褪黑素对绵羊体外培养卵泡颗粒细胞和垂体细胞分泌激素的影响
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摘要
本研究选用季节性繁殖的蒙古羊作为研究对象，采用体外细胞培养的方法，进行不同的处理，用放射免疫法(RIA)测定细胞培养液中激素的含量。结果如下：(1)卵泡颗粒细胞和垂体细胞在无血清培养体系中均可体外培养，并能够分泌相应的激素。(2)用MLT或MLT与HCG共同处理卵泡颗粒细胞，能促进细胞分泌17-β-E_2，并呈时间和剂量的依赖性，但对P_4的分泌没有影响。(3)用MLT单独处理垂体细胞，培养24h时除2000组PRL含量高于对照组外，其它各组均极显著低于对照组(P＜0.01)，但随着MLT剂量的增加，PRL的含量亦随着增加；48h时各组PRL的含量亦低于对照组。(4)用MLT单独处理垂体细胞，培养24h时除100组以外其余各组FSH含量均显著高于对照组(P＜0.05)，除100组外其余各组48hFSH的分泌量均极显著低于24h(P＜0.01)。提示MLT可能对垂体细胞FSH的分泌有促进作用，但随时间的延长而分泌功能下降。(5)用MLT单独处理垂体细胞对LH的分泌几乎没有影响。(6)用HCG和MLT共同处理垂体细胞，培养24h各组PRL的含量均低于对照组(P＜0.01)，但各处理组之间随MLT浓度的增加PRL的含量呈上升趋势；48h时2000组PRL的含量极显著高于对照组(P＜0.01)，其它组仍低于对照组，各处理组亦随MLT浓度的增加PRL的含量呈上升趋势。说明HCG和MLT共同作用并不促进垂体细胞分泌PRL。48h时对照组与HCG组PRL的含量低于24h，而各处理组48h PRL的含量高于24hPRL的含量。说明MLT和HCG共同作用可延长垂体细胞分泌PRL的功能。(7)HCG和MLT共同处理能明显促进垂体细胞FSH分泌(P＜0.01)。虽然HCG和MLT共同处理也能明显促进垂体细胞LH分泌，但这主要是HCG的作用。(8)MLT和LHRH共同处理垂体细胞时，24h和48h时均不能促进PRL的分泌，但在24h时各处理组随剂量的增加而增加，48h时随剂量的增加而减少。(9)用MLT和LHRH共同处理垂体细胞时，24h时10组和1000组LH含量比对照组高，48h时各处理组均比对照组高。说明MLT和LHRH共同作用垂体细胞时可能促进LH的分泌。
Seasonal breeding Mongolian ewes were regarded as object in this research. The study adapted the way of cell culture and then carried different treatment. The hormone concentrations in cell culture medium were analyzed by RIA. The results were as follows: (1) Granulosa cells and hypophysis cells could be cultured with free-serum culture system in vitro, and secret corresponding hormone. (2) When follicular granulosa cells were treated with MLT or MLT and HCG, it could promote secretion of 17- β-estradiol and had the reliability of time and concentration, but progesterone was not tested in follicular granulosa cells culture medium. (3) When hypophysis cells were treated with MLT singly, the concentration of PRL, except 2000 group, was higher than that of control group, others were lower than that of control group significantly (P<0.01), but the concentration of PRL increased with the increase of MLT. The concentration of PRL in all groups at 48 hour were lower than that of control group.(4) When hypophysis ce
lls were treated with MLT, the concentration of FSH, except 100 group, was higher than that of control group significantly at 24 hour (P<0.05), and its secretion when cultured for 48 hour were lower than those cultured for 24 hour significantly (P<0.01). This showed MLT could promote the secretion of FSH, but the function of secretion declined with the last of time. (5) There was no effect on the secretion of LH using only MLT to treat hypophysis cells. (6) Hypophysis cells were treated by MLT and HCG together, the concentration of PRL in treatment groups were lower than that of control group significantly (P<0.01) at 24 hour, but the concentration of PRL rose with the increase of MLT concentration in every treatment groups. PRL concentration only in 2000 group was higher than that of control group significantly in 24 hour (P<0.01), other treatment groups were lower than that of control group. And with the increase of MLT the level of
PRL rose in every treatment groups. This showed MLT and HCG together did not promote the secretion of PRL. The concentration of PRL in control and HCG group at 48 hour were lower than those of at 24 hour, however the concentration of PRL in treatment groups in 48 hour were higher than those of at 24 hour. It indicated MLT and HCG together could extend the function of PRL secretion in hypophysis cells. (7) When hypophysis cells were treated with MLT and HCG, we found it could promote the secretion of FSH significantly (P<0.01). Although MLT and HCG together could promote the secretion of LH, HCG played an important role in the process. (8) When hypophysis cells were treated with MLT and LHRH, we found there were no effect on the secretion of PRL at 24 hour and 48 hour. But in all groups the secretion increased with the increase of MLT concentration at 24 hour, and decreased with the increase of MLT concentration at 48 hour. (9) When hypophysis cells were treated with MLT and LHRH, the concentration of LH in 1
0 and 1000 group were higher than that of control group at 24 hour, and at 48 hour all treatment groups were higher than that of control group. It showed that MLT and LHRH together could possibly stimulate the secretion of LH.
