【摘要】： 隨著自由基理論的建立和發(fā)展，人們發(fā)現(xiàn)氧的某些代謝產(chǎn)物及其衍生的活性物質(zhì)可以損傷機(jī)體，這些物質(zhì)具有比氧更為活潑的化學(xué)性質(zhì)，統(tǒng)稱為活性氧(reactive oxygen species，ROS)。正常情況下，ROS的產(chǎn)生和清除保持動(dòng)態(tài)平衡，但是在某些病理情況下和衰老時(shí)，ROS產(chǎn)生增加或細(xì)胞抗氧化機(jī)制受損，可造成ROS的累積，主要導(dǎo)致大分子物質(zhì)如脫氧核糖核酸(deoxyribonucleic acid，DNA)，脂質(zhì)和蛋白質(zhì)的氧化損傷。DNA的氧化損傷可以導(dǎo)致核DNA和線粒體DNA突變或缺失，細(xì)胞膜脂質(zhì)過(guò)氧化導(dǎo)致膜流動(dòng)性改變，而蛋白的氧化損傷導(dǎo)致重要酶失活，蛋白功能受損。 研究發(fā)現(xiàn)卵泡液中存在ROS，一定水平的ROS對(duì)維持卵母細(xì)胞的正常生長(zhǎng)發(fā)揮重要作用，ROS的過(guò)度升高對(duì)卵母細(xì)胞成熟和后續(xù)胚胎發(fā)育造成不良影響。研究還發(fā)現(xiàn)氧化應(yīng)激參與胚胎發(fā)育缺陷的發(fā)生，第一天胚胎培養(yǎng)液中高水平的ROS對(duì)胚胎發(fā)育產(chǎn)生不良影響。精子冷凍和熱應(yīng)激狀態(tài)會(huì)導(dǎo)致ROS水平顯著升高。這些結(jié)果提示了人卵泡液、胚胎培養(yǎng)液和低溫冷凍精液中存在ROS所致的氧化應(yīng)激現(xiàn)象。以往的研究，主要關(guān)注ROS介導(dǎo)的脂質(zhì)過(guò)氧化損傷，但近年的研究說(shuō)明，蛋白質(zhì)的氧化修飾不容忽視。但是目前為止，關(guān)于在人卵泡液、胚胎培養(yǎng)液和低溫冷凍精液中ROS介導(dǎo)的蛋白氧化損傷的研究還未見報(bào)道，那么上述系統(tǒng)是否存在蛋白氧化應(yīng)激現(xiàn)象?其程度是否會(huì)造成不良影響?本研究擬在ROS在生殖方面的研究和蛋白氧化在人類其它組織研究的基礎(chǔ)上，以晚期氧化蛋白產(chǎn)物(advanced oxidation protein products，AOPP)為蛋白氧化損傷的指標(biāo)，觀察人卵泡液、胚胎培養(yǎng)液和低溫冷凍精液中是否存在ROS介導(dǎo)的蛋白氧化損傷，并初步探討其與卵母細(xì)胞和早期胚胎發(fā)育的關(guān)系。 第一章人卵泡液中蛋白氧化水平與IVF-ET結(jié)局參數(shù)的關(guān)系 【目的】 探討體外受精-胚胎移植(in vitro fertilization-embryo transplantation，IVF-ET)周期卵泡液中蛋白氧化水平與IVF-ET結(jié)局參數(shù)的關(guān)系。 【方法】 1．標(biāo)本來(lái)源和分組：卵泡液標(biāo)本來(lái)自南方醫(yī)院生殖中心2005年7月至2006年4月之間接受IVF-ET治療的64例不孕癥女性患者。病例選擇標(biāo)準(zhǔn)：年齡在25～38歲，平均年齡32.41±3.17歲；單純輸卵管性因素不孕；行常規(guī)IVF-ET治療；HCG日子宮內(nèi)膜在8.0 mm以上，并且無(wú)內(nèi)膜病變。根據(jù)獲卵數(shù)分為三組，獲卵數(shù)＜8個(gè)組16例，獲卵數(shù)8～15個(gè)組32例，獲卵數(shù)＞15個(gè)組16例。根據(jù)患者年齡分為三組，年齡＜30歲組11例，30歲～35歲組41例，＞35歲組12例。 2．實(shí)驗(yàn)方法：以AOPP作為蛋白氧化指標(biāo)，采用Vitko-Sarsat等介紹的方法進(jìn)行測(cè)定。 3．統(tǒng)計(jì)學(xué)方法：所有數(shù)據(jù)應(yīng)用spss10.0統(tǒng)計(jì)軟件進(jìn)行分析，P＜0.05認(rèn)為差異有統(tǒng)計(jì)學(xué)意義。結(jié)果以均數(shù)±標(biāo)準(zhǔn)差((?)±s)或百分率(％)表示，采用偏相關(guān)分析、One-way ANOVA方差分析和獨(dú)立樣本t檢驗(yàn)。 【結(jié)果】 1．卵泡液中AOPP水平與成熟卵母細(xì)胞比例(r=-0.401，P=0.001)、受精率(r=-0.257，P=0.045)、卵裂率(r=-0.290，P=0.024)、良好胚胎形成率(r=-0.520，P=0.000)均呈顯著負(fù)相關(guān)。 