摘要
为了研究发情周期牛卵巢黄体中弥散性神经内分泌系统(DNES)细胞的存在情况、DNES标志物之间是否存在协同或拮抗等互相调节作用及DNES细胞的来源,本试验采用了敏感性较高的免疫组化链霉素抗生物素蛋白-过氧化物酶法、免疫组化双标记及计算机图象分析等方法对牛发情周期卵巢组织中弥散性神经内分泌细胞标志物的分布进行了研究。主要结果如下:
1.牛黄体中部分黄体细胞胞质中含有5-羟色胺(5-HT)、S-100蛋白、神经特异性烯醇化酶(NSE)、突触素(SYP)免疫反应阳性产物。根据免疫反应复合物呈色的程度不同可分为强阳性、中等阳性、弱阳性三种。这些免疫反应阳性细胞散在或成团分布,多呈圆形、卵圆形或三角形,一些细胞具有明显的突起。与这些物质在山羊黄体中表达相比,其免疫反应产物阳性细胞在形态和分布上十分相似,但阳性细胞数量和免疫反应产物相对表达量均显著多于其在山羊黄体中的表达。神经特异性烯醇化酶(NSE)、突触素(SYP)、5-羟色胺(5-HT)、S-100蛋白均为弥散性神经内分泌系统光谱的,共同的细胞标记物,提示牛黄体中的免疫反应细胞属DNES细胞。
2.本试验进行的4种物质的6种双标记染色组合中,都发现了在同一黄体细胞中共存的情况,即每种双标记染色组合都检出了双阳性细胞。5-HT与SYP、5-HT与NSE、5-HT与S-100、NSE与SYP、NSE与S-100以及SYP与S-100等的双阳性免疫反应细胞轮廓清楚,胞质中含有深浅不等棕褐色的染色颗粒,细胞核不着色,呈圆形点状,细胞核位于细胞中央或偏于一侧。细胞形态有多样,以圆形、卵圆形、不规则形状居多。其中95%以上的SYP与NSE和5-HT共存,且与SYP/NSE、SYP/S-100在阳性细胞数量上无统计学差异;其他几组两两组合共存率也在85%以上。四种神经内分泌标志物之间相互作用,共同调节黄体功能提供了形态学依据。
3.催产素(OT)在很大程度上与神经内分泌标志物共存,OT/NSE、OT/S100、OT/5-HT双阳性细胞在数量上与OT单阳性细胞无显著差异。OT在黄体形成和退化过程中均发挥重要作用,提示黄体中DNES细胞与黄体的形成和溶解相关。
4.本试验对牛黄体中神经内分泌细胞的来源进行初步研究,结果显示,除黄体外,神经内分泌标志物在卵泡细胞和卵巢其他组织均未见SYP和NSE免疫反应阳性产物,SYP和NSE免疫反应阳性产物只有在卵巢黄体期黄体中才观察到,提示牛黄体中的神经内分泌细胞是在排卵前后,由卵泡细胞在增殖分化形成黄体的同时,发生神经内分泌分化形成。
The existence of diffused neuroendocrine system cells in corpus luteum (CL) of bovine in estrus was studied by method of immunohistochemistry streptavidin-peroxidase in present research. The co-existence and their regulation mechanics of four involved neuroendocrine markers, SYP、NSE、5-HT and S100, in one same cell were detected by means of immunohistochemical double label technique. The relation between oxytocin and diffused neuroendocrine system cells were also studied by immunohistochemical double label technique. All results were analyzed by Jiangsu JieDa cellular morphology Microsoft ware. The main results were showed following:
1. The immuno-reactive productions of four neuroendocrine markers were located in the cytoplasm of part of the luteal cells of bovine during the period of estrus. The positive cells were belonged to three groups of strongly, mid and weekly staining according to the immuno-reaction. Some of the positive cells get together while the others are diffused in the CL. There are various morphological features of the positive cells including oval and triangle and spinal in shapes and some of which have projects. In this experiment, we found that the contents of the four markers and their numbers of the positive cells in bovine CL are significantly more than which in goat CL. As the SYP、NSE、5-HT and S100 are the common markers of neuroendocrine cells, we suggest that there are a number of neuroendocrine cells in the CL of bovine in estrus.
2. The double labeled cells of two markers, 5-HT and SYP、5-HT and NSE、5-HT and S-100、NSE and SYP、NSE and S-100, SYP and S-100 were also observed in CL with colored cytoplasm of brown in present research. The double labeled cells presents the same feature as single stained ones with clear outline and round nuclear. The result shows that more than 95% SYP and NSE co-existence with 5-HT, more over, there is no significant difference between the number of SYP/NSE and SYP/S-100 double labeled cells. In addition, other pairs also have high co-existence rate. These results suggest that the four neuro-markers may affect the function of CL by regulating each other.
3. Oxytocin plays a vital role in the development and apoptosis of the CL. The result shows that oxytocin also well co-exist with neuro-markers. The numbers of double labeled cells of OT/NSE、OT/S100、OT/5-HT has no significant difference, which indicates that the luteal neuroendocrine cells involve in the regulation of the formation and regression of CL.
4. Pilot study of the origin of luteal neuroendocrine cells was also done in this research. The result shows that neuro-markers, SYP and NSE immunoreactive were detected only in corpus luteum with negative staining in other part of the ovaries. This result suggests that luteal neuroendocrine cells were generated from follicular cells by means of neuroendocrine differentiation during luteinization.
引文
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