1.呼吸率在预测D3 FET胚胎发育潜能中的应用 2.D-半乳糖诱导大鼠骨髓基质细胞衰老及其机制

发布时间：2018-05-11 09:45

本文选题：呼吸率 + 非倾入性测量　；参考：《重庆医科大学》2017年硕士论文

【摘要】：研究背景:人类辅助生殖技术(Assisted Reproductive Technology ART)的特点之一就是妊娠率在不同病人之间变化非常大,这种变化很大程度上是由于现在常用的可观测参数无法全面表现出胚胎的发育潜能。形态学一直是评估胚胎最常用也较为稳定的方法,从光学显微镜下获得的关于胚胎的信息虽然已被证实和IVF(In vitro fertilization IVF)的妊娠结局直接相关。但人们发现,即使评分相近的同父母来源的胚胎也可能有完全不同的妊娠结局。这说明胚胎形态并不能完全反应它的种植潜能。胚胎的氧耗量即胚胎的呼吸率,由于线粒体是产生三磷酸腺苷(Adenosine Triphosphate ATP)的主要细胞器,故胚胎的氧耗量与线粒体活动密切相关,测量胚胎的呼吸率可以在一定程度上反应胚胎线粒体的功能。胚胎的线粒体完全来源于卵母细胞,而卵母细胞质量对胚胎活力有很大的影响。呼吸率能够反应胚胎中线粒体的活性,从而间接反应胚胎的活力。胚胎呼吸率测量是一种非侵入性的方法,能够安全快捷测量活胚胎的活力。该方法的基础是胚胎消耗氧离子形成球形阳离子扩散,以精确到纳安级别的铂金微电极测量胚胎周围由于氧的扩散而引起的电流变化,通过电流变化反映胚胎的耗氧量(即胚胎呼吸率)。本文将非侵入性的呼吸率测量结合传统的形态学评分回顾性分析了解冻移植周期(Frozen-thawed embryo transfer FET)胚胎的氧耗量及其临床妊娠结局,观察呼吸率与临床妊娠结局之间的关系。目的:使用细胞呼吸率测量系统测量胚胎呼吸率,随访移植后病人胚胎着床情况及妊娠结局,探讨胚胎呼吸率测量能否反应胚胎发育潜能。研究对象和方法:1.入组标准及分组实验共分为三部分进行,第一部分为FET周期双胚胎移植组。该组选择FET周期患者,研究人群为:女方年龄≤35岁,排除遗传性疾病,合并卵巢和输卵管因素的不孕以及子宫因素的不孕;无输卵管积液,男方精子正常。胚胎选择IVF授精。胚胎选择基于形态学标准,胚胎质量为优于8细胞3级的可移植胚胎。实验人群按呼吸率分为三组,A组:两枚胚胎呼吸率都达到或高于3.0 fmol/s,B组:移植的两枚胚胎中只有一枚呼吸率达到3.0 fmol/s,C组:两枚胚胎呼吸率均小于3.0 fmol/s。第二部分为FET周期单胚胎移植组,该组患者纳入标准同第一部分,但移植单胚胎。胚胎根据呼吸率分为两组;A组:胚胎呼吸率小于3.0 fmol/s,B组:移植的胚胎呼吸率大于等于3.0 fmol/s。第三部分为Time-Lapse结合呼吸率选择胚胎组。该组患者纳入标准为:年龄≤35岁,月经正常,BMI:18-25,获卵数:5-15枚,采用ICSI(Intracytoplasmic sperm injection ICSI)授精。患者采用标准长方案促排卵,此次治疗为患者的首次助孕。不孕因素为单纯输卵管不孕,排除遗传性疾病,合并卵巢和输卵管因素的不孕和子宫因素的不孕。胚胎质量为优于8细胞的3级可移植胚胎。。实验分为三组,A组为形态学选择二细胞开始时间T2cb26.725 h的胚胎,B组为呼吸率大于等于3.0 fmol/s的胚胎,C组为同时满足T2cb26.725 h和呼吸率大于等于3.0 fmol/s的胚胎。三个部分的患者的胚胎呼吸率测量均于移植前进行2.研究方法(1)纳入标准的患者于周期前签署呼吸率测量相关的知情同意书。(2)于移植前进行呼吸率测量,采用SECM进行呼吸率的测量,测量方法为:1.将移植前的胚胎置于充满人输卵管缓冲液的测量槽内。测量槽为上宽下窄的圆锥形,胚胎自然沉积到底部。2.随着胚胎代谢耗氧,缓冲液顶部至底部出现递减的氧浓度差。3.调节参数后,将特殊的微电极沿垂直方向自上而下地扫描缓冲液内的电位差。在系统内转化为胚胎的呼吸率。4.记录各枚胚胎的呼吸率。全部操作在尽量短的时间内完成。(5)B超监测种植率,电话随访流产率、临床妊娠率、活产率并分析三组之间的差异。结果:FET双胚胎移植组:共测量131个周期,对共262枚胚胎进行了呼吸率测量。比较三组间年龄,BMI,子宫内膜厚度等基本情况,统计并分析了三组间种植率,临床妊娠率,流产率和活胎分娩率等妊娠结局的差异。其中A组共测量83个周期,166枚胚胎,种植率48.19%,妊娠率65.06%,流产率4.82%,活胎分娩率60.24%;B组包含36个周期,72枚胚胎,种植率43.05%,妊娠率58.33%,流产率19.4%,活胎分娩率38.89%;C组有12个周期,共测量24枚胚胎,其中种植率37.5%,妊娠率50%,流产率16.67%,活胎分娩率33.33%。统计学分析发现,三组患者年龄,BMI及子宫内膜厚度等基本情况间不存在统计学差异(P0.05)。A组拥有更低的流产率及更高的活胎分娩率,且相较于B组和C组存在明显的统计学差异(P0.05)FET周期单胚胎移植组:共移植58枚胚胎,其中呼吸率大于3.0的23枚,临床妊娠率43.48%。呼吸率小于等于3.0的35枚,临床妊娠率54.29%。两组妊娠率不存在统计学差异,但移植呼吸率大于3.0胚胎组临床妊娠率有所提高。Time-Lapse联合呼吸率组:共移植35枚胚胎,A组单纯形态学选择,移植13枚胚胎,种植率46.15%。B组单纯选择大于3.0胚胎,移植15枚,种植率53.33%。C组呼吸率联合形态学,移植7枚,种植率57.14%。提示满足形态学和呼吸率两个条件的胚胎种植率有所增加,但不存在统计学差异。结论:呼吸率测量在预测胚胎发育潜能方面还存在缺陷,但是当呼吸率大于等于3.0 fmol/s时,胚胎移植后患者的妊娠结局有所改善。提示胚胎呼吸率可以作为胚胎选择的辅助参数。研究背景:造血微环境(hematopoietic inductive microenvironment,HIM)是造血干细胞(Haemopoietic stem cells,HSCs)赖以生长发育和增殖分化的场所【1】,其结构和功能完整是维系正常造血功能的重要因素。造血基质细胞(bone marrow stromal cells,BMSCs)作为HIM的核心组分,不仅通过形成HSCs生长发育的“龛”、分泌造血生长因子和细胞外基质等方式支持和调控HSCs自我更新与分化,还与多种血液系统疾病的发生、发展和预后密切相关,因此骨髓基质细胞成为研究HIM结构与功能最为重要的研究对象。课题组前期研究证明,随着小鼠年龄增加,其HSCs也随之衰老,最终导致衰老相关性老年疾病的发生。HSCs衰老是否与造血诱导微环境的稳态有关值得思考,本研究采用D-半乳糖(D-galactose,D-gal)建立BMSCs体外和体内衰老模型,探讨D-gal诱导BMSCs衰老可能的生物学机理,为研究HSCs衰老与HIM的关系、寻找延缓HSCs衰老的有效途径奠定理论基础,提供实验室依据。目的:采用致衰剂D-gal构建大鼠骨髓基质细胞(BMSCs)体外和体内衰老模型,探讨BMSCs衰老生物学特性及其机制。材料与方法:体外对照组:常规培养大鼠骨髓BMSCs,取第三代(Passage3,P3)细胞继续培养48 h;体外衰老组:在对照组基础上加入D-gal(终浓度30 g/L),作用48 h;体内衰老组:大鼠皮下注射D-gal(120 mg/kg.d),qd×42 d;体内对照组:大鼠皮下注射等时等量0.9%Nacl溶液,模型完成第2 d,取骨髓分离培养BMSCs,取P3细胞进行实验。检测指标:CCK-8测定细胞增殖能力;流式细胞术分析细胞周期和细胞凋亡率;β-半乳糖苷酶(SA-β-Gal)染色观察BMSCs衰老百分率;DCFH-DA荧光流式细胞术检测细胞活性氧簇(ROS)水平;酶学法检测过氧化物丙二醛(MDA)含量和总超氧化物歧化酶(SOD)活性;Western blotting检测P16、P21、P53、CDK2和cyclin-D表达。结果:D-gal在体外与体内均能建立BMSCs致衰老模型。表现在:1.BMSCs增殖能力下降;细胞G0/G1期比例增高、S期比例降低(P0.05);2.SA-β-Gal染色阳性的BMSCs百分率上升(P0.05);3.胞内ROS、MDA上升,SOD下降(P0.05);4.BMSCs的P16、P21、P53表达上调,CDK2、cyclin-D下调(P0.05)。结论:D-Gal在体内与体外均能构建BMSCs衰老模型,其机制可能与D-gal诱导BMSCs氧化损伤和激活衰老信号途径相关。
