往往能达到促进动物生长的目的。考虑到使用外源性生长激素存在激素残留和使用不便的不足，当前，科学家和公众对开发和研究刺激内源性生长激素技术措施予以了特别的重视。
研究显示，动物垂体生长激素的合成和分泌主要受丘脑分泌的两种神经激素的控制。其一是生长激素释放因子(GHRF)，它刺激垂体生长激素的释放，其二是生长激素抑制因子(SS)，它抑制垂体生长激素的分泌。因此，在理论上讲，采用某些技术措施增加GHRF或减少SS的分泌就可能达到刺激或提高生长激素分泌和释放的目的。本项研究内容集中于后者，主要的试验目的是：1．生长抑素主动免疫对SS进行免疫中和对生长期湖羊生长，营养物的消化，代谢，胴体组成影响；2．对胃窦膜，基底膜和胰腺SS分泌以及SS抗体结合SS的能力影响；3．生长抑素主动免疫对食糜流通速度的影响；4，对消化道酶活及相关激素及代谢物的影响；5．对脑垂体生长激素基因的表达影响等进行研究，进而阐明其作用机理为今后这项技术在生产中的应用提供可靠的理论依据。
选用12头，6月龄公湖羊，随机地分为两组，一组为对照组，另外一组为免疫组，试验开始前，两组动物的体重差异不显著(对照和试验组分别为23.24kg和24.30kg，P〉0.05)。试验组和对照组动物分别用SS—人血清。球蛋白交联物或交联蛋白进行为期7周的免疫，其中前两次加强免疫间隔两周，最后一次免疫间隔三周。整个试验正试期为期8周。结果显示：免疫组动物在第二次加强免疫后就可诱发产生高水平，特异性免疫抗体的应答，相反，对照组绵羊不产生针对SS的特异性抗体。以光密度计，在第二次和第三次加强注射后，免疫组抗体滴度分别为0.54和0.61。血液内高水平的特异性抗体结合了大量的游离SS(抗体滴度在0.48-0.72的范围内，结合的SS的浓度范围为2.4-11.3nmol／l)，在不改变生长激素脉冲分泌各项指标和垂体生长激素mRNA丰度的情况下(对照组和免疫组动物生长激素水平总平均，基线平均，峰频率，峰强度和mRNA丰度分别为3.14ng／ml，2.25
￥内豪古农业大学搏士学位论文
ng／ml，4．3／4Hr，5＊3 ng／ml，0．48U／n g和 3＊2 ng／ml，2．24 ng／ml，3．94Hr，
4．12 ng／ml，0人SU／v g，各指标比较均差异不显著，P＞0刀5人兔疫组动物的
食糜消化道滞留时间明显增加（以指示剂累计排出50％为标准，兔疫组较对
照组大约增加 20小时），与此同时，SS免疫中和也提高了胰腺和消化道各
种消化酶的比活力（对照组和免疫组胰腺，小肠食糜总蛋白酶，胰蛋白酶，”
糜蛋白酶和淀粉酶比活分别为 1693 石7unit／g，CP，2728．33 unit／g，CP，3055．50
unit／g，CP，12．gX 106 uni屿，CPZ 2．57X 102unit／g，CP，i＊OX 103unit／g，CP，l二2
X103uni／，CP，二．98X107unit／g厂P和 2451．33 unit／g，CP，2904．17unit／g，CP，
4279．33 uni吟，CP，20石IX 106 unit／g，CP 6．45X102unit／g，2．53X103uni吨，CP，
1．83 X 103unit／g，CP，5．77X10’unit／g，CP，两组比较差异显著P＜0＊5或极显
著Prto刀l二。酶活力和消化道食糜滞留时间的延长，增加了饲料在消化道
内与消化酶作用时间，更有利于饲料的消化和营养物质的吸收，从而提高了
各种养分的表观消化率（干物质，有机物，粗蛋白，粗纤维，中性洗涤纤维，
酸性洗涤纤维的表观消化率分别由对照组的61．28％，64．11％，69二1％，
62．55％，63．87％和60．12％提高到免疫组的64．51％，66．28％，72．31％，64．66％，
65．23o，和62．98O，Prto．05＼在组织代谢层次上，SS的主动兔疫提高了
外周循环血液中mJ的水平，其中在第二次和第三次加强注射后，其浓度
分别由对照的 162．56ng／ml，180．42 ng／ml提高至 257石5 ng／ml，234石3 ng／mlo
＜0．05X但在不同的免疫期间，对胰高血糖素，甲状腺素，胰岛素，三碘甲
腺原氨酸，胃泌素和促甲状腺素水平则没有影响。SS兔疫中和可以明显地
降低血液中尿素氮水平，在总蛋白水平维持恒定的情况下，随着兔疫期的延
长，白蛋白水平逐渐地下降，这说明，随着特异性抗体的产生总蛋白中有相
当一部分于了免疫球蛋白的合成。
免疫并不改变血液中肌酸酚，谷丙转氨酶和谷草转氨酶的试验结果说明，
SS 兔疫中和并不影响动物体内，蛋白质的分解代谢过程。而游离脂肪酸，“
胆固醇和甘油三脂水平不发生变化则预示着，免疫组动物的脂肪代谢也没有
发生大的改变。
SS 免疫中和产生的消化道层次营养物质消化率的提高以及由于血液中
IGFI水平的显著增加，导致了营养物质在组织代谢层次发生改变，最终，
使得免疫组
Growth and development of animals were regulated by many factors including gene, environment,nutrition and endocrine.In endocrine system,growth hormone(GH) always taks up a centerlly regulative place.Previous studies have shown that patients with gigantism or dwarfism have abnormal high or low plasma GH levels,respectively,and animals treated with exogenous GH markedly improved its growth.Therefore,it is reasonable to enhance the levels of GH in plasma by some techniques will result in improvement of animal production performance.