2．不同獲卵數(shù)組之間AOPP水平有顯著性差異(F=3.851，P=0.027)，其中以獲卵數(shù)8～15個(gè)組AOPP水平最低，獲卵數(shù)＜8個(gè)組最高。 3．非妊娠組AOPP水平高于妊娠組，有顯著性差異(t=3.665，P=-0.001)。 4．不同年齡組AOPP水平差異有顯著性意義(F=15.919，P=0.000)，其中年齡＞35歲組AOPP水平最高，＜30歲組最低。 【結(jié)論】 1．IVF-ET周期中，卵泡液中存在蛋白氧化應(yīng)激現(xiàn)象。 2．卵泡液中高水平的AOPP可能對(duì)卵母細(xì)胞和早期胚胎發(fā)育造成不良影響，繼而影響IVF結(jié)局。 第二章第一天胚胎培養(yǎng)液中蛋白氧化水平與早期胚胎發(fā)育的關(guān)系 【目的】 探討IVF-ET周期第一天胚胎培養(yǎng)液中蛋白氧化水平與早期胚胎發(fā)育的關(guān)系。 【方法】 1．標(biāo)本來(lái)源和分組：第一天胚胎培養(yǎng)液標(biāo)本來(lái)自南方醫(yī)院生殖中心2005年7月至11月之間進(jìn)行常規(guī)IVF治療的30例患者和單精子卵細(xì)胞內(nèi)注射(intracytoplasmic sperm injection-embryo transplantation，ICSI-ET)治療的30例患者。分組：根據(jù)受精類型分為兩組，常規(guī)IVF組30例和ICSI組30例。根據(jù)不孕婦女年齡分組，常規(guī)IVF組30例中年齡＜30歲組4例，30歲～35歲組17例，＞35歲組9例；ICSI組30例中年齡＜30歲組8例，30歲～35歲組18例，＞35歲組4例。 2．實(shí)驗(yàn)方法：以AOPP作為蛋白氧化指標(biāo)，采用Vitko-Sarsat等介紹的方法進(jìn)行測(cè)定。 3．統(tǒng)計(jì)學(xué)方法：所有數(shù)據(jù)應(yīng)用spss10.0統(tǒng)計(jì)軟件進(jìn)行分析，P＜0.05認(rèn)為差異有統(tǒng)計(jì)學(xué)意義。結(jié)果以均數(shù)±標(biāo)準(zhǔn)差((?)±s)表示，采用偏相關(guān)分析，獨(dú)立樣本t檢驗(yàn)和One-way ANOVA方差分析。 【結(jié)果】 1．常規(guī)IVF周期中，第一天胚胎培養(yǎng)液中AOPP濃度在3.2μmol／l以下時(shí)，其變化與受精率(r=0.137，P=0.863)、卵裂率(r=0.623，，P=0.377)、碎片10％以下胚胎形成率(r=-0.156，P=0.844)、六細(xì)胞以上胚胎形成率(r=-0.444，P=0.556)無(wú)相關(guān)性；在3.2μmol／l以上時(shí)，AOPP水平與受精率(r=-0.226，P=0.338)、卵裂率(r=0.202，P=0.392)、碎片10％以下胚胎形成率(r=-0.232，P=0.325)、六細(xì)胞以上胚胎形成率(r=0.008，P=0.975)亦無(wú)相關(guān)性。 2．ICSI周期中，第一天胚胎培養(yǎng)液中AOPP濃度在3.2μmol／l以下時(shí)，其變化與受精率(r=-0.323，P=0.791)、卵裂率(r=0.014，P=0.991)、碎片10％以下胚胎形成率(r=0.238，P=0.847)、六細(xì)胞以上胚胎形成率(r=-0.029，P=0.982)無(wú)相關(guān)性；在3.2μmol／l以上時(shí)，AOPP水平與受精率(r=-0.472，P=0.031)、碎片10％以下胚胎形成率(r=-0.482，P=0.027)、六細(xì)胞以上胚胎形成率(r=-0.548，P=0.010)呈顯著負(fù)相關(guān)，與卵裂率(r=-0.096，P=0.679)無(wú)相關(guān)性。 3．常規(guī)IVF周期中，非妊娠組中AOPP水平高于妊娠組，有顯著性差異(t=3.160，P=0.004)；不同年齡組AOPP水平有顯著性差異(F=19.395，P=0.000)，其中不孕婦女年齡＞35歲組AOPP水平最高，＜30歲組最低。 4．ICSI周期中，非妊娠組AOPP水平高于妊娠組，有顯著性差異(t=2.421，P=0.022)；不同年齡組AOPP水平有顯著性差異(F=3.975，P=0.031)，其中不孕婦女年齡＞35歲組AOPP水平最高，＜30歲組最低。 【結(jié)論】 1．IVF-ET周期中，第一天胚胎培養(yǎng)液中存在蛋白氧化應(yīng)激現(xiàn)象。 2．