本文選題：呼吸率 + 非傾入性測量�。� 參考：《重慶醫(yī)科大學(xué)》2017年碩士論文

【摘要】：研究背景:人類輔助生殖技術(shù)(Assisted Reproductive Technology ART)的特點之一就是妊娠率在不同病人之間變化非常大,這種變化很大程度上是由于現(xiàn)在常用的可觀測參數(shù)無法全面表現(xiàn)出胚胎的發(fā)育潛能。形態(tài)學(xué)一直是評估胚胎最常用也較為穩(wěn)定的方法,從光學(xué)顯微鏡下獲得的關(guān)于胚胎的信息雖然已被證實和IVF(In vitro fertilization IVF)的妊娠結(jié)局直接相關(guān)。但人們發(fā)現(xiàn),即使評分相近的同父母來源的胚胎也可能有完全不同的妊娠結(jié)局。這說明胚胎形態(tài)并不能完全反應(yīng)它的種植潛能。胚胎的氧耗量即胚胎的呼吸率,由于線粒體是產(chǎn)生三磷酸腺苷(Adenosine Triphosphate ATP)的主要細胞器,故胚胎的氧耗量與線粒體活動密切相關(guān),測量胚胎的呼吸率可以在一定程度上反應(yīng)胚胎線粒體的功能。胚胎的線粒體完全來源于卵母細胞,而卵母細胞質(zhì)量對胚胎活力有很大的影響。呼吸率能夠反應(yīng)胚胎中線粒體的活性,從而間接反應(yīng)胚胎的活力。胚胎呼吸率測量是一種非侵入性的方法,能夠安全快捷測量活胚胎的活力。該方法的基礎(chǔ)是胚胎消耗氧離子形成球形陽離子擴散,以精確到納安級別的鉑金微電極測量胚胎周圍由于氧的擴散而引起的電流變化,通過電流變化反映胚胎的耗氧量(即胚胎呼吸率)。本文將非侵入性的呼吸率測量結(jié)合傳統(tǒng)的形態(tài)學(xué)評分回顧性分析了解凍移植周期(Frozen-thawed embryo transfer FET)胚胎的氧耗量及其臨床妊娠結(jié)局,觀察呼吸率與臨床妊娠結(jié)局之間的關(guān)系。目的:使用細胞呼吸率測量系統(tǒng)測量胚胎呼吸率,隨訪移植后病人胚胎著床情況及妊娠結(jié)局,探討胚胎呼吸率測量能否反應(yīng)胚胎發(fā)育潛能。研究對象和方法:1.入組標準及分組實驗共分為三部分進行,第一部分為FET周期雙胚胎移植組。該組選擇FET周期患者,研究人群為:女方年齡≤35歲,排除遺傳性疾病,合并卵巢和輸卵管因素的不孕以及子宮因素的不孕;無輸卵管積液,男方精子正常。胚胎選擇IVF授精。胚胎選擇基于形態(tài)學(xué)標準,胚胎質(zhì)量為優(yōu)于8細胞3級的可移植胚胎。實驗人群按呼吸率分為三組,A組:兩枚胚胎呼吸率都達到或高于3.0 fmol/s,B組:移植的兩枚胚胎中只有一枚呼吸率達到3.0 fmol/s,C組:兩枚胚胎呼吸率均小于3.0 fmol/s。第二部分為FET周期單胚胎移植組,該組患者納入標準同第一部分,但移植單胚胎。胚胎根據(jù)呼吸率分為兩組;A組:胚胎呼吸率小于3.0 fmol/s,B組:移植的胚胎呼吸率大于等于3.0 fmol/s。第三部分為Time-Lapse結(jié)合呼吸率選擇胚胎組。該組患者納入標準為:年齡≤35歲,月經(jīng)正常,BMI:18-25,獲卵數(shù):5-15枚,采用ICSI(Intracytoplasmic sperm injection ICSI)授精。患者采用標準長方案促排卵,此次治療為患者的首次助孕。不孕因素為單純輸卵管不孕,排除遺傳性疾病,合并卵巢和輸卵管因素的不孕和子宮因素的不孕。胚胎質(zhì)量為優(yōu)于8細胞的3級可移植胚胎。。實驗分為三組,A組為形態(tài)學(xué)選擇二細胞開始時間T2cb26.725 h的胚胎,B組為呼吸率大于等于3.0 fmol/s的胚胎,C組為同時滿足T2cb26.725 h和呼吸率大于等于3.0 fmol/s的胚胎。三個部分的患者的胚胎呼吸率測量均于移植前進行2.研究方法(1)納入標準的患者于周期前簽署呼吸率測量相關(guān)的知情同意書。(2)于移植前進行呼吸率測量,采用SECM進行呼吸率的測量,測量方法為:1.將移植前的胚胎置于充滿人輸卵管緩沖液的測量槽內(nèi)。測量槽為上寬下窄的圓錐形,胚胎自然沉積到底部。2.隨著胚胎代謝耗氧,緩沖液頂部至底部出現(xiàn)遞減的氧濃度差。3.調(diào)節(jié)參數(shù)后,將特殊的微電極沿垂直方向自上而下地掃描緩沖液內(nèi)的電位差。在系統(tǒng)內(nèi)轉(zhuǎn)化為胚胎的呼吸率。4.記錄各枚胚胎的呼吸率。全部操作在盡量短的時間內(nèi)完成。(5)B超監(jiān)測種植率,電話隨訪流產(chǎn)率、臨床妊娠率、活產(chǎn)率并分析三組之間的差異。結(jié)果:FET雙胚胎移植組:共測量131個周期,對共262枚胚胎進行了呼吸率測量。比較三組間年齡,BMI,子宮內(nèi)膜厚度等基本情況,統(tǒng)計并分析了三組間種植率,臨床妊娠率,流產(chǎn)率和活胎分娩率等妊娠結(jié)局的差異。其中A組共測量83個周期,166枚胚胎,種植率48.19%,妊娠率65.06%,流產(chǎn)率4.82%,活胎分娩率60.24%;B組包含36個周期,72枚胚胎,種植率43.05%,妊娠率58.33%,流產(chǎn)率19.4%,活胎分娩率38.89%;C組有12個周期,共測量24枚胚胎,其中種植率37.5%,妊娠率50%,流產(chǎn)率16.67%,活胎分娩率33.33%。