抑制素主动免疫对雌性大鼠生殖激素和卵巢发育的影响：FA和Montanide佐剂效应的比较
发布时间：2018-09-10 06:10
【摘要】： 抑制素的基本生物学作用是抑制FSH合成和分泌,利用抑制素主动免疫可以提高家畜的排卵率和产仔数,从而提高繁殖力。近年来国内外研究者利用抑制素的这一特性,主动免疫猪、牛、羊等家畜,在提高繁殖力方面取得了一定的研究进展。目前它作为一种多胎疫苗已成为提高动物生殖能力的新途径,其研究和应用前景非常广阔。然而,大多数研究者使用只含有一个抗原决定簇的抑制素合成肽片段或重组蛋白作为免疫原,并使用传统弗氏佐剂,其免疫效果仍不堪理想,且不同研究者研究结果差异较大。与此同时,弗氏佐剂产生严重的炎症反应,出于动物福利的考虑,弗氏佐剂不宜于动物生产中应用。因此,本研究首次将含有两个抗原决定簇的新疆细毛羊抑制素α亚基成熟区序列的重组融合蛋白(roINH)作为免疫原,并比较了Montanide(MON)佐剂和传统弗氏佐剂(FA)在roINH主动免疫大鼠后的免疫效果。以寻求一种免疫效果好,炎症反应小(副作用小),能在动物实际生产中应用的佐剂。所得研究结果为抑制素α亚基成熟区序列的重组融合蛋白主动免疫提供理论基础和数据资料,具有重要的科研价值和实践意义。 主要的研究工作和取得的结果如下: 为测定抗体,激素等指标,将60只体重为220.6±17.2 g的性成熟雌性SD大鼠随机分成三组,每组20只,各处理组分别为:(1)FA佐剂组(CFA+roINH用于首次免疫;IFA+roINH用于加强免疫),(2)MON佐剂组(MON+roINH),(3)对照组(注射0.9%NaCl),试验期共免疫三次,每次皮下注射0.5 mL,含1 mg免疫原。以第一次免疫当天为试验的0天,分别在试验的第20和40天进行加强免疫,方式和剂量同首次免疫。在试验的0,10,20,30,40,50天从大鼠尾静脉采血以测定抗体滴度;从第50天开始,每隔6小时采一次血,连续采血5天,用于测定血液中促卵泡素(FSH),促黄体素(LH)和孕酮(P)的含量。分别在第二次和第三次免疫10天后的动情期,用乙醚麻醉后将其处死,观察卵泡发育情况。为观察动物的炎症反应,将50只体重为220.6±17.2 g的性成熟雌性SD大鼠随机分成四组:(1)FA佐剂组(CFA+roINH)(n=20),(2)MON佐剂组(MON+roINH)(n=20),(3)蛋白组(roINH)(n=5),(4)对照组(0.9%NaCl)(n=5)。1—2组分别在腹侧面皮下注射0.1 mL,含1 mg的免疫原,3组注射0.1 mL,只含1 mg roINH的免疫原,4组只注射0.1 mL 0.9%NaCl。分别在注射后第2,5,7,14,28天取注射部位用于组织切片,并同时测量脓疱(炎症反应)直径。 免疫组(MON佐剂组和FA佐剂组)血清中抗体滴度在首次免疫后持续增加,在第50天到达高峰,在第20和40天,MON佐剂组抗体滴度显著高于FA佐剂组(P＜0.05)。在抑制素第二次免疫10天后动情期,FA佐剂组成熟卵泡数(32.83±4.49个)显著高于对照组(26.83±4.36个)(P＜0.05);而MON佐剂组成熟卵泡数(28.80±3.96个)和对照组相差不多(P＞0.05)。在抑制素第三次免疫10天后动情期,MON佐剂组成熟卵泡数和FA佐剂组成熟卵泡数(分别为35.00±4.12个和35.2±1.64个)均显著高于对照组(28.67±1.58个)(P＜0.05)。两次加强免疫后,抑制素抗体阳性鼠抗体滴度与成熟卵泡数(r=0.727)和卵巢重量(r=0.716)均呈极显著正相关(P＜0.01)。在抑制素第三次免疫10天后的整个发情周期内,MON佐剂组血清中FSH的平均浓度(9.96±1.01mIU/mL)和FA佐剂组(11.34±1.42 mIU/mL)是对照组FSH平均浓度(3.20±0.56mIU/mL)的3-4倍(P＜0.01),两免疫组间FSH平均浓度均差异不显著,峰值处MON佐剂组和FA佐剂组FSH浓度(分别为:25.36±2.62 mIU/mL和30.24±1.04 mIU/mL)均显著高于对照组(P＜0.01)。MON佐剂组和FA佐剂组血液中LH平均浓度(分别为:5.81±0.58 mIU/mL和6.52±0.77 mIU/mL)与对照组(6.09±0.78 mIU/mL)相比均差异不显著,孕酮(P)平均浓度的结果和LH相似,两免疫组和对照组P平均浓度均无差异(MON佐剂组为3.39±0.48 ng/mL;FA佐剂组为2.90±0.48 ng/mL;对照组为3.19±0.51ng/mL)。 