游离DNA能够影响胚胎质量机制的探讨

发布时间：2018-01-03 15:17

本文关键词：游离DNA能够影响胚胎质量机制的探讨　出处：《郑州大学》2016年硕士论文　论文类型：学位论文

【摘要】：随着不孕症患者的逐年增多,对于人类辅助生殖技术(ART)来说,如何提高临床妊娠率非常重要。而妊娠的达成需要较好的胚胎质量和优良的子宫环境。目前对于胚胎质量来说,较多的评价依赖于胚胎的形态学标准,然而,依靠胚胎形态学的主观观察来预测妊娠结局是受限制的。最近的许多研究都集中于研究来源于卵母细胞微环境的非侵入性生物学标志物,来提高胚胎选择的精确性。游离DNA(cf DNA),是游离核酸的一种,为双链DNA分子,且有着比基因组DNA更低的分子量,主要来源于坏死或者凋亡的进程。最早是1948年被Mendel和Metais在血浆中发现。近年来被广泛研究于肿瘤、妇产科等领域。多项研究发现血液中cf DNA水平在一些癌症和严重疾病的患者体内是升高的,基于此,cf DNA已经被用于非侵入性的生物学标志物来早期诊断和判断某些疾病的预后。而基于母体血液中胎儿cf DNA的非侵入的产前诊断检测,也构成了妇产科的一种很有前景的方法。目前,有研究发现卵泡液中的cf DNA水平与胚胎质量存在着负相关。如果卵泡液中的cf DNA含量确实与胚胎质量存在着密不可分的关系,应用其对胚胎质量进行非侵入性的预测将会是一种很有前景的方法。本研究通过对卵泡液中cf DNA含量与胚胎质量的关系探讨,首先验证了国外关于卵泡液中cf DNA水平与胚胎质量关系的研究,确实呈现负相关。进而通过cf DNA与氧化应激及凋亡的关系,进一步探讨cf DNA与胚胎质量存在关联性的原因。目的人卵泡液中cf DNA含量与胚胎质量之间是否存在相关性,并探讨cf DNA能够影响胚胎质量的原因。材料与方法1.实验材料:研究选取了从2014.09-2015.02于郑州大学第三附属医院生殖中心行新鲜周期体外受精(in vitro fertilization IVF)或卵胞浆内单精子显微注射技术(Intracytoplasmic sperm injection,ICSI)助孕治疗的189位患者,取卵日分别收集每位患者的所有卵泡液。入选患者纳入标准为:1.年龄≤40岁;2.体重指数:18-25kg/m2;3.月经周期21-35天,内分泌正常;4.未发现有卵巢或子宫器质性病变者;5.治疗方案为黄体中期短效长方案。剔除标准:女方不孕原因为多囊卵巢综合症、反复流产史、粘膜下层肌瘤,子宫粘连、子宫内膜异位症、HPV感染等的患者。2.方法:(1)分别使用酚氯仿异丙醇法(方法A)、改良酚氯仿异丙醇法(方法B)、Qiagen试剂盒法(方法C)量化其中48份卵泡液的cf DNA含量,得出最佳检测方法。余下的141份卵泡液选用最佳法进行检测;(2)在189份卵泡液中随机选取20份卵泡液,使用淋巴细胞分离液梯度分离出其中的颗粒细胞,将颗粒细胞随机分为四组,每组5例,分别加入不同浓度cf DNA进行共培养24h;(3)使用QRTPCR分别检测每组氧化应激相关因子的m RNA表达量;(4)使用Western-Blot检测加入cf DNA后不同时间段相关凋亡因子的蛋白表达量;(5)使用流式细胞仪分别检测每组的凋亡率。3.统计学方法:用SPSS17.0统计软件进行统计学分析,计数资料用t检验,计量资料用X2检验,相关分析用Spearman相关检验,P0.05为差异有统计学差异。结果1.卵泡液中cf DNA提取方法比较的结果:酚氯仿异丙醇法(方法A)、改良酚氯仿异丙醇法(方法B)、Qiagen试剂盒法(方法C)这三种方法的OD值分别为(1.72±0.06),(1.69±0.05),(1.75±0.03),没有明显差异;方法A所测得的cf DNA含量(1.518±0.095mg/ml)明显低于方法B(1.825±0.114 mg/ml)和C(1.838±0.106mg/ml),差异有统计学意义(P0.05),方法B和方法C效果相当,没有显著差异,但方法C花费较之方法B昂贵。2.优胚率与cf DNA的相关性分析:优胚率与cf DNA含量呈负相关,相关系数为-0.865,且P0.05。3.氧化应激相关因子的m RAN表达量:FOXO1、TRAIL、Caspase-3、Fas L、Fas五个因子的m RAN表达量随着培养基中cf DNA浓度的升高(从0mg/ml到6mg/ml)逐渐升高,且与对照组比较,均有统计学意义(p0.05)。4.死亡受体途径中相关凋亡因子的蛋白表达量:当培养基中cf DNA浓度为2mg/ml时,随着培养时间增加(0h、2h、4h、8h),活化的Caspase-8、Fas L、Fas的蛋白相对表达量增加,活化的Caspase-3虽然在培养时间2h时,蛋白相对表达量有稍许下降,但在随后4h、8h的检测时,其相对表达量呈上升趋势,且与对照组相比,均有统计学意义(p0.05)。5.凋亡率的比较:当在培养基中加入cf DNA浓度分别为0mg/ml、2mg/ml、4mg/ml、6mg/ml时,共培养24h后,凋亡率分别为13%、24%、34%、48%,且与对照组相比,均有统计学意义(p0.05)。结论1.改良酚氯仿异丙醇法(方法B)是三种方法中性价比最高的量化卵泡液中cf DNA的方法,而Qiagen试剂盒法是最为简单易操作的方法。2.cf DNA与优胚率负相关,可能是由于卵泡发生过程中cf DNA导致氧化应激,进一步触发颗粒细胞凋亡,使颗粒细胞凋亡率增高,卵母细胞质量下降,进而导致胚胎质量下降引起的。3.cf DNA可能是一个很有潜力的预测胚胎质量的非侵入性检测指标。
[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.

【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R714.8

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