統(tǒng)計學(xué)分析發(fā)現(xiàn),三組患者年齡,BMI及子宮內(nèi)膜厚度等基本情況間不存在統(tǒng)計學(xué)差異(P0.05)。A組擁有更低的流產(chǎn)率及更高的活胎分娩率,且相較于B組和C組存在明顯的統(tǒng)計學(xué)差異(P0.05)FET周期單胚胎移植組:共移植58枚胚胎,其中呼吸率大于3.0的23枚,臨床妊娠率43.48%。呼吸率小于等于3.0的35枚,臨床妊娠率54.29%。兩組妊娠率不存在統(tǒng)計學(xué)差異,但移植呼吸率大于3.0胚胎組臨床妊娠率有所提高。Time-Lapse聯(lián)合呼吸率組:共移植35枚胚胎,A組單純形態(tài)學(xué)選擇,移植13枚胚胎,種植率46.15%。B組單純選擇大于3.0胚胎,移植15枚,種植率53.33%。C組呼吸率聯(lián)合形態(tài)學(xué),移植7枚,種植率57.14%。提示滿足形態(tài)學(xué)和呼吸率兩個條件的胚胎種植率有所增加,但不存在統(tǒng)計學(xué)差異。結(jié)論:呼吸率測量在預(yù)測胚胎發(fā)育潛能方面還存在缺陷,但是當呼吸率大于等于3.0 fmol/s時,胚胎移植后患者的妊娠結(jié)局有所改善。提示胚胎呼吸率可以作為胚胎選擇的輔助參數(shù)。研究背景:造血微環(huán)境(hematopoietic inductive microenvironment,HIM)是造血干細胞(Haemopoietic stem cells,HSCs)賴以生長發(fā)育和增殖分化的場所【1】,其結(jié)構(gòu)和功能完整是維系正常造血功能的重要因素。造血基質(zhì)細胞(bone marrow stromal cells,BMSCs)作為HIM的核心組分,不僅通過形成HSCs生長發(fā)育的“龕”、分泌造血生長因子和細胞外基質(zhì)等方式支持和調(diào)控HSCs自我更新與分化,還與多種血液系統(tǒng)疾病的發(fā)生、發(fā)展和預(yù)后密切相關(guān),因此骨髓基質(zhì)細胞成為研究HIM結(jié)構(gòu)與功能最為重要的研究對象。課題組前期研究證明,隨著小鼠年齡增加,其HSCs也隨之衰老,最終導(dǎo)致衰老相關(guān)性老年疾病的發(fā)生。HSCs衰老是否與造血誘導(dǎo)微環(huán)境的穩(wěn)態(tài)有關(guān)值得思考,本研究采用D-半乳糖(D-galactose,D-gal)建立BMSCs體外和體內(nèi)衰老模型,探討D-gal誘導(dǎo)BMSCs衰老可能的生物學(xué)機理,為研究HSCs衰老與HIM的關(guān)系、尋找延緩HSCs衰老的有效途徑奠定理論基礎(chǔ),提供實驗室依據(jù)。目的:采用致衰劑D-gal構(gòu)建大鼠骨髓基質(zhì)細胞(BMSCs)體外和體內(nèi)衰老模型,探討B(tài)MSCs衰老生物學(xué)特性及其機制。材料與方法:體外對照組:常規(guī)培養(yǎng)大鼠骨髓BMSCs,取第三代(Passage3,P3)細胞繼續(xù)培養(yǎng)48 h;體外衰老組:在對照組基礎(chǔ)上加入D-gal(終濃度30 g/L),作用48 h;體內(nèi)衰老組:大鼠皮下注射D-gal(120 mg/kg.d),qd×42 d;體內(nèi)對照組:大鼠皮下注射等時等量0.9%Nacl溶液,模型完成第2 d,取骨髓分離培養(yǎng)BMSCs,取P3細胞進行實驗。檢測指標:CCK-8測定細胞增殖能力;流式細胞術(shù)分析細胞周期和細胞凋亡率;β-半乳糖苷酶(SA-β-Gal)染色觀察BMSCs衰老百分率;DCFH-DA熒光流式細胞術(shù)檢測細胞活性氧簇(ROS)水平;酶學(xué)法檢測過氧化物丙二醛(MDA)含量和總超氧化物歧化酶(SOD)活性;Western blotting檢測P16、P21、P53、CDK2和cyclin-D表達。結(jié)果:D-gal在體外與體內(nèi)均能建立BMSCs致衰老模型。表現(xiàn)在:1.BMSCs增殖能力下降;細胞G0/G1期比例增高、S期比例降低(P0.05);2.SA-β-Gal染色陽性的BMSCs百分率上升(P0.05);3.胞內(nèi)ROS、MDA上升,SOD下降(P0.05);4.BMSCs的P16、P21、P53表達上調(diào),CDK2、cyclin-D下調(diào)(P0.05)。結(jié)論:D-Gal在體內(nèi)與體外均能構(gòu)建BMSCs衰老模型,其機制可能與D-gal誘導(dǎo)BMSCs氧化損傷和激活衰老信號途徑相關(guān)。
[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.

【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R714.8

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