[Abstract]:Research background: one of the characteristics of Assisted Reproductive Technology ART is that the pregnancy rate varies greatly between different patients. This change is largely due to the fact that the commonly used observable parameters are unable to fully demonstrate the developmental potential of the embryo. Morphology has always been the most common and more commonly used evaluation of embryos. In a stable way, information about embryos obtained from an optical microscope has been directly related to the pregnancy outcome of the IVF (In vitro fertilization IVF). But it is found that even the similar parents of the same parent may have a completely different pregnancy outcome. This suggests that the embryo's form does not fully respond to its species. The oxygen consumption of the embryo, that is, the respiratory rate of the embryo, because mitochondria are the main organelles that produce Adenosine Triphosphate ATP, so the oxygen consumption of the embryo is closely related to the mitochondrial activity. The measurement of the embryo's respiration rate can reflect the function of embryo embryo mitochondria to a certain extent. The mitochondria of embryo are completely derived. In oocytes, the quality of oocytes has a great influence on the vitality of the embryo. The respiration rate can react to the activity of the mitochondria in the embryo and indirectly reflect the vitality of the embryo. The measurement of the embryo respiratory rate is a noninvasive method to measure the vitality of the living embryos safely and quickly. The basis of this method is the formation of oxygen ions from embryos. Spherical cation diffusion, measuring the current changes caused by oxygen diffusion around the embryo by the platinum microelectrode of the precise to the ANN level, reflects the oxygen consumption of the embryo through the current change (i.e., the embryo respiratory rate). This article reviews the non invasive respiration rate and the traditional morphologic score to understand the frozen transplantation cycle (Froze N-thawed embryo transfer FET) oxygen consumption of embryo and clinical pregnancy outcome, and observe the relationship between respiratory rate and clinical pregnancy outcome. Objective: to measure the rate of embryo respiration by measuring the cell respiration rate system, follow up the implantation situation and pregnancy outcome of the patients after transplantation, and explore whether the embryo respiratory rate measurement can respond to the developmental potential of embryo. Research subjects and methods: the standard of 1. entry group and group experiment were divided into three parts. The first part was FET cycle double embryo transplantation group. The group selected FET cycle patients. The study group was women aged less than 35 years old, excluding hereditary diseases, infertility with ovarian and fallopian tube factors, and infertility of uterus; and no oviduct effusion, male Sperm is normal. Embryo chooses IVF fertilization. Embryo selection is based on morphological criteria. Embryo quality is superior to 8 cell 3. The experimental population is divided into three groups according to respiratory rate, group A: two embryo respiration rate is up to or higher than 3 fmol/s, group B: only one respiratory rate of two embryos of transplant is 3 fmol/s, C group: two embryo call The rate of aspiration was less than 3 fmol/s. and second parts for the single embryo transfer group of FET cycle. The patients were included in the standard and the first part, but the embryo transfer was divided into two groups according to the respiratory rate; the A group: the embryo respiration rate was less than 3 fmol/s, and the B group: the embryo respiration rate was greater than 3 fmol/s. third part of the Time-Lapse combined respiration rate selection. The group