本文關(guān)鍵詞：游離DNA能夠影響胚胎質(zhì)量機(jī)制的探討　出處：《鄭州大學(xué)》2016年碩士論文　論文類(lèi)型：學(xué)位論文

  更多相關(guān)文章： 卵泡液 胚胎質(zhì)量 顆粒細(xì)胞 氧化應(yīng)激 凋亡

【摘要】：隨著不孕癥患者的逐年增多,對(duì)于人類(lèi)輔助生殖技術(shù)(ART)來(lái)說(shuō),如何提高臨床妊娠率非常重要。而妊娠的達(dá)成需要較好的胚胎質(zhì)量和優(yōu)良的子宮環(huán)境。目前對(duì)于胚胎質(zhì)量來(lái)說(shuō),較多的評(píng)價(jià)依賴(lài)于胚胎的形態(tài)學(xué)標(biāo)準(zhǔn),然而,依靠胚胎形態(tài)學(xué)的主觀(guān)觀(guān)察來(lái)預(yù)測(cè)妊娠結(jié)局是受限制的。最近的許多研究都集中于研究來(lái)源于卵母細(xì)胞微環(huán)境的非侵入性生物學(xué)標(biāo)志物,來(lái)提高胚胎選擇的精確性。游離DNA(cf DNA),是游離核酸的一種,為雙鏈DNA分子,且有著比基因組DNA更低的分子量,主要來(lái)源于壞死或者凋亡的進(jìn)程。最早是1948年被Mendel和Metais在血漿中發(fā)現(xiàn)。近年來(lái)被廣泛研究于腫瘤、婦產(chǎn)科等領(lǐng)域。多項(xiàng)研究發(fā)現(xiàn)血液中cf DNA水平在一些癌癥和嚴(yán)重疾病的患者體內(nèi)是升高的,基于此,cf DNA已經(jīng)被用于非侵入性的生物學(xué)標(biāo)志物來(lái)早期診斷和判斷某些疾病的預(yù)后。而基于母體血液中胎兒cf DNA的非侵入的產(chǎn)前診斷檢測(cè),也構(gòu)成了婦產(chǎn)科的一種很有前景的方法。目前,有研究發(fā)現(xiàn)卵泡液中的cf DNA水平與胚胎質(zhì)量存在著負(fù)相關(guān)。如果卵泡液中的cf DNA含量確實(shí)與胚胎質(zhì)量存在著密不可分的關(guān)系,應(yīng)用其對(duì)胚胎質(zhì)量進(jìn)行非侵入性的預(yù)測(cè)將會(huì)是一種很有前景的方法。本研究通過(guò)對(duì)卵泡液中cf DNA含量與胚胎質(zhì)量的關(guān)系探討,首先驗(yàn)證了國(guó)外關(guān)于卵泡液中cf DNA水平與胚胎質(zhì)量關(guān)系的研究,確實(shí)呈現(xiàn)負(fù)相關(guān)。進(jìn)而通過(guò)cf DNA與氧化應(yīng)激及凋亡的關(guān)系,進(jìn)一步探討cf DNA與胚胎質(zhì)量存在關(guān)聯(lián)性的原因。目的人卵泡液中cf DNA含量與胚胎質(zhì)量之間是否存在相關(guān)性,并探討cf DNA能夠影響胚胎質(zhì)量的原因。材料與方法1.實(shí)驗(yàn)材料:研究選取了從2014.09-2015.02于鄭州大學(xué)第三附屬醫(yī)院生殖中心行新鮮周期體外受精(in vitro fertilization IVF)或卵胞漿內(nèi)單精子顯微注射技術(shù)(Intracytoplasmic sperm injection,ICSI)助孕治療的189位患者,取卵日分別收集每位患者的所有卵泡液。入選患者納入標(biāo)準(zhǔn)為:1.年齡≤40歲;2.體重指數(shù):18-25kg/m2;3.月經(jīng)周期21-35天,內(nèi)分泌正常;4.未發(fā)現(xiàn)有卵巢或子宮器質(zhì)性病變者;5.治療方案為黃體中期短效長(zhǎng)方案。剔除標(biāo)準(zhǔn):女方不孕原因?yàn)槎嗄衣殉簿C合癥、反復(fù)流產(chǎn)史、粘膜下層肌瘤,子宮粘連、子宮內(nèi)膜異位癥、HPV感染等的患者。2.方法:(1)分別使用酚氯仿異丙醇法(方法A)、改良酚氯仿異丙醇法(方法B)、Qiagen試劑盒法(方法C)量化其中48份卵泡液的cf DNA含量,得出最佳檢測(cè)方法。余下的141份卵泡液選用最佳法進(jìn)行檢測(cè);(2)在189份卵泡液中隨機(jī)選取20份卵泡液,使用淋巴細(xì)胞分離液梯度分離出其中的顆粒細(xì)胞,將顆粒細(xì)胞隨機(jī)分為四組,每組5例,分別加入不同濃度cf DNA進(jìn)行共培養(yǎng)24h;(3)使用QRTPCR分別檢測(cè)每組氧化應(yīng)激相關(guān)因子的m RNA表達(dá)量;(4)使用Western-Blot檢測(cè)加入cf DNA后不同時(shí)間段相關(guān)凋亡因子的蛋白表達(dá)量;(5)使用流式細(xì)胞儀分別檢測(cè)每組的凋亡率。3.統(tǒng)計(jì)學(xué)方法:用SPSS17.0統(tǒng)計(jì)軟件進(jìn)行統(tǒng)計(jì)學(xué)分析,計(jì)數(shù)資料用t檢驗(yàn),計(jì)量資料用X2檢驗(yàn),相關(guān)分析用Spearman相關(guān)檢驗(yàn),P0.05為差異有統(tǒng)計(jì)學(xué)差異。結(jié)果1.卵泡液中cf DNA提取方法比較的結(jié)果:酚氯仿異丙醇法(方法A)、改良酚氯仿異丙醇法(方法B)、Qiagen試劑盒法(方法C)這三種方法的OD值分別為(1.72±0.06),(1.69±0.05),(1.75±0.03),沒(méi)有明顯差異;方法A所測(cè)得的cf DNA含量(1.518±0.095mg/ml)明顯低于方法B(1.825±0.114 mg/ml)和C(1.838±0.106mg/ml),差異有統(tǒng)計(jì)學(xué)意義(P0.05),方法B和方法C效果相當(dāng),沒(méi)有顯著差異,但方法C花費(fèi)較之方法B昂貴。2.優(yōu)胚率與cf DNA的相關(guān)性分析:優(yōu)胚率與cf DNA含量呈負(fù)相關(guān),相關(guān)系數(shù)為-0.865,且P0.05。3.氧化應(yīng)激相關(guān)因子的m RAN表達(dá)量:FOXO1、TRAIL、Caspase-3、Fas L、Fas五個(gè)因子的m RAN表達(dá)量隨著培養(yǎng)基中cf DNA濃度的升高(從0mg/ml到6mg/ml)逐漸升高,且與對(duì)照組比較,均有統(tǒng)計(jì)學(xué)意義(p0.05)。4.死亡受體途徑中相關(guān)凋亡因子的蛋白表達(dá)量:當(dāng)培養(yǎng)基中cf DNA濃度為2mg/ml時(shí),隨著培養(yǎng)時(shí)間增加(0h、2h、4h、8h),活化的Caspase-8、Fas L、Fas的蛋白相對(duì)表達(dá)量增加,活化的Caspase-3雖然在培養(yǎng)時(shí)間2h時(shí),蛋白相對(duì)表達(dá)量有稍許下降,但在隨后4h、8h的檢測(cè)時(shí),其相對(duì)表達(dá)量呈上升趨勢(shì),且與對(duì)照組相比,均有統(tǒng)計(jì)學(xué)意義(p0.05)。5.凋亡率的比較:當(dāng)在培養(yǎng)基中加入cf DNA濃度分別為0mg/ml、2mg/ml、4mg/ml、6mg/ml時(shí),共培養(yǎng)24h后,凋亡率分別為13%、24%、34%、48%,且與對(duì)照組相比,均有統(tǒng)計(jì)學(xué)意義(p0.05)。結(jié)論1.改良酚氯仿異丙醇法(方法B)是三種方法中性?xún)r(jià)比最高的量化卵泡液中cf DNA的方法,而Qiagen試劑盒法是最為簡(jiǎn)單易操作的方法。2.cf DNA與優(yōu)胚率負(fù)相關(guān),可能是由于卵泡發(fā)生過(guò)程中cf DNA導(dǎo)致氧化應(yīng)激,進(jìn)一步觸發(fā)顆粒細(xì)胞凋亡,使顆粒細(xì)胞凋亡率增高,卵母細(xì)胞質(zhì)量下降,進(jìn)而導(dǎo)致胚胎質(zhì)量下降引起的。3.cf DNA可能是一個(gè)很有潛力的預(yù)測(cè)胚胎質(zhì)量的非侵入性檢測(cè)指標(biāo)。
