本文選題：Real-Time + PCR��； 參考：《大連醫(yī)科大學(xué)》2005年碩士論文

【摘要】：巖藻糖基轉(zhuǎn)移酶(fucosyltransferases,FUTs),是一類參與巖藻化寡糖合成的關(guān)鍵酶,研究表明 LeY,sLex和 Lex等巖藻化寡糖的階段特異性表達(dá)在胚胎發(fā)育和著床過程起十分重要的作用。因此,對巖藻糖基轉(zhuǎn)移酶表達(dá)的分析和研究不僅有助于探討寡糖在著床過程中的作用機(jī)制,并為對著床過程進(jìn)行干預(yù)提供了新的思路和手段。 選擇適當(dāng)?shù)?高靈敏度的檢測方法是基因表達(dá)定量分析的關(guān)鍵,本文通過瓊脂糖凝膠電泳,微流控芯片,熒光定量 PCR 等定量方法的比較,選擇 Real-Time PCR 對小鼠受孕早期胚胎、子宮內(nèi)膜 FUT2 基因和胚胎 FUTs(FUT1, FUT4, FUT7, FUT8,FUT9)基因表達(dá)進(jìn)行定量分析。 白血病抑制因子(LIF)是一類具有廣泛生物學(xué)功能的分泌型糖蛋白,已被證實(shí)是胚胎著床必需的重要細(xì)胞因子,已知其對著床網(wǎng)絡(luò)中的多種因子(如基質(zhì)金屬蛋白酶 MMPs,表皮生長因子 EGF 等)均有調(diào)節(jié)作用,但其對巖藻糖基轉(zhuǎn)移酶的調(diào)控機(jī)理尚不清楚。本文主要研究了LIF 因子對 FUT7 的影響作用。 本論文的具體方法和結(jié)果如下: (一)收集小鼠未孕及孕 D4 天子宮內(nèi)膜,應(yīng)用 RT-PCR 技術(shù)進(jìn)行 2 FUT2 基因和內(nèi)參 GAPDH 基因的擴(kuò)增,對不同循環(huán)數(shù)的 PCR 擴(kuò)增產(chǎn)物進(jìn) 行瓊脂糖凝膠電泳和微流控芯片技術(shù)檢測,同時應(yīng)用 Real-Time PCR 技術(shù)對上述基因進(jìn)行分析,并比較三種方法的靈敏度和最低檢測限度。 結(jié)果顯示,FUT2 基因瓊脂糖凝膠電泳可檢測最低限度為 25 個循環(huán)的 PCR 擴(kuò)增產(chǎn)物,而 20 個循環(huán)擴(kuò)增的產(chǎn)物無法檢測到;微流控芯片技術(shù) 可以檢測到稀釋 5 倍的經(jīng) 20 個循環(huán)擴(kuò)增的 PCR 產(chǎn)物,其靈敏度提高 103 倍。Real-Time PCR 有較高靈敏度和特異性,對于單胚胎的基因擴(kuò)增可 在 18.88 個循環(huán)檢測到產(chǎn)物,并可實(shí)時監(jiān)測,準(zhǔn)確定量,在重復(fù)性和 穩(wěn)定性有明顯優(yōu)勢。 (二)應(yīng)用 Real-Time PCR 技術(shù)對小鼠受孕不同天數(shù)子宮內(nèi)膜和 不同發(fā)育時期單個胚胎 FUT2 基因的表達(dá)進(jìn)行定量分析,并改進(jìn)了單胚 胎基因定量分析方法。結(jié)果表明,Real-Time PCR 方法可以準(zhǔn)確的對目 的基因進(jìn)行定量,靈敏度高,對 2-cell 期的單個胚胎的基因表達(dá)即可 進(jìn)行檢測。 FUT2 基因在受孕子宮內(nèi)膜和胚胎均有表達(dá),但孕 D4 天子 宮內(nèi)膜和胚胎都有降低趨勢,子宮內(nèi)膜拷貝數(shù)約從 1011降至 1010個,胚 胎拷貝數(shù)約從 108降至 107個。結(jié)果尚提示 FUT2 基因可能與著床前胚胎 的發(fā)育成熟及子宮內(nèi)膜接受態(tài)的建立有關(guān),但具體機(jī)制尚不清楚,其 基因表達(dá)量的改變?yōu)檫M(jìn)一步開展其功能研究提供了一定的實(shí)驗依據(jù)。 (三)對小鼠不同發(fā)育時期單個胚胎表達(dá) FUT 基因家族(FUT1, FUT4, FUT7, FUT8,FUT9)進(jìn)行 Real-Time PCR 定量分析。結(jié)果顯 示:在發(fā)育不同時期 FUT1 基因, FUT4 基因 和 FUT7 基因的表達(dá)逐漸 升高,到桑椹期達(dá)到高峰并持續(xù)至囊胚期;FUT8 基因在各發(fā)育時期的 表達(dá)相對穩(wěn)定,無明顯變化趨勢;FUT9 基因在 8-cell 高表達(dá),桑椹期 和囊胚期開始降低。FUT 基因家族各時期表達(dá)量及變化趨勢各不相同, 但卻和各自合成的相關(guān)寡糖抗原變化趨勢一致,提示對 FUT 基因選擇 性調(diào)控可以影響相映寡糖的合成和表達(dá),由此影響胚胎的發(fā)育。 (四)應(yīng)用 PCR、間接免疫熒光以及 Dot-blot 等分析方法分別研 究了 LIF 因子對體外培養(yǎng)胚胎和子宮內(nèi)膜細(xì)胞 FUT7 基因表達(dá)和酶蛋白 分泌以及胚胎培養(yǎng)液中其產(chǎn)物 sLeX寡糖分泌的影響。胚胎和細(xì)胞分別 在含 LIF 因子(0.1,1,10,100ng/ml)或 LIF-Ab (3μg/ml)的培養(yǎng)
[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)

【學(xué)位授予單位】：大連醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2005
【分類號】：Q789

【參考文獻(xiàn)】

相關(guān)期刊論文 前2條

1 陳繼華,陳惠黎;α1,3-巖藻糖基轉(zhuǎn)移酶及其相關(guān)產(chǎn)物[J];生命的化學(xué)(中國生物化學(xué)會通訊);1997年04期

2 張煒,周劍萍,劉銀坤,張俊慧;白血病抑制因子在小鼠圍著床期子宮內(nèi)膜的表達(dá)[J];生殖醫(yī)學(xué)雜志;2000年02期

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本文編號：1884408