Seasonal breeding Mongolian ewes were regarded as object in this research. The study adapted the way of cell culture and then carried different treatment. The hormone concentrations in cell culture medium were analyzed by RIA. The results were as follows: (1) Granulosa cells and hypophysis cells could be cultured with free-serum culture system in vitro, and secret corresponding hormone. (2) When follicular granulosa cells were treated with MLT or MLT and HCG, it could promote secretion of 17- β-estradiol and had the reliability of time and concentration, but progesterone was not tested in follicular granulosa cells culture medium. (3) When hypophysis cells were treated with MLT singly, the concentration of PRL, except 2000 group, was higher than that of control group, others were lower than that of control group significantly (P<0.01), but the concentration of PRL increased with the increase of MLT. The concentration of PRL in all groups at 48 hour were lower than that of control group.(4) When hypophysis ce
lls were treated with MLT, the concentration of FSH, except 100 group, was higher than that of control group significantly at 24 hour (P<0.05), and its secretion when cultured for 48 hour were lower than those cultured for 24 hour significantly (P<0.01). This showed MLT could promote the secretion of FSH, but the function of secretion declined with the last of time. (5) There was no effect on the secretion of LH using only MLT to treat hypophysis cells. (6) Hypophysis cells were treated by MLT and HCG together, the concentration of PRL in treatment groups were lower than that of control group significantly (P<0.01) at 24 hour, but the concentration of PRL rose with the increase of MLT concentration in every treatment groups. PRL concentration only in 2000 group was higher than that of control group significantly in 24 hour (P<0.01), other treatment groups were lower than that of control group. And with the increase of MLT the level of
PRL rose in every treatment groups. This showed MLT and HCG together did not promote the secretion of PRL. The concentration of PRL in control and HCG group at 48 hour were lower than those of at 24 hour, however the concentration of PRL in treatment groups in 48 hour were higher than those of at 24 hour. It indicated MLT and HCG together could extend the function of PRL secretion in hypophysis cells. (7) When hypophysis cells were treated with MLT and HCG, we found it could promote the secretion of FSH significantly (P<0.01). Although MLT and HCG together could promote the secretion of LH, HCG played an important role in the process. (8) When hypophysis cells were treated with MLT and LHRH, we found there were no effect on the secretion of PRL at 24 hour and 48 hour. But in all groups the secretion increased with the increase of MLT concentration at 24 hour, and decreased with the increase of MLT concentration at 48 hour. (9) When hypophysis cells were treated with MLT and LHRH, the concentration of LH in 1
0 and 1000 group were higher than that of control group at 24 hour, and at 48 hour all treatment groups were higher than that of control group. It showed that MLT and LHRH together could possibly stimulate the secretion of LH.
引文
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2．王建辰．动物生殖调控[M]．合肥：安徽科技出版社，1998．
3．马明新．褪黑素对哺乳动物繁殖生理的作用[J]．国外畜牧科技，1989(16)4：19-21
4．杨世华．几种生殖激素对体外培养的牦牛卵泡颗粒细胞凋亡的影响．[学位论文]．兰州：甘肃农业大学，2002．
5．张家骅．家畜生殖内分泌研究方法学[M]．杨陵：天则出版社，1993．
6. HadleyME. Endocrine role of the pineal gland. In Hadley ME Eds. Endocrinology, 2nd, New Jersey, prentice Hall, Inc, 1998, 474-495.
7. Preslock JP. The pineal gland: basic and clinical correlations. Endocrinal Rev, 984, 5: 282-287.
8．马明新．褪黑素对哺乳动物繁殖生理的作用[J]．国外畜牧科技，1989，16(4)：19-21．
9．戴兰馥．松果体褪黑素的研究概况[J]．国外医药学分册，1986，6：321-327．
10．陈北亨，王建辰主编．兽医产科学．北京：中国农业出版社，2001．
11．丁长海，魏伟，沈玉先，等．松果体-胸腺轴在衰老的免疫学机制中的作用[J]．中国药理学通报，1993，9：231-234．
12．徐峰，李金才，张迎暴，等．褪黑素对光照制度改变影响的对抗作用[J]，中国药理学通报，1994，10：448-451．
13．蔡晓宏，刘鸿．褪黑素的抗肿瘤研究进展[J]．国外医学肿瘤学分册，1998，25(2)：90-92．
14．魏伟，徐淑云．褪黑素的神经内分泌免疫作用及其应用前景[J]．中国药理学通报，1997 13(3)：196-200．
15．应水旺，张家驹．褪黑素对脑机能的影响[J]．生理科学进展，1987，18(3)：251-255．
16．秦立强，童建．褪黑素与抗氧化作用[J]．中国生化药物杂志，1999，20(3)：153-160．
17．肖建辉，梁宗琦，刘爱英，等．褪黑素的生理活性研究概况[J]，中草药，2001，32(9)：850-852．
18．郭志勤．家畜胚胎工程[M]．北京：中国科学技术出版社，1998．
19．姚丽娟，李华峰，程瑞禾，等．褪黑素的研究进展[J]．家畜生态，1996，17(4)：47-49．
20. Reither RJ. The mammalian pineal gland structure and fuction. Am J Ant. 1981, 162, 287-313.
21．张勇．双峰驼和牦牛繁殖季节褪黑素与促性腺激素之间的关系．[学位论文]．兰州：甘肃农业大学，2000．
22. Kennaway DJ, Dwnstan EA. Photoperiodic control of the onset of breeding activity and fecundity in ewes. J. Reprod Fertil Suppl. 1987, 34: 187-199.
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