抑制素主动免疫后,MON佐剂组和FA佐剂组免疫后炎症反应程度有所不同,在免疫后第2和7天时MON佐剂组脓包直径小于FA佐剂组(P＜0.05),在第28天时,MON佐剂组脓包直径显著小于FA佐剂组(P＜0.01)。整个试验期间蛋白组(只注射roINH)和生理盐水组均没有组织学上可见损伤。 以上结果表明:应用抑制素α亚基成熟区蛋白作为免疫原主动免疫大鼠时,MON佐剂和FA佐剂均可产生较好的生物学效应,促进了大鼠卵泡发育和提高了大鼠血中FSH、P激素水平;与此同时,MON佐剂在免疫后第20和40天产生的抗体滴度显著高于FA佐剂,且引起的炎症反应比FA佐剂组弱。因此,MON佐剂更适宜于动物生产。
[Abstract]:The basic biological function of inhibin is to inhibit the synthesis and secretion of FSH. Active immunization with inhibin can improve the ovulation rate and litter size of domestic animals and improve their fecundity. In recent years, researchers at home and abroad have made * some progress in improving the fecundity of pigs, cattle and sheep by using this characteristic of inhibin. At present, it has become a new way to improve the reproductive capacity of animals as a multi-fetal vaccine, and its research and application prospects are very broad. However, most researchers use an inhibitor-synthesized peptide fragment or recombinant protein containing only one antigenic determinant as an immunogen, and use traditional Freund's adjuvant, its immune effect is still unsatisfactory. At the same time, Freund's adjuvant produces severe inflammation and is not suitable for animal production because of animal welfare considerations. Therefore, the recombinant fusion protein (roINH) containing two antigenic determinants in the mature region of inhibin alpha subunit of Xinjiang fine wool sheep was first used in this study. In order to find an adjuvant with good immune effect, less inflammatory reaction (side effect) and practical application in animal production, the immunogenic effects of Montanide (MON) adjuvant and traditional Freund's adjuvant (FA) on roINH active immunization in rats were compared. Immunization provides theoretical basis and data, which has important scientific research value and practical significance.