A series of studies indicated that the pituitary release of GH was regulated by two hypothalamically derived neurohormones GHRF ,which stimulated secretion of GH, and somatostatin(SS),which inhibated pituitary secretion of GH.So theoretically,!! is inferred that immunoneutralization of SS would increase secretion of GH ,and finally get the goal of improvement performance.
The objective of this study is to evaluate the effects of active immunization against SS on nutrient digestion and metabolism,growth and carcass composition of the growing Hu-sheep;2,on the SS secretion in fundus, antrum and pancreas and the ablity of the antibodies to integrate free SS;3,on passage rates of digesta;4,on activities of digestive enzymes in pancreas and digesta;4,on gene expression of GH in pituitary.
Twelve male Hu-sheep,aged six-month,were randomly assigned to one of two treatment groups,!.e.control group,and immunized group.The animals in the immunized or control groups were immunized with human a globin-conjugated SS or conjugated a globin alone,respectively. Whole immunization period went on 7 weeks,for that time primary and boosted immunization were carried out at 0, 2, 4, 7 week, respectively.The results shown that antibodies titer of immunized animals were 0.54 and 0.61(OD)at 4, 7 week, respectively,and control animals had no production of specific antibodies. High levels antibodies in plasma of immunized animals draw on large numbers of free SS, and its titer or integrated free SS concentration were 0.48-0.72 or 2.4-11.3nmol/l, respectively. Active immunization against SS did not alter pattern of GH secretion(over mean, basline mean, peak frequency, peak amplitude) and mRNA abundant of GH secration. Compared to control, retention times of digesta in whole alimentary tract of immunized animals inc
reased by 20 hours(to use cumlative excretion of 5%marker as reference). Immunoneutralization of SS significantly augmented activities of digestive enzymes(proteolytic ,trypsin,chymotrypsin, amylase) in pancreas and the small intestine(control and immunized animals were 1693.67unit/g,CP, 2728.33 unit/g,CP, 3055.50 unit/g,CP, 12.9X106 unit/g,CP; 2.57X
102unit/g,CP, 1.20X103unit/g,CP, 1.12X 103unit/g,CP, 2.98X 107unit/g,CP ft 2451.33 unit/g,CP, 2904.17 unit/g,CP, 4279.33 unit/g,CP, 20.61 X 106 unit/g,CP; 6.45 X 102unit/g, CP, 2.53 X 103unit/g,CP, 1-83 X 103unit/g,CP, 5.77 X 107unit/g,CP, respectively, P<0.05 or P<0.01).