ICSI周期中，第一天胚胎培養(yǎng)液中高水平的AOPP可能對(duì)早期胚胎發(fā)育和IVF結(jié)局造成不良影響。 3．常規(guī)IVF周期中，第一天胚胎培養(yǎng)液中高水平的AOPP雖然不影響早期胚胎發(fā)育，但對(duì)IVF結(jié)局造成不良影響。 第三章低溫冷凍和孵育對(duì)人精子氧化應(yīng)激水平的影響 【目的】 探討低溫冷凍和孵育對(duì)人精子氧化應(yīng)激水平的影響。 【方法】 1．標(biāo)本來(lái)源和分組：60例正常人精液標(biāo)本取自南方醫(yī)院生殖醫(yī)學(xué)中心因女性因素行試管嬰兒治療的男性患者。將每一例精液標(biāo)本，梯度離心法處理后，取得沉淀之精子，加人輸卵管培養(yǎng)液(human tubal fluid，HTF)，配制成精子密度在(20～30)×10~6／mL的懸液，取2.5 mL均分五份，分為五組，分別進(jìn)行以下處理： A組為處理前對(duì)照組，不進(jìn)行低溫冷凍處理或孵育處理。 B組為低溫冷凍空白實(shí)驗(yàn)組，去除精子后的精子懸液進(jìn)行低溫冷凍處理。 C組為低溫冷凍樣本組，含精子的精子懸液進(jìn)行低溫冷凍處理。 D組為孵育空白實(shí)驗(yàn)組，去除精子后的精子懸液進(jìn)行孵育處理。 E組為孵育樣本組，含精子的精子懸液進(jìn)行孵育處理。 2．實(shí)驗(yàn)方法：以AOPP作為蛋白氧化指標(biāo)，采用Vitko-Sarsat等介紹的方法進(jìn)行測(cè)定。以丙二醛(malondialdehyde，MDA)作為脂質(zhì)過(guò)氧化指標(biāo)，采用硫代巴比妥酸(thiobarbituri，TBA)比色法。 3．統(tǒng)計(jì)學(xué)方法：所有數(shù)據(jù)以均數(shù)±標(biāo)準(zhǔn)差((?)±s)表示，采用SPSS10.0統(tǒng)計(jì)軟件進(jìn)行分析，P＜0.05為差異顯著。采用One-way ANOVA方差分析，AOPP水平的多重比較采用Tamhane's T2方法，MDA水平的多重比較采用最小有意義差異(least significant difference，LSD)t檢驗(yàn)方法。 【結(jié)果】 1．不同處理組之間AOPP水平(F=19.994，P=0.000)和MDA水平(F=31.837，P=0.000)均有顯著性差異。 2．低溫冷凍樣本組MDA水平顯著高于處理前對(duì)照組(P=0.000)和低溫冷凍空白實(shí)驗(yàn)組(P=0.000)，但AOPP水平無(wú)顯著性差異(P_1=0.663；P_2=0.829)。 3．孵育樣本組AOPP和MDA水平均顯著高于處理前對(duì)照組和孵育空白實(shí)驗(yàn)組(AOPP：P_1=0.000；P_2=0.000。MDA：P_1=0.000；P_2=0.000)。 4．孵育樣本組與低溫冷凍樣本組比較，AOPP(P=0.000)和MDA(P=0.046)水平均顯著升高。 【結(jié)論】 1．精子的低溫冷凍和孵育，均存在氧化應(yīng)激損傷。 2．低溫冷凍主要造成精子的脂質(zhì)過(guò)氧化損傷，孵育可致精子的脂質(zhì)過(guò)氧化損傷和蛋白氧化損傷。
[Abstract]:With the establishment and development of free radical theory, it has been found that some metabolites of oxygen and their derivatives can damage the body. These substances have more active chemical properties than oxygen. They are collectively called reactive oxygen species (ROS). Under normal conditions, the production and removal of ROS maintain a dynamic balance, but in some pathological conditions, ROS is more active than oxygen. In both cases and senescence, ROS production increases or cellular antioxidant mechanisms are impaired, resulting in the accumulation of ROS, mainly leading