The main research work and results are as follows:
To determine antibodies and hormones, 60 adult female SD rats weighing 220.6 (+ 17.2 g) were randomly divided into three groups, 20 rats in each group. The treatment groups were: (1) FA adjuvant group (CFA + roINH for the first time immunization; IFA + roINH for strengthening immunity), (2) MON adjuvant group (MON + roINH), (3) control group (injection of 0.9% NaCl) for three times each trial period. 0.5 mL, containing 1 mg of immunogen, was subcutaneously injected. On the 20th and 40th days of the first immunization, the immunization was strengthened in the same manner and dose as the first immunization. To determine the levels of follicle stimulating hormone (FSH), luteinizing hormone (LH) and progesterone (P) in blood, 50 adult female SD rats weighing 220.6 65507 FA adjuvant group (CFA + roINH) (n = 20), (2) MON adjuvant group (MON + roINH) (n = 20), (3) protein group (roINH) (n = 5), (4) control group (0.9% NaCl) (n = 5). 1 - 2 groups were subcutaneously injected with 0.1 mL, containing 1 mg of immunogen, 3 groups were injected with 0.1 mL, containing only 1 mg of immunogen, 4 groups were injected with 0.1% NaCl. The site was used for tissue sections and the diameter of pustules (inflammatory response) was measured simultaneously.
The antibody titers in serum of the immunized group (MON adjuvant group and FA adjuvant group) increased continuously after the first immunization and reached the peak on the 50th day. On the 20th and 40th days, the antibody titers in the MON adjuvant group were significantly higher than those in the FA adjuvant group (P < 0.05). In estrus stage, the number of mature follicles in the FA adjuvant group (32.83 (+4.49)) was significantly higher than that in the control group (26.83 (+4.49)). The number of mature follicles in MON adjuvant group (28.80 + 3.96) was similar to that in control group (P > 0.05). The number of mature follicles in MON adjuvant group and that in FA adjuvant group (35.00 + 4.12 and 35.2 + 1.64 respectively) were significantly higher than that in control group (28.67 + 1.58) (P < 0.05). After two times of intensive immunization, the titer of antibody against inhibin was positively correlated with the number of mature follicles (r = 0.727) and the weight of ovary (r = 0.716) (P The mean concentration of FSH in the control group was 3-4 times (P < 0.01). The mean concentration of FSH in the MON adjuvant group and the FA adjuvant group was significantly higher than that in the control group (P < 0.01). The results of progesterone (P) concentration were similar to that of LH. There was no significant difference in P concentration between the two groups (MON adjuvant group was 3.39 + 0.48 ng / mL; FA adjuvant group was 2.90 + 0.48 ng / mL; control group was 3.19 + 0.51 ng / mL).
After active immunization with inhibin, the degree of inflammatory reaction was different between MON adjuvant group and FA adjuvant group. The diameter of pus in MON adjuvant group was smaller than that in FA adjuvant group at 2 and 7 days after immunization (P < 0.05). On 28 days, the diameter of pus in MON adjuvant group was significantly smaller than that in FA adjuvant group (P < 0.01). There was no histological damage in the water group.
The results showed that both MON adjuvant and FA adjuvant could produce better biological effects when inhibin-alpha subunit mature region protein was used as immunogen to actively immunize rats, which promoted follicular development and increased the levels of FSH and P hormones in rat blood. Meanwhile, the antibody titers produced by MON adjuvant on the 20th and 40th days after immunization were significantly increased. The FA adjuvant was weaker than the FA adjuvant group, and the MON adjuvant was more suitable for animal production.
【学位授予单位】：四川农业大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392
本文编号：2233603
[Abstract]:The basic biological function of inhibin is to inhibit the synthesis and secretion of FSH. Active immunization with inhibin can improve the ovulation rate and litter size of domestic animals and improve their fecundity. In recent years, researchers at home and abroad have made * some progress in improving the fecundity of pigs, cattle and sheep by using this characteristic of inhibin. At present, it has become a new way to improve the reproductive capacity of animals as a multi-fetal vaccine, and its research and application prospects are very broad. However, most researchers use an inhibitor-synthesized peptide fragment or recombinant protein containing only one antigenic determinant as an immunogen, and use traditional Freund's adjuvant, its immune effect is still unsatisfactory. At the same time, Freund's adjuvant produces severe inflammation and is not suitable for animal production because of animal welfare considerations. Therefore, the recombinant fusion protein (roINH) containing two antigenic determinants in the mature region of inhibin alpha subunit of Xinjiang fine wool sheep was first used in this study. In order to find an adjuvant with good immune effect, less inflammatory reaction (side effect) and practical application in animal production, the immunogenic effects of Montanide (MON) adjuvant and traditional Freund's adjuvant (FA) on roINH active immunization in rats were compared. Immunization provides theoretical basis and data, which has important scientific research value and practical significance.