The enhancement of digestive enzyme activeies and retention times of digesta resulted in a significant improvement of apparent digestibilities of DM, OM, CP, CF, ADF, NDF, which were obviously enhanced from 61.28%, 64.11%, 69.11%, 62.55%, 63.87% ft 60.12%to 64.51%, 66.28%, 72.31%, 64.66%, 65.23%, 62.98%, respectively( P<0.05).
Active immunization against SS significantly increased IGF-I level,but had no effect on GH,insulin,,glucagon,T4,T3,TSH, gastrin concentrations in plasma,at the same time, the content of plasma urea for immunized animals clearly decreased.
As the time of immunization time went on, total protein level in plasma gradually increased,and albuman level gradually decreased. This indicated that a part of plasma protein were used for synthesis of IgG.
Immunoneutralization of SS had also no influence on creatinine, NEFA, Tg, Glu, Ch, GPT and GOT activities, which indicated that in immunized animals compared to control animals, the fat metabolism and protein catalism had no change.
As a result.immunized animals had high growth rate and feed conversion effic
研究显示，动物垂体生长激素的合成和分泌主要受丘脑分泌的两种神经激素的控制。其一是生长激素释放因子(GHRF)，它刺激垂体生长激素的释放，其二是生长激素抑制因子(SS)，它抑制垂体生长激素的分泌。因此，在理论上讲，采用某些技术措施增加GHRF或减少SS的分泌就可能达到刺激或提高生长激素分泌和释放的目的。本项研究内容集中于后者，主要的试验目的是：1．生长抑素主动免疫对SS进行免疫中和对生长期湖羊生长，营养物的消化，代谢，胴体组成影响；2．对胃窦膜，基底膜和胰腺SS分泌以及SS抗体结合SS的能力影响；3．生长抑素主动免疫对食糜流通速度的影响；4，对消化道酶活及相关激素及代谢物的影响；5．对脑垂体生长激素基因的表达影响等进行研究，进而阐明其作用机理为今后这项技术在生产中的应用提供可靠的理论依据。
选用12头，6月龄公湖羊，随机地分为两组，一组为对照组，另外一组为免疫组，试验开始前，两组动物的体重差异不显著(对照和试验组分别为23.24kg和24.30kg，P〉0.05)。试验组和对照组动物分别用SS—人血清。球蛋白交联物或交联蛋白进行为期7周的免疫，其中前两次加强免疫间隔两周，最后一次免疫间隔三周。整个试验正试期为期8周。结果显示：免疫组动物在第二次加强免疫后就可诱发产生高水平，特异性免疫抗体的应答，相反，对照组绵羊不产生针对SS的特异性抗体。以光密度计，在第二次和第三次加强注射后，免疫组抗体滴度分别为0.54和0.61。血液内高水平的特异性抗体结合了大量的游离SS(抗体滴度在0.48-0.72的范围内，结合的SS的浓度范围为2.4-11.3nmol／l)，在不改变生长激素脉冲分泌各项指标和垂体生长激素mRNA丰度的情况下(对照组和免疫组动物生长激素水平总平均，基线平均，峰频率，峰强度和mRNA丰度分别为3.14ng／ml，2.25
￥内豪古农业大学搏士学位论文
ng／ml，4．3／4Hr，5＊3 ng／ml，0．48U／n g和 3＊2 ng／ml，2．24 ng／ml，3．94Hr，
4．12 ng／ml，0人SU／v g，各指标比较均差异不显著，P＞0刀5人兔疫组动物的
食糜消化道滞留时间明显增加（以指示剂累计排出50％为标准，兔疫组较对
照组大约增加 20小时），与此同时，SS免疫中和也提高了胰腺和消化道各
种消化酶的比活力（对照组和免疫组胰腺，小肠食糜总蛋白酶，胰蛋白酶，”
糜蛋白酶和淀粉酶比活分别为 1693 石7unit／g，CP，2728．33 unit／g，CP，3055．50
unit／g，CP，12．gX 106 uni屿，CPZ 2．57X 102unit／g，CP，i＊OX 103unit／g，CP，l二2