to oxidative damage to macromolecules such as deoxyribonucleic acid (DNA), lipids and proteins. DNA oxidative damage can lead to mutations or deletions in nuclear DNA and mitochondrial DNA, and lipid peroxidation in cell membranes leads to the formation of ROS. Membrane fluidity changes, and protein oxidative damage leads to inactivation of important enzymes and impaired protein function.
It was found that ROS was present in follicular fluid, and a certain level of ROS played an important role in maintaining the normal growth of oocytes. Over-elevation of ROS had adverse effects on oocyte maturation and subsequent embryonic development. These results suggest that ROS-induced oxidative stress exists in human follicular fluid, embryonic culture fluid and cryopreserved semen. Previous studies have focused on ROS-mediated lipid peroxidation damage, but recent studies have shown that protein oxygen is present in human follicular fluid, embryonic culture fluid and cryopreserved semen. Chemical modification should not be neglected. However, up to now, there is no report on ROS-mediated protein oxidative damage in human follicular fluid, embryo culture medium and cryopreserved semen. Is there any protein oxidative stress in the above-mentioned system? Is it harmful to the extent? This study intends to study the reproductive effects of ROS and eggs. Based on the study of other human tissues, advanced oxidation protein products (AOPP) were used as an indicator of protein oxidative damage. ROS-mediated protein oxidative damage was observed in human follicular fluid, embryo culture medium and cryopreserved semen, and its relationship with oocytes and early embryos was preliminarily discussed. Developmental relationship.
Chapter 1 Relationship between protein oxidation level in human follicular fluid and IVF-ET outcome parameters
[Objective]
Objective To investigate the relationship between protein oxidation level in follicular fluid and outcome parameters of IVF-ET during in vitro fertilization-embryo transfer (IVF-ET) cycles.
