Ⅰ、低剂量棉酚和甾体激素联合应用对大鼠睾丸Cyclin D1、P63和PKC-α表达的影响　Ⅱ、部分切除鱼尾后的尾鳍再生

【作者】 邵金辉；

【导师】 许增禄；

【作者基本信息】 中国协和医科大学 ， 人体解剖与组织胚胎学， 2010， 博士

【摘要】 男性避孕药发展远远落后于女性避孕药,至今仍无安全、有效、无副作用的男性避孕药面市。我室经多年研究发现,低剂量棉酚和甾体激素联合用药可基本满足了副作用低和生殖能力可逆性恢复的要求。与单独使用棉酚和甾体激素相比,棉甾联合用药起效更快,两者在抗生育效果方面具有协同作用,但是尚不明确两者发挥协同作用位点。棉甾联合用药具有抗生育作用,能够影响睾丸中细胞的增殖、凋亡和分化等过程。而Cyclin D1和P63在细胞的增殖过程中起作用,所以棉甾联合用药可能影响Cyclin D1和P63的表达。PKC分布和作用广泛,棉甾联合用药的抗生育作用,也可能影响PKC-α在睾丸中的表达。成年雄性大鼠被随机分为四组,喂服棉酚12.5mg／kg·d+去氧孕烯0.125mg／kg·d+炔雌醇0.025mg／kg·d+十一酸睾酮100mg／kg·d的联合用药组(GH组),单独喂服相同剂量棉酚12.5mg／kg·d的棉酚组(G组)、单独喂服相同剂量激素,去氧孕烯0.125mg／kg-d+炔雌醇0.025mg/kg·d+十一酸睾酮100mg／kg·d的激素组(H组)以及喂服载体溶剂1%甲基纤维素的阴性对照组(C组)。用药后6、8和10周取睾丸和附睾组织,应用免疫组化和Western blot技术,检测(?)cyclin D1、P63和PKC-a的表达变化,以寻找联合用药对精原细胞的作用分子。研究结果显示,棉甾联合用药可有效地抑制精子发生,减少附睾成熟精子的数量,并且使睾丸和附睾的重量和体积明显变小。在本实验中,Cyclin D1蛋白免疫组化的结果为,Cyclin D1蛋白主要在生精小管管腔面的残余体中表达也在生精小管之间的睾丸间质中表达。在棉甾联合用药4w和10w后,各组间无明显差异。而Western blot结果显示4w各组中,C组的表达量明显高于其它三组, (P＜0.05)；10w各组中,C组的表达量明显低于其它三组, (P＜0.05)。在棉甾联合用药4w后,H和GH组的P63蛋白质在生精小管之间的睾丸间质中的表达,与C和G组对比有所减弱,用药10w后明显减弱,而对其Western blot结果没有明显影响。在棉甾联合用药10w后,免疫组织化学结果显示,H和GH组的PKC-α蛋白质在生精小管之间的睾丸间质中的表达,与C和G组对比明显减弱。PKC-α蛋白表达的Western blot结果为,4w的GH组与4w的C、G和GH组比较,PKC-a蛋白表达明显下降(P＜0.05)。10w的H和GH组与10w的C和G组比较,PKC-a蛋白表达明显下降(P＜0.05),C和G两组间没有明显的差异。以PBS代替一抗的阴性对照,均未见阳性着色区,可见对三种蛋白的免疫组化的结果是特异性的总之,棉甾联合用药可有效地抑制精子发生,减少附睾成熟精子的数量,并且使睾丸和附睾的重量和体积明显变小。免疫组化的结果提示,在棉甾联合用药10w后,H和GH组的P63和PKC-α蛋白质在相应部位的表达,与C和G组对比明显减弱。以PBS代替一抗的阴性对照,均未见阳性着色区,可见免疫组化的结果是特异性的。Western blot结果提示,在棉甾联合用药10w后,三种基因在整个大鼠睾丸的总体蛋白表达量中,只有PKC-α的下降。所有的生物体对损伤都有生物反应,但是它们再生的能力却大不相同。这种自身稳定的更新机制是固有的特殊谱系干细胞所介导的。在这方面,硬骨鱼和两栖动物受到特别的关注,因为能够再生受损伤的器官包括部分心脏,脊髓,视网膜,和肢体／鳍。相对简单和易获得的尾鳍成为解析骨等再生分子机制的良好体系。在内骨骼水平切除鳍将不会有再生,在真皮鳍条切除将引起丢失结构大约3周内完全恢复。许多基因在鳍再生过程中的作用已经被研究。视黄酸(RA)能够改变再生尾鳍的生长情况和形态。有报道观察到鲚鱼的含有肌肉和鱼鳞的尾部。但是,有关经过断尾切除术后的鳍再生实验还没有报道。断尾再生的尾鳍再生于具有内骨骼和肌肉组织的鱼尾部分,这与现有的认识不同。但是当鲚鱼被捕到网里后都拼命挣扎而死去,所以不能够用鲚鱼做断尾再生的实验。四种鱼,包括草鱼、普通鲤鱼、鲤鱼和斑马鱼,用于研究鱼的断尾后尾鳍再生。鱼尾切断术是指切除部位在具有肌肉、内骨骼和鱼鳞的鱼尾的远侧边缘内侧1-2 mm处；近侧尾鳍切断术是指切除部位在具有肌肉、内骨骼和鱼鳞的鱼尾的远侧边缘外侧1-2 mm处；远侧尾鳍切断术是指切除部位恰好在尾鳍分岔前。鱼鳍经过固定、脱钙、包埋、切片。切片再经过脱蜡、梯度水化,进行H-E染色。将切下的鱼尾或者尾鳍固定清洗,在过滤的0.1%阿辛蓝溶液中染色、脱水,然后转入溶于0.5%KOH的0.1%茜素红溶液中染色。在应用BrdU进行染色的实验中,将鱼腹腔注射BrdU。固定后切片脱蜡,应用鼠源BrdU一抗处理,DAB显色。再生尾鳍中的细胞凋亡情况,应用TUNEL检测试剂盒,按照说明书中的方法进行检测。在鱼缸中加入RA至终浓度为10-6M,每天更换水和RA,连续4天。尾鳍切断术后,再生的锦鲤尾鳍比普通鲤鱼的分岔早。有的红色普通鲤鱼在它们再生的尾鳍上有较多的黑色素。在鱼尾或者尾鳍切断术后的尾鳍再生有区别。断尾后的尾鳍再生率约为50%,而尾鳍切断术后的再生率为100%。只有一部分断尾后再生尾鳍比较完整,而所有尾鳍切断术后再生的尾鳍均完整。在鱼尾切断术后2-4周,才有肉眼可见的尾鳍再生,而在尾鳍切断术后3天就可以见到。前者从具有肌肉、内骨骼和鱼鳞的尾部部分再生,而后者再生源于具有鳍条的尾鳍部位。一些草鱼的尾部经过切断术后2个多月,再生出不规则的部分尾鳍。断尾术后42天,单纯用肉眼仍然不能够看出明显的鳍条,鳍条样条状物是血管。这条普通鲤鱼已经断尾后3个月,再生出从形状上看完整的尾鳍已经1个月了,但是也不能够在再生的尾鳍上看到鳍条。在普通鲤鱼具有肌肉的尾后缘远侧1-2 mm处切断尾鳍,在一个月后再生出从形状上看完整的尾鳍,2个月后的再生尾鳍具有肉眼可见的鳍条。锦鲤断尾再生的尾鳍中有丰富的血管,但是没有明显的鳍条。断尾鳍后尾鳍再生的概率为100%,多数都能够再生出完整的尾鳍。斑马鱼断尾后再生尾鳍的概率约为50%,而再生的尾鳍中,只有一部分再生比较规则和完整。当断尾再生的尾鳍达到2 mm时,就可以看到鳍条,而不能够看到血管。在普通鲤鱼断尾后,很少的情况下尾部断端被鱼鳞样物质覆盖。经过阿辛蓝和茜素红染色,断尾再生锦鲤和斑马鱼的尾鳍与正常的以及断鳍再生的锦鲤和斑马鱼尾鳍显示出明显的区别。断尾再生的普通鲤鱼尾鳍经过H-E染色,显示在含有肌肉的尾部区域年和再生的尾鳍之间有不光滑的部分。断尾再生的尾鳍经过BrdU整合处理后,应用DAB检测显示增殖细胞位于再生尾鳍的边缘。TUNEL检测结果显示凋亡细胞主要存在于尾鳍的边缘。对斑马鱼进行断尾处理和RA处理,2个月以后,仅仅有一条再生出很小的尾鳍,用肉眼很难看到,再过了一个月也没有发现进一步的生长。斑马鱼经过断尾处理后,再生为期的概率约为50%,然而,经过断尾处理和RA处理后,没有斑马鱼能够再生出肉眼容易看到的尾鳍。与断尾后再生尾鳍的实验不同,经常有人做断尾鳍后再生尾鳍的相关实验。实验显示四种鱼：草鱼、普通鲤鱼、锦鲤和斑马鱼,在断尾后能够再生尾鳍。断尾再生的尾鳍起源自具有肌肉、内骨骼和鳞片的尾部。这与当前的认识—在内骨骼水平切断处理后不会再生—是不同的。在断尾再生的尾鳍中,应用TUNEL方法也检测到细胞凋亡。这样,可能去分化和干细胞激活都对芽基的形成起作用。尽管细胞的去分化现象只在两栖动物肢体和尾巴再生过程中得到证实,经过对斑马鱼再生尾鳍的芽基的形态学,本体论和基因表达研究,提示斑马鱼鳍再生存在相似的机制。鱼经过断尾处理后的横切面处的尾部组织与四足动物再生部位有相似的结构。因此,断尾再生尾鳍有可能揭示一个普遍的原理,即在所有脊椎动物的含有内骨骼组织部位的再生机制。而其它的“鳍再生”研究仅仅限于鳍条处的再生,这种再生与内骨骼和肌肉组织无关。对于断尾再生,RA表现出显著的抑制作用,而断鳍再生的斑马鱼,在年RA处理后都能够再生出畸形的尾鳍。更多还原