X103uni／，CP，二．98X107unit／g厂P和 2451．33 unit／g，CP，2904．17unit／g，CP，
4279．33 uni吟，CP，20石IX 106 unit／g，CP 6．45X102unit／g，2．53X103uni吨，CP，
1．83 X 103unit／g，CP，5．77X10’unit／g，CP，两组比较差异显著P＜0＊5或极显
著Prto刀l二。酶活力和消化道食糜滞留时间的延长，增加了饲料在消化道
内与消化酶作用时间，更有利于饲料的消化和营养物质的吸收，从而提高了
各种养分的表观消化率（干物质，有机物，粗蛋白，粗纤维，中性洗涤纤维，
酸性洗涤纤维的表观消化率分别由对照组的61．28％，64．11％，69二1％，
62．55％，63．87％和60．12％提高到免疫组的64．51％，66．28％，72．31％，64．66％，
65．23o，和62．98O，Prto．05＼在组织代谢层次上，SS的主动兔疫提高了
外周循环血液中mJ的水平，其中在第二次和第三次加强注射后，其浓度
分别由对照的 162．56ng／ml，180．42 ng／ml提高至 257石5 ng／ml，234石3 ng／mlo
＜0．05X但在不同的免疫期间，对胰高血糖素，甲状腺素，胰岛素，三碘甲
腺原氨酸，胃泌素和促甲状腺素水平则没有影响。SS兔疫中和可以明显地
降低血液中尿素氮水平，在总蛋白水平维持恒定的情况下，随着兔疫期的延
长，白蛋白水平逐渐地下降，这说明，随着特异性抗体的产生总蛋白中有相
当一部分于了免疫球蛋白的合成。
免疫并不改变血液中肌酸酚，谷丙转氨酶和谷草转氨酶的试验结果说明，
SS 兔疫中和并不影响动物体内，蛋白质的分解代谢过程。而游离脂肪酸，“
胆固醇和甘油三脂水平不发生变化则预示着，免疫组动物的脂肪代谢也没有
发生大的改变。
SS 免疫中和产生的消化道层次营养物质消化率的提高以及由于血液中
IGFI水平的显著增加，导致了营养物质在组织代谢层次发生改变，最终，
使得免疫组
Growth and development of animals were regulated by many factors including gene, environment,nutrition and endocrine.In endocrine system,growth hormone(GH) always taks up a centerlly regulative place.Previous studies have shown that patients with gigantism or dwarfism have abnormal high or low plasma GH levels,respectively,and animals treated with exogenous GH markedly improved its growth.Therefore,it is reasonable to enhance the levels of GH in plasma by some techniques will result in improvement of animal production performance.
A series of studies indicated that the pituitary release of GH was regulated by two hypothalamically derived neurohormones GHRF ,which stimulated secretion of GH, and somatostatin(SS),which inhibated pituitary secretion of GH.So theoretically,!! is inferred that immunoneutralization of SS would increase secretion of GH ,and finally get the goal of improvement performance.
The objective of this study is to evaluate the effects of active immunization against SS on nutrient digestion and metabolism,growth and carcass composition of the growing Hu-sheep;2,on the SS secretion in fundus, antrum and pancreas and the ablity of the antibodies to integrate free SS;3,on passage rates of digesta;4,on activities of digestive enzymes in pancreas and digesta;4,on gene expression of GH in pituitary.