of patients was included in the group: age less than 35 years old, normal menstruation, BMI:18-25, the number of eggs obtained: 5-15, using ICSI (Intracytoplasmic sperm injection ICSI) insemination. The patient used the standard rectangle to promote ovulation. The treatment was the first pregnancy for the patients. The infertility factor was simple fallopian tube infertility, excluded hereditary diseases, combined ovaries and ovary. Oviduct factor infertility and uterine factor infertility. The embryo quality is superior to 8 cell 3 grade portable embryo.. the experiment is divided into three groups, the A group is the morphological selection of two cell start time T2cb26.725 h embryo, the B group is the embryo with the respiration rate greater than 3 fmol/s, the C group is full full foot T2cb26.725 h and the respiratory rate is greater than equal 3 fmol/s The embryo respiration rate of three parts of the patients was measured by 2. research methods before transplantation (1). The standard patients signed the informed consent of the respiratory rate measurement before the period. (2) the respiration rate was measured before transplantation and the SECM was used to measure the respiratory rate. The method of measurement was: 1. put the pre transplant embryo in full of people. In the measurement tank of the fallopian tube buffer. The measurement slot is a narrow cone-shaped under the upper width. The embryo naturally deposits to the bottom.2. with the oxygen consumption of the embryo and the decreasing oxygen concentration.3. adjustment parameters at the top to the bottom of the buffer. The potential difference in the buffer solution is scanned from the vertical direction from the vertical to the vertical direction. The respiratory rate of the embryo.4. recorded the respiratory rate of each embryo. All the operations were completed in the shortest possible time. (5) B ultrasonic monitoring implantation rate, telephone follow-up abortion rate, clinical pregnancy rate, survival rate and analysis of the difference between the three groups. Results: a total of 131 cycles were measured in the FET double embryo transplantation group, and a total of 262 embryos were measured with a respiratory rate measurement. Three The intergroup age, BMI, endometrium thickness and other basic conditions, statistics and analysis of the difference between three groups of implant rates, clinical pregnancy rate, abortion rate and live birth rate, including 83 cycles, 166 embryos, 48.19% implantation rate, 65.06% pregnancy rate, 4.82% flow rate, and 60.24% live birth rate, and 36 cycles, 72 embryos in group B. The pregnancy rate was 43.05%, the pregnancy rate was 58.33%, the abortion rate was 19.4%, the birth rate of the live fetus was 38.89%, and the group C had 12 cycles, and 24 embryos were measured. Among them, the planting rate was 37.5%, the pregnancy rate was 50%, the abortion rate was 16.67%, and the birth rate of the fetus was 33.33%. statistically analysis found that there was no statistical difference between the age of the three groups, BMI and the thickness of the endometrium (P0.05) .A group had lower abortion rate and higher birth rate, compared with group B and C group, there was significant difference between group B and C group (P0.05) FET cycle single embryo transplantation group: a total of 58 embryos were transplanted, including 23 of the respiratory rate more than 3, the clinical pregnancy rate was less than 35 of 3, and the pregnancy rate of 54.29%. two in clinical pregnancy rate did not exist. In the statistical difference, the clinical pregnancy rate in the 3 embryo group was higher than that of the 3 embryo group. 