子宫内膜和单个胚胎FUT基因表达的定量分析及LIF因子对FUT7表达调控作用的研究

发布时间：2018-05-13 19:00

本文选题：Real-Time + PCR　；参考：《大连医科大学》2005年硕士论文

【摘要】：岩藻糖基转移酶(fucosyltransferases,FUTs),是一类参与岩藻化寡糖合成的关键酶,研究表明 LeY,sLex和 Lex等岩藻化寡糖的阶段特异性表达在胚胎发育和着床过程起十分重要的作用。因此,对岩藻糖基转移酶表达的分析和研究不仅有助于探讨寡糖在着床过程中的作用机制,并为对着床过程进行干预提供了新的思路和手段。选择适当的,高灵敏度的检测方法是基因表达定量分析的关键,本文通过琼脂糖凝胶电泳,微流控芯片,荧光定量 PCR 等定量方法的比较,选择 Real-Time PCR 对小鼠受孕早期胚胎、子宫内膜 FUT2 基因和胚胎 FUTs(FUT1, FUT4, FUT7, FUT8,FUT9)基因表达进行定量分析。白血病抑制因子(LIF)是一类具有广泛生物学功能的分泌型糖蛋白,已被证实是胚胎着床必需的重要细胞因子,已知其对着床网络中的多种因子(如基质金属蛋白酶 MMPs,表皮生长因子 EGF 等)均有调节作用,但其对岩藻糖基转移酶的调控机理尚不清楚。本文主要研究了LIF 因子对 FUT7 的影响作用。本论文的具体方法和结果如下: (一)收集小鼠未孕及孕 D4 天子宫内膜,应用 RT-PCR 技术进行 2 FUT2 基因和内参 GAPDH 基因的扩增,对不同循环数的 PCR 扩增产物进行琼脂糖凝胶电泳和微流控芯片技术检测,同时应用 Real-Time PCR 技术对上述基因进行分析,并比较三种方法的灵敏度和最低检测限度。结果显示,FUT2 基因琼脂糖凝胶电泳可检测最低限度为 25 个循环的 PCR 扩增产物,而 20 个循环扩增的产物无法检测到;微流控芯片技术可以检测到稀释 5 倍的经 20 个循环扩增的 PCR 产物,其灵敏度提高 103 倍。Real-Time PCR 有较高灵敏度和特异性,对于单胚胎的基因扩增可在 18.88 个循环检测到产物,并可实时监测,准确定量,在重复性和稳定性有明显优势。 (二)应用 Real-Time PCR 技术对小鼠受孕不同天数子宫内膜和不同发育时期单个胚胎 FUT2 基因的表达进行定量分析,并改进了单胚胎基因定量分析方法。结果表明,Real-Time PCR 方法可以准确的对目的基因进行定量,灵敏度高,对 2-cell 期的单个胚胎的基因表达即可进行检测。 FUT2 基因在受孕子宫内膜和胚胎均有表达,但孕 D4 天子宫内膜和胚胎都有降低趋势,子宫内膜拷贝数约从 1011降至 1010个,胚胎拷贝数约从 108降至 107个。结果尚提示 FUT2 基因可能与着床前胚胎的发育成熟及子宫内膜接受态的建立有关,但具体机制尚不清楚,其基因表达量的改变为进一步开展其功能研究提供了一定的实验依据。 (三)对小鼠不同发育时期单个胚胎表达 FUT 基因家族(FUT1, FUT4, FUT7, FUT8,FUT9)进行 Real-Time PCR 定量分析。结果显示:在发育不同时期 FUT1 基因, FUT4 基因和 FUT7 基因的表达逐渐升高,到桑椹期达到高峰并持续至囊胚期;FUT8 基因在各发育时期的表达相对稳定,无明显变化趋势;FUT9 基因在 8-cell 高表达,桑椹期和囊胚期开始降低。FUT 基因家族各时期表达量及变化趋势各不相同, 但却和各自合成的相关寡糖抗原变化趋势一致,提示对 FUT 基因选择性调控可以影响相映寡糖的合成和表达,由此影响胚胎的发育。 (四)应用 PCR、间接免疫荧光以及 Dot-blot 等分析方法分别研究了 LIF 因子对体外培养胚胎和子宫内膜细胞 FUT7 基因表达和酶蛋白分泌以及胚胎培养液中其产物 sLeX寡糖分泌的影响。胚胎和细胞分别在含 LIF 因子(0.1,1,10,100ng/ml)或 LIF-Ab (3μg/ml)的培养
[Abstract]:Fucosyltransferases (FUTs), a class of key enzymes involved in the synthesis of glycosylated oligosaccharides, shows that the specific expression of LeY, sLex and Lex oligosaccharide oligosaccharides plays an important role in embryo development and implantation. Therefore, the analysis and study of the expression of fucosyltransferase is not only helpful to explore the expression of fucosyltransferase. The mechanism of oligosaccharide in implantation process provides new ideas and means for intervening in the process of bed.
Selecting appropriate, highly sensitive detection methods is the key to quantitative analysis of gene expression. By comparing the quantitative methods such as agarose gel electrophoresis, microfluidic chip and fluorescence quantitative PCR, Real-Time PCR is selected for the gene table of early pregnancy, FUT2 gene of endometrium and FUTs (FUT1, FUT4, FUT7, FUT8, FUT9) in mice. Quantitative analysis is carried out.
Leukemic inhibitory factor (LIF) is a kind of secretory glycoprotein with extensive biological functions. It has been proved to be an important cytokine which is essential for embryo implantation. It is known to regulate a variety of factors in the bed network, such as matrix metalloproteinase MMPs, epidermal growth factor EGF and so on, but it regulates the fucoidase. The mechanism is not clear yet. This paper mainly studies the influence of LIF factor on FUT7.
The specific methods and results of this paper are as follows:
(1) collecting the endometrium of unpregnant mice and D4 days of pregnancy, using RT-PCR technology.
Two