[Abstract]:With infertility increased year by year, for human assisted reproductive technology (ART), how to improve the clinical pregnancy rate and pregnancy is very important. A need for good embryo quality and excellent environment. The quality of embryo uterine, morphologic criteria, more evaluation depends on the embryo however, rely on subjective observation of embryo morphology to predict the outcome of pregnancy is limited. Many recent studies have focused on the study from the oocyte microenvironment noninvasive biomarkers to improve the accuracy of embryo selection. Free DNA (CF DNA), is a kind of free nucleic acid, as a double stranded DNA molecule, and compared with the molecular genomic DNA lower, mainly from necrosis or apoptosis process. The first is 1948 Mendel and Metais found in the plasma. In recent years has been widely studied in obstetrics and gynecology tumor, several research fields. The CF DNA level in the blood is elevated, in some serious diseases in patients with cancer and based on this, CF DNA has been used for non invasive biological markers for the early diagnosis and judging the prognosis of the disease. But some non-invasive prenatal diagnosis of fetal CF in maternal blood detection based on DNA, which a promising method of Obstetrics and gynecology. At present, studies have found that CF DNA levels in follicular fluid and the embryo quality. If there is a negative correlation between the CF content of DNA in follicular fluid and the embryo quality indeed there is a close relationship, the application of non invasive embryo quality predicting is a a very promising approach. This study explored the relationship between follicular fluid CF DNA contents and the quality of embryos, the first to verify the research on the relationship between CF DNA level and the quality of embryos in follicular fluid abroad, does show a negative phase Then through CF DNA. The relationship with oxidative stress and apoptosis, to further explore the reason for the existence of the relevance of the CF DNA and the quality of embryos. Whether there is a correlation between the embryo and the content of DNA and CF in follicular fluid quality objective, and to explore CF DNA can affect the quality of embryo. Materials and methods 1. experimental materials: study from 2014.09-2015.02 in the reproductive center of the Third Affiliated Hospital of Zhengzhou University, the fresh cycle in vitro fertilization (in vitro fertilization IVF) or intracytoplasmic sperm injection (Intracytoplasmic sperm injection, ICSI) 189 infertile patients, all follicular fluid on the day of oocyte retrieval were collected from each patient. Patients were included: 1. aged less than 40 years; 2. body mass index: 18-25kg/m2; 3. 21-35 days of menstrual cycle, endocrine normal; 4. found no ovarian or uterine lesions; 5. treatment regimens of corpus luteum Mid short acting rectangular case. Exclusion criteria: the causes of infertility of polycystic ovary syndrome, recurrent abortion, submucosa myoma, uterine adhesions, endometriosis,.2. in patients with HPV infection methods: (1) using phenol chloroform isopropanol (A method), modified phenol chloroform isopropanol method (method B), Qiagen Kit Method (C method) to quantify 48 follicular fluid CF DNA contents, the optimum detection method. 