【Abstract】 The development of male contraceptive pill has lagged far behind that of female contraceptive pill. Up to date, no even one safe, effective, reliable and reversible male contraceptive pill is on sale. Studing for many years, we have found that combined regimen of low-dose gossypol and steroid hormones has reversible antifertility action with low side effects. The combined regimen has faster effect than that of low dose gossypol or steroid hormones alone,although these results demonstrate that low-dose gossypol and steroid hormones have synergistic effect on antiferlity, the sites of synergistic effect remained unknown. Combined regimen of low-dose gossypol and steroid hormones has antifertility action and can affect proliferation, apoptosis and differentiation of cells in testis. Cyclin D1 and P63 have effect on cellular proliferation, so combined regimen of low-dose gossypol and steroid hormones may affect the expression of Cyclin D1 and P63. PKC have widespread distribution and effect, so combined regimen of low-dose gossypol and steroid hormones may also affect the expression of PKC.Adult male rats were divided into four groups randomly, group GH:rats were fed orally with Gossypol acetic acid (GA,12.5mg/kg·d) and Desogestrel (DSG,0.125mg/kg·d)/Ethinylestradiol (E,0.025mg/kg·d)/Testosterone undecanoate (TU,100mg/kg·d); group G:a single does of GA (12.5mg/kg·d) was given; group H:DSG(0.125mg/kg·d)/E(0.025mg/kg·d)/TU(100mg/kg·d) were administered; group C:rats only received vehicle(1% methyl cellulose). The testis and epididymis were removed at 6、8 and 10 week for detection. The expressions of cyclin D1, P63 and PKC-a, were detected by immunohistochemical method and western blot in order to find the effector molecules of the combined regiman.Our results show that combined low-dose gossypol and steroid hormones can supress spermatogenesis and reduce obviously the weight and volume of testis and epididymis.In this experiment, immunochemistry results show that protein Cyclin D1 is expressed mainly in residual body of seminiferous tubule cavosurface and interstitial tissue of testis among seminiferous tubule. There are no distinguished difference between group of C, G, H and GH at 4w or 10w. Western blot results of protein Cyclin D1 show that the expression of group C is higher greatly than any other group at 4w (P<0.05). While, the expression of group C is lower greatly than any other group at lOw (P＜0.05) Immunochemistry results show that protein P63 is expressed mainly in interstitial tissue of testis among seminiferous tubule. The expression of P63 of group H and GH at 4w descend little compared with that of group C and G. While, the expression of P63 of group H and GH at10w descend greatly compared with that of group C and G. Western blot results of P63 show that the expression is similar among each group at 10w. Immunochemistry results show that protein PKC-a is expressed mainly in interstitial tissue