mmu-miR-141在早孕小鼠胚胎着床前后子宫内膜中的表达及作用

发布时间：2018-03-18 21:20

本文选题：胚胎着床　切入点：子宫内膜　出处：《重庆医科大学》2012年硕士论文　论文类型：学位论文

【摘要】：胚胎能否成功着床取决于子宫内膜和胚泡间的同步协调反应。在此过程中，子宫内膜生理变化所依赖的一系列细胞因子的差异表达起着关键作用。有研究表明MicroRNAs （miRNAs）在基因差异表达的精细调控中发挥了重要作用。课题组前期应用miRNAs芯片技术对早孕小鼠胚胎着床前后子宫内膜miRNAs的表达进行研究，发现孕6天（d6）和孕4天（d4）相比mmu-miR-141明显下调。本文以mmu-miR-141为研究对象深入探讨mmu-miR-141在早孕小鼠胚胎着床前、后子宫内膜中的表达及作用，，为进一步了解胚胎着床的分子机制提供实验依据。方法 1、荧光定量PCR（FQ-PCR）验证芯片结果，原位杂交方法检测mmu-miR-141在早孕小鼠胚胎着床前、后子宫内膜组织中的定位表达； 2、MTT法检测mmu-miR-141抑制剂对小鼠子宫内膜基质细胞增殖的影响，流式细胞术检测mmu-miR-141抑制剂对小鼠子宫内膜基质细胞周期和细胞凋亡的影响； 3、应用miRNAs的靶基因预测数据库检索mmi-miR-141调控的靶基因； 4、在子宫内膜基质细胞中，分别瞬时转染mmu-miR-141的模拟物和抑制剂，并用Western Blot检测PTEN的表达； 5、于孕d2天行小鼠子宫角注射mmu-miR-141的模拟物和抑制剂，孕d7天观察并计数胚胎数。实验结果 1、FQ-PCR结果显示mmu-miR-141在着床后（d6）小鼠子宫内膜组织中的表达较着床前（d4）下调（P0.05），且与芯片结果一致； 2、原位杂交结果显示mmu-miR-141主要定位于基质细胞，在腔上皮和腺上皮中几乎没有表达。着床后（d6）表达强度较着床前（d4）降低（P0.05）； 3、瞬时转染mmu-miR-141的抑制剂作用于小鼠子宫内膜基质细胞后，MTT检测结果显示下调mmu-miR-141的表达，小鼠子宫内膜基质细胞增殖活性随之下降（P0.05）。流式细胞术检测结果显示下调mmu-miR-141表达时，细胞被阻滞在S期，且细胞凋亡率明显增加（P0.01）； 5、经生物信息学分析和综合前期的实验结果分析得到mmu-miR-141作用的靶基因有PTEN、PDCD4和KLF6，且PTEN可能性大； 4、采用mmu-miR-141的模拟物或抑制剂分别上调或下调mmu-miR-141表达时，PTEN的mRNA表达水平均不受影响。但是，当下调mmu-miR-141的表达时，PTEN蛋白表达显著增强，而上调mmu-miR-141的表达时，PTEN蛋白表达水平明显降低； 6、采用子宫角注射mmu-miR-141的抑制剂和模拟物，均导致胚胎着床数明显下降。结论 1、mmu-miR-141在早孕小鼠胚胎着床前（d4）后（d6）子宫内膜组织中存在着明显差异的表达，胚胎着床后较胚胎着床前显著降低。 2、mmu-miR-141可能通过靶向作用PTEN基因的表达来调控子宫内膜基质细胞的增殖和凋亡，从而影响小鼠胚胎的着床。
[Abstract]:The successful implantation of an embryo depends on the synchronous and coordinated response between the endometrium and the blastocyst. The differential expression of a series of cytokines dependent on the physiological changes of endometrium plays a key role. Some studies have shown that MicroRNAs miRNAsplays an important role in the fine regulation of gene differential expression. MiRNAs microarray technique was used in the early stage of the study. The expression of miRNAs in endometrium of early pregnant mice before and after implantation was studied. It was found that the expression and role of mmu-miR-141 in the endometrium before and after embryo implantation in early pregnant mice were significantly down-regulated by mmu-miR-141 compared with that on day 6 and day 4 of gestation. It provides experimental basis for further understanding the molecular mechanism of embryo implantation. Method. 1. Fluorescence quantitative PCR FQ-PCRR was used to verify the results of microarray, and in situ hybridization was used to detect the localization of mmu-miR-141 in the endometrium of early pregnant mice before and after implantation. 2the effect of mmu-miR-141 inhibitor on the proliferation of mouse endometrial stromal cells was detected by MTT assay, and the effect of mmu-miR-141 inhibitor on the cell cycle and apoptosis of mouse endometrial stromal cells was detected by flow cytometry. 3. Target genes regulated by mmi-miR-141 were searched by target gene prediction database of miRNAs. (4) in endometrial stromal cells, the mimics and inhibitors of mmu-miR-141 were transiently transfected, and the expression of PTEN was detected by Western Blot. 5. On the 2nd day of gestation, mice were injected with mmu-miR-141 mimics and inhibitors, and the number of embryos was observed and counted on the 7th day of gestation. Experimental results. 1the results of FQ-PCR showed that the expression of mmu-miR-141 in endometrial tissue of postimplantation mice was lower than that of preimplantation day 4 (P 0.05), which was consistent with the results of microarray. 2. The results of in situ hybridization showed that mmu-miR-141 was mainly localized in stromal cells, but hardly expressed in luminal epithelium and glandular epithelium. 3After the transient transfection of mmu-miR-141 inhibitor on mouse endometrial stromal cells, the expression of mmu-miR-141 was down-regulated, and the proliferative activity of mouse endometrial stromal cells decreased (P0.05N). Flow cytometry showed that the expression of mmu-miR-141 was down-regulated. The cells were blocked in S phase and the apoptosis rate was increased significantly (P 0.01). 5. Through bioinformatics analysis and comprehensive analysis of experimental results, we found that the target genes of mmu-miR-141 were PTEN PDCD4 and KLF6, and the possibility of PTEN was high. (4) the expression level of mRNA was not affected when mmu-miR-141 was up-regulated or down-regulated by mmu-miR-141 mimics or inhibitors respectively. However, the expression of mmu-miR-141 protein increased significantly when the expression of mmu-miR-141 was down-regulated, but the expression of mmu-miR-141 protein decreased significantly when the expression of mmu-miR-141 was upregulated. 6. Injection of mmu-miR-141 inhibitor and analogue into uterine horn resulted in a significant decrease in the number of embryo implantation. Conclusion. 1the expression of mmu-miR-141 in endometrium of early pregnant mice was significantly lower than that before implantation, and the expression of mmu-miR-141 in endometrium was significantly lower than that before implantation. 2mmu-miR-141 may regulate the proliferation and apoptosis of endometrial stromal cells by targeting the expression of PTEN gene, thus affecting the implantation of mouse embryos.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R321

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