牦牛输卵管上皮细胞的分离培养及FSH对其分泌输卵管特异性糖蛋白的影响

发布时间：2018-03-06 23:16

本文选题：牦牛　切入点：输卵管上皮细胞　出处：《甘肃农业大学》2015年硕士论文　论文类型：学位论文

【摘要】：本研究以牦牛为研究对象,首先改进了输卵管上皮细胞体外培养方法;其次在此基础上,探讨了促卵泡生成素(FSH)对牦牛输卵管上皮细胞分泌特异性糖蛋白的影响。以几种不同消化酶分离、纯化,最终成功地体外培养了牦牛输卵管上皮细胞,并对该细胞进行了免疫学鉴定和分析。体外分离培养的牦牛输卵管上皮细胞,在细胞中添加不同浓度的FSH;运用荧光定量PCR技术,分析输卵管特异性糖蛋白的m RNA的表达水平,并对其分泌输卵管蛋白的部位,用细胞免疫标记进行分析。研究结果表明:(1)牦牛输卵管上皮细胞的最佳培养方法是胰蛋白酶和胶原蛋白酶共同作用分离纯化细胞,用该方法培养的细胞株较纯,纯度接近90%.(2)从生长曲线可以看出,该细胞在第一天到第三天生长缓慢,第四天到第五天呈指数生长趋势,从第六天开始,增值又变缓。(3)所培养的所有输卵管上皮细胞细胞都表达输卵管特异性糖蛋白,且主要由细胞质表达。(4)当FSH的浓度为0.5~5μg/mL时,输卵管蛋白的表达量随着FSH浓度的上升而增加;在5μg/mL的FSH作用后,输卵管蛋白的m RNA表达量最高,浓度超过10μg/mL时,输卵管蛋白基因的表达量降低。本研究成功地体外培养了牦牛输卵管上皮细胞,并发现FSH具有促进输卵管上皮细胞分泌输卵管特异性糖蛋白的作用,且具有剂量依赖性。该结果为解决牦牛的繁殖率和胚胎移植的存活率低,以及为了给进一步开展牦牛生殖生理研究提供了理论依据。
[Abstract]:In this study, yak was used as the research object. Firstly, the culture method of oviduct epithelial cells in vitro was improved; secondly, on the basis of this, The effects of follicle stimulating hormone (FSH) on the secretion of specific glycoproteins by yak oviductal epithelial cells were studied. The yak oviduct epithelial cells were successfully cultured in vitro by the isolation and purification of several digestive enzymes. The cells were identified and analyzed by immunology. Different concentrations of FSHs were added to the cultured yak oviductal epithelial cells in vitro, and the expression level of m RNA of oviduct specific glycoprotein was analyzed by fluorescence quantitative PCR technique. The results showed that the best culture method of yak oviduct epithelial cells was to isolate purified cells by the interaction of trypsin and collagenase. From the growth curve, we can see that the cells grow slowly from the first day to the third day, and the growth trend is exponential from 4th days to 5th days, starting from 6th days. Oviductal specific glycoprotein was expressed in all oviductal epithelial cells cultured in vitro, and mainly expressed by cytoplasm. When the concentration of FSH was 0.5 渭 g / mL, the expression of tubal protein increased with the increase of FSH concentration. After the treatment of 5 渭 g / mL of FSH, the expression of m RNA in oviduct protein was the highest, and when the concentration was more than 10 渭 g / mL, the expression of oviduct protein gene decreased. In this study, yak oviduct epithelial cells were successfully cultured in vitro. It was found that FSH could promote the secretion of oviduct specific glycoprotein by oviductal epithelial cells in a dose-dependent manner. In order to provide theoretical basis for further research on reproductive physiology of yak.
【学位授予单位】：甘肃农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S823.85

【相似文献】