The main research work and results are as follows:
To determine antibodies and hormones, 60 adult female SD rats weighing 220.6 (+ 17.2 g) were randomly divided into three groups, 20 rats in each group. The treatment groups were: (1) FA adjuvant group (CFA + roINH for the first time immunization; IFA + roINH for strengthening immunity), (2) MON adjuvant group (MON + roINH), (3) control group (injection of 0.9% NaCl) for three times each trial period. 0.5 mL, containing 1 mg of immunogen, was subcutaneously injected. On the 20th and 40th days of the first immunization, the immunization was strengthened in the same manner and dose as the first immunization. To determine the levels of follicle stimulating hormone (FSH), luteinizing hormone (LH) and progesterone (P) in blood, 50 adult female SD rats weighing 220.6 65507 FA adjuvant group (CFA + roINH) (n = 20), (2) MON adjuvant group (MON + roINH) (n = 20), (3) protein group (roINH) (n = 5), (4) control group (0.9% NaCl) (n = 5). 1 - 2 groups were subcutaneously injected with 0.1 mL, containing 1 mg of immunogen, 3 groups were injected with 0.1 mL, containing only 1 mg of immunogen, 4 groups were injected with 0.1% NaCl. The site was used for tissue sections and the diameter of pustules (inflammatory response) was measured simultaneously.
The antibody titers in serum of the immunized group (MON adjuvant group and FA adjuvant group) increased continuously after the first immunization and reached the peak on the 50th day. On the 20th and 40th days, the antibody titers in the MON adjuvant group were significantly higher than those in the FA adjuvant group (P < 0.05). In estrus stage, the number of mature follicles in the FA adjuvant group (32.83 (+4.49)) was significantly higher than that in the control group (26.83 (+4.49)). The number of mature follicles in MON adjuvant group (28.80 + 3.96) was similar to that in control group (P > 0.05). The number of mature follicles in MON adjuvant group and that in FA adjuvant group (35.00 + 4.12 and 35.2 + 1.64 respectively) were significantly higher than that in control group (28.67 + 1.58) (P < 0.05). After two times of intensive immunization, the titer of antibody against inhibin was positively correlated with the number of mature follicles (r = 0.727) and the weight of ovary (r = 0.716) (P The mean concentration of FSH in the control group was 3-4 times (P < 0.01). The mean concentration of FSH in the MON adjuvant group and the FA adjuvant group was significantly higher than that in the control group (P < 0.01). The results of progesterone (P) concentration were similar to that of LH. There was no significant difference in P concentration between the two groups (MON adjuvant group was 3.39 + 0.48 ng / mL; FA adjuvant group was 2.90 + 0.48 ng / mL; control group was 3.19 + 0.51 ng / mL).
After active immunization with inhibin, the degree of inflammatory reaction was different between MON adjuvant group and FA adjuvant group. The diameter of pus in MON adjuvant group was smaller than that in FA adjuvant group at 2 and 7 days after immunization (P < 0.05). On 28 days, the diameter of pus in MON adjuvant group was significantly smaller than that in FA adjuvant group (P < 0.01). There was no histological damage in the water group.
The results showed that both MON adjuvant and FA adjuvant could produce better biological effects when inhibin-alpha subunit mature region protein was used as immunogen to actively immunize rats, which promoted follicular development and increased the levels of FSH and P hormones in rat blood. Meanwhile, the antibody titers produced by MON adjuvant on the 20th and 40th days after immunization were significantly increased. The FA adjuvant was weaker than the FA adjuvant group, and the MON adjuvant was more suitable for animal production.
【学位授予单位】：四川农业大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392
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