[method]
1. Source and grouping: Follicular fluid specimens were collected from 64 infertile women treated with IVF-ET from July 2005 to April 2006 in the Reproductive Center of Southern Hospital. Case selection criteria: age ranged from 25 to 38 years with an average age of 32.41 (+ 3.17); tubal factor infertility alone; conventional IVF-ET treatment; endometrium on HCG days. The patients were divided into three groups according to the number of eggs harvested: 16 cases in 8 groups, 32 cases in 8-15 groups and 16 cases in 15 groups.
2. experimental method: AOPP was used as protein oxidation index, and the method was introduced by Vitko-Sarsat.
3. Statistical methods: All data were analyzed by SPSS 10.0 statistical software, P < 0.05, and the difference was statistically significant. The results were expressed as mean (?) + standard deviation (?) + s) or percentage (%). Partial correlation analysis, one-way ANOVA variance analysis and independent sample t test were used.
[results]
1. AOPP levels in follicular fluid were negatively correlated with the ratio of mature oocytes (r = - 0.401, P = 0.001), fertilization rate (r = - 0.257, P = 0.045), cleavage rate (r = - 0.290, P = 0.024), and good embryo formation rate (r = - 0.520, P = 0.000).
2. There were significant differences in AOPP levels among different egg-harvesting groups (F=3.851, P=0.027), among which the lowest AOPP level was found in 8-15 egg-harvesting groups and the highest AOPP level was found in 8 egg-harvesting groups.
3. the level of AOPP in non pregnant group was higher than that in pregnancy group, with significant difference (t=3.665, P=-0.001).
4. There was significant difference in AOPP levels among different age groups (F = 15.919, P = 0.000), and AOPP levels were the highest in the group over 35 years old and the lowest in the group under 30 years old.
[Conclusion]
Oxidative stress is present in follicular fluid during 1.IVF-ET cycle.
2. High levels of AOP in follicular fluid may have adverse effects on oocyte and early embryonic development, and then affect the outcome of IVF.
The second chapter is about the relationship between the level of protein oxidation in embryonic culture medium and the development of early embryos.
[Objective]
Objective to investigate the relationship between protein oxidation level and embryonic development in the first day embryo of IVF-ET cycle.
[method]
1. Sample Sources and Groups: Embryo Culture Medium Samples from the first day of the Southern Hospital Reproduction Center from July to November 2005 routine IVF treatment of 30 patients and intracytoplasmic sperm injection-embryo transplantation (ICSI-ET) treatment of 30 patients. Two groups, 30 cases in the conventional IVF group and 30 cases in the ICSI group. According to the age of infertile women, there were 4 cases in the conventional IVF group, 17 cases in the 30-35-year-old group, 9 cases in the over-35-year-old group, 8 cases in the 30-year-old group, 18 cases in the 30-year-old group and 4 cases in the over-35-year-old group.
2. experimental method: AOPP was used as protein oxidation index, and the method was introduced by Vitko-Sarsat.
3. Statistical methods: All the data were analyzed by SPSS 10.0 statistical software, P < 0.05, and the difference was statistically significant. The results were expressed as mean (?) + standard deviation (?) + s), partial correlation analysis, independent sample t test and one-way ANOVA analysis of variance.
[results]
1. There was no correlation between AOPP concentration in the first day of IVF cycle and fertilization rate (r = 0.137, P = 0.863), cleavage rate (r = 0.623, P = 0.377), embryo formation rate (r = - 0.156, P = 0.844) and embryo formation rate (r = - 0.444, P = 0.556) above 6 cells in the first day of IVF cycle. There was no correlation between fertilization rate (r = - 0.226, P = 0.338), cleavage rate (r = 0.202, P = 0.392), embryo formation rate below 10% of fragments (r = - 0.232, P = 0.325) and embryo formation rate above six cells (r = 0.008, P = 0.975).
2. During the ICSI cycle, when AOPP concentration in the first day of embryo culture was below 3.2 micromol/l, there was no correlation between AOPP level and fertilization rate (r = - 0.323, P = 0.791), cleavage rate (r = 0.014, P = 0.991), embryo formation rate below 10% of fragments (r = 0.238, P = 0.847), and embryo formation rate above 6 cells (r = - 0.029, P = 0.982). Fertilization rate (r = - 0.472, P = 0.031), embryo formation rate below 10% of fragments (r = - 0.482, P = 0.027), embryo formation rate above six cells (r = - 0.548, P = 0.010) was negatively correlated with cleavage rate (r = - 0.096, P = 0.679).