35 embryos were transplanted in a total of 35 embryos, 13 embryos were transplanted in group A, 13 embryos were transplanted, 15 transplanted, 15 transplantation in group 53.33%.C and 7 transplantation. The rate of 57.14%. suggests that the implantation rate of two conditions satisfying the morphology and respiration rate has increased, but there is no statistical difference. Conclusion: there is still a defect in the prediction of the developmental potential of the embryo, but when the respiration rate is greater than 3 fmol/s, the pregnancy outcome after the embryo transfer is improved. The research background: hematopoietic inductive microenvironment (HIM) is a place for the growth and proliferation of Haemopoietic stem cells (HSCs) (1), and its structure and function are an important factor in maintaining normal hematopoiesis. Hematopoietic stromal cells (b) One marrow stromal cells, BMSCs), as the core component of HIM, not only supports and regulates the self-renewal and differentiation of HSCs, but also is closely related to the occurrence, development and prognosis of various blood system diseases by forming the "niche" of the growth and development of HSCs, secreting the hematopoietic growth factor and extracellular matrix, as well as the development and prognosis of various blood system diseases. The most important research object of HIM structure and function. The previous study of the group showed that as the age of mice increased, the HSCs also senescent, which eventually led to the consideration of whether.HSCs senescence of senescence related senile diseases was related to the homeostasis of hematopoiesis induced microenvironment. This study adopted D- galactose (D-galactose, D-gal) to establish BMSCs body. In vitro and in vivo aging model, the biological mechanism of D-gal induction of BMSCs senescence is explored, which lays a theoretical foundation for studying the relationship between HSCs senescence and HIM and finding an effective way to postpone HSCs senescence. Objective: to construct a rat bone marrow stromal cell (BMSCs) in vitro and in vivo aging model, and to explore BMSCs failure. Old biological characteristics and its mechanism. Materials and methods: in vitro control group: conventional culture of rat bone marrow BMSCs, third generations (Passage3, P3) cells continue to culture 48 h; in vitro aging group: on the basis of the control group, adding D-gal (final concentration 30 g/L), the effect of 48 h; the body senescence group: rat subcutaneous injection D-gal (120 mg/kg.d), QD * 42 d; in vivo control group: large control group: big control group: big group: big group: big group: big group: larger group: larger group: larger group: Large The mice were given the same amount of 0.9%Nacl solution at the time of subcutaneous injection. The model completed second D. The bone marrow was isolated and cultured for BMSCs, and P3 cells were taken for experiment. Detection index: CCK-8 assay cell proliferation ability; flow cytometry analysis cell cycle and apoptosis rate; beta galactosidase (SA- beta -Gal) staining to observe BMSCs senescence percentage; DCFH-DA fluorescence flow cytometry Detection of cell active oxygen cluster (ROS) level; enzymology detection of peroxidase (MDA) content and total superoxide dismutase (SOD) activity; Western blotting to detect P16, P21, P53, CDK2 and cyclin-D expression. Results: D-gal in vitro and in vivo can establish a BMSCs induced aging model. The percentage of S phase decreased (P0.05), and the percentage of BMSCs positive by 2.SA- beta -Gal increased (P0.05), and ROS, MDA increased and SOD decreased (P0.05) in 3. cells. The signal pathway of living senescence is related.

【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R714.8

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