141 samples of follicular fluid remaining the best method; (2) randomly selected 20 samples in 189 samples of follicular fluid in follicular fluid using lymphocyte separation liquid gradient isolated granulosa cells among them, the granulosa cells were randomly divided into four groups, 5 cases in each group, respectively with different concentrations of CF DNA were co cultured with 24h; (3) the use of QRTPCR m RNA were detected in each group of oxidative stress related factor expression; (4) using the Western-Blot assay at different time after adding CF DNA The expression of apoptosis related protein; (5) using the.3. statistical method in each group the apoptosis rate were detected by flow cytometry: statistical analysis was performed using SPSS17.0 statistical software, count data using t test, measurement data using X2 test, correlation analysis using Spearman correlation test, P0.05 was a statistically significant difference. The results of comparison of extraction methods 1. CF in follicular fluid DNA results: phenol chloroform isopropanol (A method), modified phenol chloroform isopropanol (B), Qiagen Kit Method (C method) the OD value of the three methods respectively (1.72 + 0.06), (1.69 + 0.05), (1.75 + 0.03). There was no significant difference between A measured by CF method; the content of DNA (1.518 + 0.095mg/ml) was significantly lower than that of B (1.825 + 0.114 mg/ml) and C (1.838 + 0.106mg/ml), the difference was statistically significant (P0.05), a method of B and C, there is no significant difference, but C costs compared with method of B expensive.2. embryo The rate of correlation with CF DNA analysis: excellent embryo rate was negatively correlated with CF content of DNA, the correlation coefficient is -0.865, P0.05.3. and oxidative stress related factors of M RAN expression: FOXO1, TRAIL, Caspase-3, Fas, L, Fas five factor M RAN expression with CF DNA concentration in medium high rise (from 0mg/ml to 6mg/ml) increased gradually, and compared with the control group, there was statistically significant (P0.05).4. death receptor pathway in apoptosis related protein expression: when cultured CF DNA concentration in medium was 2mg/ml, as the culture time increased (0h, 2h, 4h, 8h), activated Caspase-8, Fas L, the relative expression of Fas protein increased, the activation of Caspase-3 in the training time of 2h, the relative expression amount slightly decreased, but in the following 4h, 8h detection, the relative expression increased, and compared with the control group, were statistically significant (P0.05) compared the apoptosis rate of.5. when: The medium added CF DNA concentration were 0mg/ml, 2mg/ml, 4mg/ml, 6mg/ml, 24h after co culture, the apoptosis rates were 13%, 24%, 34%, 48%, and compared with the control group, there was statistically significant (P0.05). Conclusion 1. modified phenol chloroform isopropanol (B) is three quantification of follicular fluid in the highest price method in CF DNA, and Qiagen kit method is the most simple and easy operation method of.2.cf DNA with excellent embryo rate negative correlation may be due to folliculogenesis in CF DNA induced oxidative stress, triggering further granulosa cell apoptosis, granulosa cell apoptosis rate increased, decreased egg oocyte quality, leading to the decline in the quality of.3.cf embryos induced by DNA may be an embryo quality prediction promising non-invasive detection index.

【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：R714.8

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8 第四軍醫(yī)大學(xué)基礎(chǔ)醫(yī)學(xué)部生物化學(xué)與分子生物學(xué)教研室教授 李福洋;破除法老DNA的咒語(yǔ)[N];東方早報(bào);2011年

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