of testis among seminiferous tubule.The expression of PKC-a of group H and GH at lOw descend greatly compared with that of group C and G Western blot results of PKC-a show that the expression of PKC-a of group GH at 4w compared with that of group C, G and H (P＜0.05). The expression of PKC-a of group H and GH at 10w descend compared with that of group C and G (P<0.05);While there are no difference between group C and G at 10w. When antibodies are replaced by PBS, results of immunochemistry are all negtive. So those results are specific. In conclusion, our results show that combined low-dose gossypol and steroid hormones can supress spermatogenesis and reduce obviously the weight and volume of testis and epididymis. Immunochemistry results indicate that the expression of P63 and PKC-a of group H and GH descend compared with group C and G in corresponding region after use of low-dose gossypol and steroid hormones for 10 w. When antibodies are replaced by PBS, results of immunochemistry are all negtive. The results are specific. Results of western blot indicate that expression of PKC-a only among the three kinds of gene in whole rat testis descend after use of low-dose gossypol and steroid hormones for 10 w. All organisms mount biological responses to damage, but they vary widely in the degree to which they can recover from damage. These types of homeostatic renewals are thought to be mediated by resident stem cells of specific lineages. In this context, teleost fish and amphibian are of particular interest, as they have a remarkable capacity to regenerate damaged organs, including heart, spinal cord, retina and limbs/fins. The relative simplicity and the easy access of the caudal fin and of its skeletal elements make it an excellent system for dissecting the molecular pathways involved in bone regeneration. While resection of the fin at the level of the endoskeleton is not followed by regeneration, amputation of the dermal fin rays results in complete replacement of the lost structures within approximately 3 weeks. Many functional genes in fin regeneration have been researched. Retinoic acid(RA) might alter growth and morphogenesis of the regenerating fins. Fin regeneration from anchovy tail segments with musculature and scales was observed and reported in Freshwater Fisheries. But, no experiment about fin regeneration after tail amputation was reported. These caudal fins arose from segments of the endoskeleton, which contrast with currently accepted knowledge. While, they always struggle excessively when they are in fishing nets, consequently, it is difficult to use anchovies as experimental animals.Four kinds of fish, including grass carp, common carp, koi carp, and zebrafish (Danio rerio) were used in the present study to research further into fin regeneration from fish tail segments with musculature and scales. Tail amputation was carried out at the proximal 1-2 mm of the far margins of the tail that had musculature, endoskeleton, and scales. Proximal caudal fin amputations were done at the distal 1-2 mm of the far margin of the tail that had musculature, endoskeleton, and scales. Distal caudal fin amputations were carried out in the region just before