动态血管发育模型的建立及其miRNA的变化

发布时间：2018-06-09 01:28

本文选题：miRNA芯片 + 鸡胚绒毛尿囊膜　；参考：《南华大学》2007年硕士论文

【摘要】： 目的:建立鸡胚绒毛尿囊膜动态血管发育模型,观察和分析血管发育过程中miRNA的表达变化,为确定血管发育关键调控miRNA及其下游基因提供实验依据。方法:利用鸡胚绒毛尿囊膜血管发育周期短,可操作性强的特点,选取新鲜、大小均匀的种蛋,自行孵育,原位观察第一天到第二十一天的鸡胚绒毛尿囊膜血管发育情况,确定尿囊膜血管初生、旺盛和消退的三个时间点,建立动态血管发育模型。选取不同时间点的尿囊膜血管,提取蛋白,用Western blotting技术检测FGF2和TGF-β2蛋白在血管发育的这三个阶段的表达量的变化,判断动态血管发育模型是否建立成功。用Trizol法提取总RNA,紫外吸收测定法测RNA浓度和纯度,琼脂糖凝胶电泳观察28s、18s带,检测提取的RNA的质量,制作miRNA芯片,分析在血管发育过程中miRNA的变化。结果:研究发现第四天、第十三天、第十六天分别为血管发育的初生、旺盛和消退的三个时间点;Western blotting检测结果与文献报道相符;血管发育过程中miRNA的表达变化:⑴、旺盛期与初生期比较:表达上调的有5个miRNA,表达下调的有1个miRNA;⑵、消退期与初生期比较:表达上调的有37个miRNA,表达下调的有4个miRNA;⑶、消退期与旺盛期比较:表达上调的有27个miRNA,表达下调的有2个miRNA。结论:1、利用鸡胚绒毛尿囊膜血管动态发育周期短,血管发育过程中存在明显的初生期、旺盛期、消退期不同阶段,且可重复性强的特点,可建立一种新型动态血管发育模型。 2、血管发育的初生期、旺盛期、消退期不同阶段,相对应出现的miRNA表达谱的显著变化,为确定血管发育关键调控miRNA及其下游基因提供了新的思路和方向。
[Abstract]:Objective: to establish a dynamic angiogenesis model of chicken chorioallantoic membrane and to observe and analyze the expression of miRNA during the development of chorioallantoic membrane. In order to provide experimental basis for determining the key regulation of miRNA and its downstream genes in vascular development methods: the chorioallantoic membrane vascular development cycle of chicken embryo was short and maneuverability was strong. Fresh and evenly sized eggs were selected and incubated by themselves. The angiogenesis of chorioallantoic membrane of chorioallantoic embryo was observed in situ from the first day to the 21 day, and the dynamic vascular development model was established by determining the three time points of the origin, exuberance and regression of the allantoic membrane vessels. The expression of FGF2 and TGF- 尾 2 proteins in the three stages of vascular development were detected by Western blotting to determine whether the dynamic vascular development model was established successfully. The total RNAs were extracted by Trizol method, the concentration and purity of RNA were measured by ultraviolet absorption assay, the 28s ~ (18 s) band was observed by agarose gel electrophoresis, the quality of extracted RNA was detected, the miRNA microarray was made, and the changes of miRNA during vascular development were analyzed. On the thirteenth and sixteenth days, the results of Western blotting were consistent with those of the literature at the three time points of exuberant, exuberant and regression, respectively, and the expression of miRNA was changed at 1: 1 during vascular development. There were 5 miRNAs up-regulated, 1 miRNA-2 down-regulated, 37 miRNAs upregulated and 4 miRNAs down-regulated. There were 27 miRNAs upregulated and 2 miRNAs down-regulated in extinction period and exuberant phase. Conclusion: 1. The dynamic development cycle of allantoic membrane of chicken embryo chorioallantoic membrane is short, and there are obvious primary and exuberant phases in the process of vascular development. A new type of dynamic vascular development model can be established with different stages of regression period and strong reproducibility. 2. The significant changes of miRNA expression profiles in different stages of vascular development, such as the primary stage, the exuberant stage, and the extinction stage, can be established. It provides a new idea and direction for determining the key regulation of miRNA and its downstream genes in vascular development.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R-332;R346

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