Amplification of the FUT2 gene and the GAPDH gene of the internal reference gene were used to expand the yield of PCR with different cycle numbers.
The detection was performed by agarose gel electrophoresis and microfluidic chip technology, while Real-Time PCR was applied.
The above genes were analyzed by the technology, and the sensitivity and minimum detection limit of the three methods were compared.
The results showed that the FUT2 gene could be detected by agarose gel electrophoresis with a minimum of 25 cycles.
PCR amplification products, while 20 loop amplification products were not detected; microfluidic chip technology.
It is possible to detect 5 times diluted PCR products amplified by 20 cycles, and their sensitivity is increased by 103.
Double.Real-Time PCR has higher sensitivity and specificity, and can be used for gene amplification of single embryo.
The products are detected in 18.88 cycles, and can be monitored in real time, accurately and quantitatively, in repeatability and
Stability has obvious advantages.
(two) application of Real-Time PCR technology to mouse endometrium with different days of conception.
The expression of FUT2 gene in single embryo at different developmental stages was quantitatively analyzed, and single embryo was improved.
Quantitative analysis of fetal genes. The results show that the Real-Time PCR method is accurate.
The gene is quantified and highly sensitive to gene expression in 2-cell stage single embryos.
The FUT2 gene was expressed in the endometrium and embryo of pregnancy, but D4 was born in pregnancy.
The number of endometrium copy decreased from 1011 to 1010 embryos.
The number of fetal copies decreased from 108 to 107. The results suggest that the FUT2 gene may be associated with preimplantation embryos.
The development and maturation of endometrium are related to the establishment of endometrial receptivity, but the specific mechanism is not yet clear.
The change of gene expression provides some experimental evidence for further research on its function.
(three) expression of FUT gene family (FUT1) in mouse embryos at different developmental stages.
FUT4, FUT7, FUT8, FUT9) conducted quantitative analysis of Real-Time PCR.
Expression of FUT1 gene, FUT4 gene and FUT7 gene at different developmental stages
The peak of FUT8 gene reached its peak in the mulberry stage and continued to blastocyst stage.
The expression of FUT9 gene was relatively stable, and there was no obvious change trend; the expression of 8-cell gene was high in the mulberry stage.
And the blastocyst stage began to decrease, and the expression and variation trend of.FUT gene family were different.
However, the change trend of the oligosaccharide antigen was consistent with each other, suggesting the selection of FUT gene.
Sexual regulation can affect the synthesis and expression of the corresponding oligosaccharides, thereby affecting the development of embryos.
(four) applied PCR, indirect immunofluorescence and Dot-blot analysis methods respectively.
The expression of FUT7 gene and enzyme protein in embryo and endometrial cells were studied by LIF factor in vitro.
Secretion and the effect of sLeX oligosaccharide secretion in embryo culture medium.
Culture in the presence of LIF factor (0.1,1,10100ng/ml) or LIF-Ab (3 g/ml)

【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：Q789

【参考文献】