Twelve male Hu-sheep,aged six-month,were randomly assigned to one of two treatment groups,!.e.control group,and immunized group.The animals in the immunized or control groups were immunized with human a globin-conjugated SS or conjugated a globin alone,respectively. Whole immunization period went on 7 weeks,for that time primary and boosted immunization were carried out at 0, 2, 4, 7 week, respectively.The results shown that antibodies titer of immunized animals were 0.54 and 0.61(OD)at 4, 7 week, respectively,and control animals had no production of specific antibodies. High levels antibodies in plasma of immunized animals draw on large numbers of free SS, and its titer or integrated free SS concentration were 0.48-0.72 or 2.4-11.3nmol/l, respectively. Active immunization against SS did not alter pattern of GH secretion(over mean, basline mean, peak frequency, peak amplitude) and mRNA abundant of GH secration. Compared to control, retention times of digesta in whole alimentary tract of immunized animals inc
reased by 20 hours(to use cumlative excretion of 5%marker as reference). Immunoneutralization of SS significantly augmented activities of digestive enzymes(proteolytic ,trypsin,chymotrypsin, amylase) in pancreas and the small intestine(control and immunized animals were 1693.67unit/g,CP, 2728.33 unit/g,CP, 3055.50 unit/g,CP, 12.9X106 unit/g,CP; 2.57X
102unit/g,CP, 1.20X103unit/g,CP, 1.12X 103unit/g,CP, 2.98X 107unit/g,CP ft 2451.33 unit/g,CP, 2904.17 unit/g,CP, 4279.33 unit/g,CP, 20.61 X 106 unit/g,CP; 6.45 X 102unit/g, CP, 2.53 X 103unit/g,CP, 1-83 X 103unit/g,CP, 5.77 X 107unit/g,CP, respectively, P<0.05 or P<0.01).
The enhancement of digestive enzyme activeies and retention times of digesta resulted in a significant improvement of apparent digestibilities of DM, OM, CP, CF, ADF, NDF, which were obviously enhanced from 61.28%, 64.11%, 69.11%, 62.55%, 63.87% ft 60.12%to 64.51%, 66.28%, 72.31%, 64.66%, 65.23%, 62.98%, respectively( P<0.05).
Active immunization against SS significantly increased IGF-I level,but had no effect on GH,insulin,,glucagon,T4,T3,TSH, gastrin concentrations in plasma,at the same time, the content of plasma urea for immunized animals clearly decreased.
As the time of immunization time went on, total protein level in plasma gradually increased,and albuman level gradually decreased. This indicated that a part of plasma protein were used for synthesis of IgG.
Immunoneutralization of SS had also no influence on creatinine, NEFA, Tg, Glu, Ch, GPT and GOT activities, which indicated that in immunized animals compared to control animals, the fat metabolism and protein catalism had no change.
As a result.immunized animals had high growth rate and feed conversion effic
引文
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2． Abe, H. , Molitch, J. J. Van Wyk and L. E. Underwood. 1983． Human growth hormone and somatomedin-C suppress the spontaneous release of growth hormone in unanesthetized rats, andocrinology 113:1319．
3． Abrams, R. L. , Grumbach, M. M. Kaplan, S. L. THe effect of administration of human growth hormone on the plasma growth hormone, cortisol. glucose and free fatty acid response to insulin:Evidence for growth hormone autoregulation in man. J. Clin. Invest. 50: 940-950; 1971．
4． Adams, T. E. , Baker, L. , Fiddes, R. J. and Brandon, M. R. 1990． The sheep growth hormone receptormolecular cloning and ontogeny of mRNA expression in the liver. Molecular and Cellular Endocrinology 73:135-145．
5． Aguila, M. C. , and S. M. McCann. 1987． Evidence that growth hormone-releasing factor stimulates somatostatin release in vitro via beta-endorphin. endocrinology 120:341．
6． Aguila, M. C. and S. M. McCann. 1985． Stimulation of somatostatin release in vitro by synthetic human growth hormone-releasing factor by a nondopaminergic mechanism. Endocrinology 117:762．
7． Alpert L C, Brawer J R, Patel Y C. Reichlin S. Somatostatinergic neurons in anteriorhypothalamus:immunohistochemieal localization. Endocrinology 1976: 98: 255-8．
8． Alumets J. Sundler F. Hakanson R. (1977) Distribution, ontogency and ultrastructure somatostatin-immunoreactive cells in the pancreas and gut. Cell Tissue Res 185, 165-179
9． Anderson LL. Development in calves and heifers after hypophysial stalk transection or hypophysectomy. Am J Physiol 1977;232:E497-503．
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