【作者】 李伟；

【导师】 陈树林；

【作者基本信息】 西北农林科技大学 ， 神经生物学， 2009， 硕士

【摘要】 为了研究小鼠卵巢黄体中弥散性神经内分泌系统（DNES）细胞的存在情况、DNES细胞的来源;GnRH、LH和黄体细胞对神经胶质瘤细胞C₆生长的影响。本试验采用了免疫组化链霉素抗生物素蛋白-过氧化物酶法、免疫组织荧光化学法对小鼠卵巢黄体中是否存在神经内分泌细胞及其来源进行了研究;采用细胞培养法、免疫细胞荧光化学法、HE染色法、MTT比色法、嗜银蛋白分析法、细胞共培养法和计算机图象分析法对黄体细胞和神经胶质瘤细胞C₆的培养及二者的共培养;GnRH、LH和黄体细胞对神经胶质瘤细胞C₆生长的影响进行了深入的研究。主要结果如下:1.小鼠黄体中部分细胞中含神经元特异性烯醇化酶（NSE）、S100蛋白和催产素（OT）免疫反应阳性产物。根据免疫反应产物呈色的程度不同可分为强阳性、中等阳性和弱阳性三种。这些免疫反应阳性细胞散在或成团分布,多呈圆形、卵圆形,一些细胞具有明显的突起。NSE和S100蛋白均为DNES系统广谱的,共同的细胞标记物,提示小鼠黄体中的免疫反应细胞属DNES细胞。在小鼠次级卵泡的卵泡膜外膜也存在NSE和S100蛋白的表达,提示小鼠卵巢在排卵前就已经开始了神经内分泌化;DNES物质参与了卵泡发育、黄体形成与退化的整个过程。2.GnRH、LH和黄体细胞对神经胶质瘤细胞C₆的影响。结果显示: 35μg/mL的GnRH和100IU的LH作用24小时后显著抑制神经胶质瘤细胞C₆生长。3. GnRH、LH和黄体细胞对神经胶质瘤细胞C₆中p21基因、NSE和S100蛋白表达的影响。GnRH可以显著降低p21基因、NSE和S100蛋白的表达;LH可以显著降低p21基因和NSE的表达,而对S100的表达无显著影响。4.黄体细胞和神经胶质瘤细胞C₆共培养之间的作用。黄体细胞对神经胶质瘤细胞C₆的生长起了一定的抑制作用。结果提示:小鼠黄体中存在DNES细胞;小鼠黄体细胞对混合共培养的神经胶质瘤细胞C₆的生长有一定的抑制作用。更多还原

【Abstract】 The distribution properties and source of diffused neuroendocrine system cells in corpus luteum（CL） of mouse during different physiological periods was studied by method of immunohistochemistry streptavidin-peroxidase and immunohistofluorescence in present research; and in order to investigate the effect of GnRH、LH and luteal cell on glioma cell C₆, we used the cell culture in vitro, immunocytefluorescence, HE staining method, MTT assay method, AgNORs analytical method, cell coculture to detect the cell morphous change of leteal cell culture, glioma cell C₆ culture and their coculture; and to research the expression diversity of p21 protein, NSE and S100 protein in glioma cell C₆ when we exert GnRH, LH and luteal cell culture into the culture of glioma cell C₆ or not. All results were analyzed by Jiangsu JieDa cellular morphology Microsoft ware. The main contents and results of this studies are as follows:1.The immuno-reactive productions of NSE, S100 protein and OT were located in the cytoplasm of part of the luteal cells of mouse during different physiological periods. 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. As the NSE, S100 protein are the common markers of neuroendocrine cells, this experiment show that there are a number of neuroendocrine cells in the CL of mouse. This experiment also found that there are expression of NSE, S100 protein in tunica externa of secondary follicle. It could be infer that DNES cells exist in the mouse ovary, and the muose ovary develop neuroendocrine preovulation.DNES markers exert different roles in the process of ovarian follicular development, corpus luteum development and luteolysis. Some cells develop DNES cells during ovary development.2. The effect of GnRH, LH and luteal cell on glioma cell C₆ in vitro. Results show that 35μg/mL GnRH and 100 IU LH for 24h inhibited the growth of glioma cells.3. The effect of GnRH, LH and luteal cell on the expression of p21 protein, NSE and S100 protein in glioma cell C₆. The result indicated that the expression of p21 protein, NSE and S100 protein were down regulated dominantly in glioma cells C₆ after GnRH treatment compared with the control group. And also show that the expression of p21 protein and NSE decreased in LH treatment group compared with the control group. However, LH has no different effect on the S100 protein expression in glioma cells compared with the control group.4. The interaction between luteal cell and glioma cell by means of their coculture. The result show that luteal cell makes a significant inhibition of luteal cells growth after coculture for 24 h.From the results above, we draw the following conclusions: there are neuroendocrine cells in the CL of mouse; and mouse luteal cells have a remarkably effect on the growth of glioma cells.更多还原