the first bifurcation of the fin rays. Fins were fixed, decalcified, embedded, and cut into sections. Sections were dewaxed, hydrated in a gradient and stained by a routine hematoxylin-eosin(H-E) staining technique. Portions of tail and caudal fins were fixed and washed, then stained with a filtered 0.1% Alcian blue, and dehydrated. Bones were stained by placing the fins in a fresh solution of 0.1% Alizarin red in 0.5% KOH. For studies involving bromodeoxyuridine (BrdU), The fish were injected BrdU. fixed. Slides treated with primary mouse anti-BrdU antibody 3,3’-Diaminobenzidine (DAB) was used to visualize and photograph BrdU incorporation. Apoptosis in fish caudal fins on tissue slides was detected by enzymatic labeling of DNA strand breaks using terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL). TUNEL was carried out using a cell death detection kit (Roche, Mannheim, Germany) according to the manufacturer’s instructions. An appropriate amount of RA at a final concentration of 10-6 M was added directly into the tank water following amputation for 4 continuous days, and the water and RA were refreshed every day.Regenerated koi carp caudal fins bifurcate earlier than common carp. Some red common carp have a fair amount of melanin pigment in their regenerated caudal fins. There are some differences in caudal fin regeneration when caudal fins or tails are anesthetized for amputation, which was carried out at the proximal 1-2 mm of the rear margin of the tail with musculature, endoskeleton, and scales. The regeneration rate.of the caudal fin after tail amputation is about 50%, while the rate after caudal fin amputation is 100%. Only some of the tail amputations regenerated completely, while all of the caudal fin amputations completely regenerated. Caudal fins can be seen with the naked eye about 2 weeks to 4 weeks after tail amputation and about 3 days after caudal fin amputation. The former regeneration comes from the segment of the tail with musculature, endoskeleton, and scales, while the latter regeneration comes from the caudal fins with fin rays. Some grass carp whose tails had been cut two months prior regenerated irregularly only part of the caudal fin. After 42 days following tail amputation, distinct fin rays still could not be seen with the naked eye and the existing fin ray was more similar to a blood vessel.When the common carp tail was cut about three months previously, it regenerated a complete caudal fin according to its shape after one month. However, the regenerated caudal fins had no fin rays that could be seen with the naked eye. When the common carp caudal fin was cut distally 1-2 mm behind the margin of tail with musculature about two months prior, it regenerated a complete caudal fin according to its shape after one month. These regenerated caudal fins had fin rays that could be seen with the naked eye. Regenerated caudal fins of Koi carp had abundant blood vessels but no distinct fin rays. The probability ratio of caudal fin regeneration after tail amputation was 100% and most of these regenerated caudal fins were complete according to their shape. The probability ratio of zebrafish caudal fin regeneration after tail amputation