3. In the conventional IVF cycle, the AOPP level in the non-pregnant group was significantly higher than that in the pregnant group (t = 3.160, P = 0.004); the AOPP level in the different age groups was significantly different (F = 19.395, P = 0.000), and the AOPP level was the highest in the infertile women aged over 35 and the lowest in the women aged under 30.
4. In ICSI cycle, the AOPP level in non-pregnant women was higher than that in pregnant women (t = 2.421, P = 0.022), and there was a significant difference among different age groups (F = 3.975, P = 0.031). The AOPP level in infertile women aged over 35 was the highest, and that in women aged under 30 was the lowest.
[Conclusion]
In the 1.IVF-ET cycle, there was protein oxidative stress on the first day of embryo culture.
2. During the ICSI cycle, high levels of AOP in the first day of embryo culture may have adverse effects on early embryonic development and IVF outcomes.
3. High levels of AOP in the first day of IVF cycle did not affect early embryonic development, but negatively affected the outcome of IVF.
The third chapter is the effect of cryopreservation and incubation on the level of oxidative stress in human sperm.
[Objective]
Objective to investigate the effects of cryopreservation and incubation on oxidative stress in human spermatozoa.
[method]
1. Source and grouping of specimens: 60 normal human semen specimens were taken from male patients treated with in vitro fertilization for female factors in the Reproductive Medical Center of Southern Hospital. The suspension of ~6 / mL was divided into five groups according to 2.5 mL and divided into five groups.
Group A was treated with pre control group without cryopreservation or incubation.
Group B was cryopreserved blank group, and sperm suspension after cryopreservation was cryopreserved.
Group C was cryopreserved, and sperm containing sperm suspension was cryopreserved.
Group D was incubated in blank experimental group, and sperm suspension after spermatozoa was incubated.
Group E was incubated with sperm samples and incubated with sperm suspension.
2. Experimental method: AOPP was used as the index of protein oxidation, and Vitko-Sarsat was used to determine it. Malondialdehyde (MDA) was used as the index of lipid peroxidation, and thiobarbituri (TBA) colorimetry was used.
3. Statistical methods: All data were expressed as mean (?) + standard deviation ((?) + s) and analyzed with SPSS10.0 statistical software, P < 0.05 as significant difference. One-way ANOVA analysis of variance showed that multiple comparisons of AOPP levels were performed with Tamhane's T2 method, and multiple comparisons of MDA levels were performed with least significant difference (LSD). T test method.
[results]
1. There were significant differences in AOPP level (F = 19.994, P = 0.000) and MDA level (F = 31.837, P = 0.000) among different treatment groups.
2. The level of MDA in cryopreservation sample group was significantly higher than that in control group (P = 0.000) and cryopreservation blank group (P = 0.000), but the level of AOPP had no significant difference (P_1 = 0.663; P_2 = 0.829).
3. The levels of AOPP and MDA in incubation sample group were significantly higher than those in control group and incubation blank group before treatment (AOPP: P_1 = 0.000; P_2 = 0.000. MDA: P_1 = 0.000; P_2 = 0.000).
4. the levels of AOPP (P=0.000) and MDA (P=0.046) in the incubated sample group were significantly higher than those in the cryopreservation group.
[Conclusion]
1. there is oxidative stress damage in cryopreservation and incubation of spermatozoa.
2. Cryopreservation mainly causes lipid peroxidation damage of sperm, and incubation can cause lipid peroxidation damage and protein oxidation damage of sperm.
【學(xué)位授予單位】：第一軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類號(hào)】：R321-33

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相關(guān)期刊論文 前1條

1 余月明,侯凡凡,周華,楊燕,張訓(xùn),楊凌,胡敏燕;慢性腎衰竭患者同型半胱氨酸血癥與動(dòng)脈粥樣硬化的關(guān)系[J];中華內(nèi)科雜志;2002年08期

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