抑制素主動免疫對雌性大鼠生殖激素和卵巢發(fā)育的影響：FA和Montanide佐劑效應的比較
發(fā)布時間：2018-09-10 06:10
【摘要】： 抑制素的基本生物學作用是抑制FSH合成和分泌,利用抑制素主動免疫可以提高家畜的排卵率和產(chǎn)仔數(shù),從而提高繁殖力。近年來國內(nèi)外研究者利用抑制素的這一特性,主動免疫豬、牛、羊等家畜,在提高繁殖力方面取得了一定的研究進展。目前它作為一種多胎疫苗已成為提高動物生殖能力的新途徑,其研究和應用前景非常廣闊。然而,大多數(shù)研究者使用只含有一個抗原決定簇的抑制素合成肽片段或重組蛋白作為免疫原,并使用傳統(tǒng)弗氏佐劑,其免疫效果仍不堪理想,且不同研究者研究結果差異較大。與此同時,弗氏佐劑產(chǎn)生嚴重的炎癥反應,出于動物福利的考慮,弗氏佐劑不宜于動物生產(chǎn)中應用。因此,本研究首次將含有兩個抗原決定簇的新疆細毛羊抑制素α亞基成熟區(qū)序列的重組融合蛋白(roINH)作為免疫原,并比較了Montanide(MON)佐劑和傳統(tǒng)弗氏佐劑(FA)在roINH主動免疫大鼠后的免疫效果。以尋求一種免疫效果好,炎癥反應小(副作用小),能在動物實際生產(chǎn)中應用的佐劑。所得研究結果為抑制素α亞基成熟區(qū)序列的重組融合蛋白主動免疫提供理論基礎和數(shù)據(jù)資料,具有重要的科研價值和實踐意義。 主要的研究工作和取得的結果如下: 為測定抗體,激素等指標,將60只體重為220.6±17.2 g的性成熟雌性SD大鼠隨機分成三組,每組20只,各處理組分別為:(1)FA佐劑組(CFA+roINH用于首次免疫;IFA+roINH用于加強免疫),(2)MON佐劑組(MON+roINH),(3)對照組(注射0.9%NaCl),試驗期共免疫三次,每次皮下注射0.5 mL,含1 mg免疫原。以第一次免疫當天為試驗的0天,分別在試驗的第20和40天進行加強免疫,方式和劑量同首次免疫。在試驗的0,10,20,30,40,50天從大鼠尾靜脈采血以測定抗體滴度;從第50天開始,每隔6小時采一次血,連續(xù)采血5天,用于測定血液中促卵泡素(FSH),促黃體素(LH)和孕酮(P)的含量。分別在第二次和第三次免疫10天后的動情期,用乙醚麻醉后將其處死,觀察卵泡發(fā)育情況。為觀察動物的炎癥反應,將50只體重為220.6±17.2 g的性成熟雌性SD大鼠隨機分成四組:(1)FA佐劑組(CFA+roINH)(n=20),(2)MON佐劑組(MON+roINH)(n=20),(3)蛋白組(roINH)(n=5),(4)對照組(0.9%NaCl)(n=5)。1—2組分別在腹側面皮下注射0.1 mL,含1 mg的免疫原,3組注射0.1 mL,只含1 mg roINH的免疫原,4組只注射0.1 mL 0.9%NaCl。分別在注射后第2,5,7,14,28天取注射部位用于組織切片,并同時測量膿皰(炎癥反應)直徑。 免疫組(MON佐劑組和FA佐劑組)血清中抗體滴度在首次免疫后持續(xù)增加,在第50天到達高峰,在第20和40天,MON佐劑組抗體滴度顯著高于FA佐劑組(P＜0.05)。在抑制素第二次免疫10天后動情期,FA佐劑組成熟卵泡數(shù)(32.83±4.49個)顯著高于對照組(26.83±4.36個)(P＜0.05);而MON佐劑組成熟卵泡數(shù)(28.80±3.96個)和對照組相差不多(P＞0.05)。在抑制素第三次免疫10天后動情期,MON佐劑組成熟卵泡數(shù)和FA佐劑組成熟卵泡數(shù)(分別為35.00±4.12個和35.2±1.64個)均顯著高于對照組(28.67±1.58個)(P＜0.05)。兩次加強免疫后,抑制素抗體陽性鼠抗體滴度與成熟卵泡數(shù)(r=0.727)和卵巢重量(r=0.716)均呈極顯著正相關(P＜0.01)。在抑制素第三次免疫10天后的整個發(fā)情周期內(nèi),MON佐劑組血清中FSH的平均濃度(9.96±1.01mIU/mL)和FA佐劑組(11.34±1.42 mIU/mL)是對照組FSH平均濃度(3.20±0.56mIU/mL)的3-4倍(P＜0.01),兩免疫組間FSH平均濃度均差異不顯著,峰值處MON佐劑組和FA佐劑組FSH濃度(分別為:25.36±2.62 mIU/mL和30.24±1.04 mIU/mL)均顯著高于對照組(P＜0.01)。MON佐劑組和FA佐劑組血液中LH平均濃度(分別為:5.81±0.58 mIU/mL和6.52±0.77 mIU/mL)與對照組(6.09±0.78 mIU/mL)相比均差異不顯著,孕酮(P)平均濃度的結果和LH相似,兩免疫組和對照組P平均濃度均無差異(MON佐劑組為3.39±0.48 ng/mL;FA佐劑組為2.90±0.48 ng/mL;對照組為3.19±0.51ng/mL)。 抑制素主動免疫后,MON佐劑組和FA佐劑組免疫后炎癥反應程度有所不同,在免疫后第2和7天時MON佐劑組膿包直徑小于FA佐劑組(P＜0.05),在第28天時,MON佐劑組膿包直徑顯著小于FA佐劑組(P＜0.01)。整個試驗期間蛋白組(只注射roINH)和生理鹽水組均沒有組織學上可見損傷。 以上結果表明:應用抑制素α亞基成熟區(qū)蛋白作為免疫原主動免疫大鼠時,MON佐劑和FA佐劑均可產(chǎn)生較好的生物學效應,促進了大鼠卵泡發(fā)育和提高了大鼠血中FSH、P激素水平;與此同時,MON佐劑在免疫后第20和40天產(chǎn)生的抗體滴度顯著高于FA佐劑,且引起的炎癥反應比FA佐劑組弱。因此,MON佐劑更適宜于動物生產(chǎn)。
[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.
【學位授予單位】：四川農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2009
【分類號】：R392
本文編號：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.
【學位授予單位】：四川農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2009
【分類號】：R392
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