was about 50%. Only part of the regenerated caudal fins was regular and complete. When the regenerated caudal fin after tail amputation reached 2 mm, fin rays could be seen with the naked eye, although blood vessels could not be seen. In a few cases among common carp, the broken ends of the tails that had been operated on were covered with a scale-like substance. After Alcian blue and Alizarin staining, tails of Koi carp and zebrafish with regenerated caudal fins after tail amputations showed dissection evidence that was comparable to that of normal Koi carp and zebrafish. This clearly indicates that tail amputation greatly differed from caudal fin amputation. H-E staining of regenerated common carp caudal fin after tail amputation indicated that the area between the tail with musculature and the regenerated caudal fin is not smooth. Proliferated cells stained by DAB are located at the margin of the regenerated caudal fin treated with BrdU after tail amputation. Apoptotic cells occurred primarily at the margin of the caudal fin. Zebrafish were treated with RA and with tail amputation. After 2 months, only one regenerated small caudal fin appeared, which could not easily be seen with the naked eye and which showed no further growth after another month. The regeneration ratio of zebrafish after tail amputation was about 50%, while no zebrafish regenerated caudal fins that could be seen easily with the naked eye after identical tail amputation and RA treatment.Caudal fin regeneration after fin amputation has been researched repeatedly, in contrast to experiments on caudal fin regeneration after tail amputation. Fin regeneration after tail amputation was demonstrated in four species of fish, including grass carp, common carp, Koi carp, and zebrafish. Fin regeneration after tail amputation come from tail segment with musculature, endoskeleton, and scales. This is different from currently held views that resection of the fin at the level of the endoskeleton is not followed by regeneration. In caudal fin regeneration after tail amputation, cell apoptosis was also found by TUNEL. Thus, it is likely that de-differentiation and stem cell activation both contribute to formation of the blastema. Although de-differentiation of cells has not yet been demonstrated in regenerating structures other than amphibian limbs and tails, the morphology, ontology, and gene expression profile of the zebrafish blastema in the regenerating caudal fin suggests that zebrafish fin regeneration occurs by similar mechanisms. Tail tissue that formed after tail amputation in the region around the cross-section had homologous structure to that seen in tetrapods. Therefore, fin regeneration after tail amputation has the potential to reveal general principles that are common to regeneration from tissue with endoskeleton in all vertebrate appendages. Other "fin regeneration" studies performed on actinopterygians have been limited to the lepidotrichia, which do not have an endoskeleton or musculature. RA was shown to have a very powerful inhibitory effect on caudal fin regeneration after tail amputation. Zebrafish with caudal fin amputation after RA treatment always regenerated deformed fins.更多还原