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研究生:謝詠函
研究生(外文):Yung-Han Hsieh
論文名稱:血清以及血清替代物在保護小鼠著床前胚胎和胚胎幹細胞免於活性氧所誘導的細胞凋亡中所扮演的角色
論文名稱(外文):Roles of Serum and Serum Substitutes in the Protection of ROS-induced Apoptosis in Mouse Pre-implantation
指導教授:宋晏仁宋晏仁引用關係李新揚李新揚引用關係
指導教授(外文):Yen-Jen SungHsin-Yang Li
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:解剖暨細胞生物學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:英文
論文頁數:67
中文關鍵詞:著床前胚胎活性氧自由基胚胎幹細胞血清替代物血清替代物胚胎發育
外文關鍵詞:pre-implantationROSembryonic stem cellserum substitutesserum supplementembryo development
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人工生殖技術(assisted reproductive technology)可幫助不孕症夫婦孕育下一代,然而人工生殖技術的成功關鍵在高品質的胚胎培養。在體外胚胎培養的過程中,培養箱的高氧氣濃度(20%)及胚胎操作時的光線暴露等傷害都可能會導致胚胎的活性氧(reactive oxygen species)濃度上升,而活性氧累積所造成的氧化壓力和胚胎發育不良有關。有研究顯示血清以及血清替代物不但可以提升胚胎於體外發育至囊胚的比例,也可以增進胚胎著床率及活產率,但是其機
轉並不清處,並且目前多數的人工生殖機構只於培養液中加入人類血清白蛋白(human serum albumin)而未加入血清替代物。我們假設血清以及血清替代物可以降低體外胚胎培養過程中的氧化壓力,避免胚胎細胞凋亡,進而促進胚胎發育。我們使用小鼠著床前胚胎和胚胎幹細胞作為研究材料,以過氧化氫(H2O2)提供外源性活性氧,而以丁硫氨酸-亞碸亞胺(L-Buthioninesulfoximine,一種γ-glutamylcysteine synthetase inhibitor)使胚胎細胞內生性活性
氧升高。結果發現過氧化氫會誘導胚胎細胞凋亡及胚胎發育遲滯,而培養液中加入0.5%胎牛血清或10%血漿注射劑(Plasmanate,一種血清替代物)可以降低過氧化氫引起之胚胎細胞活性氧的累積並保護細胞免於凋亡,進而促進胚胎發育;但是培養液中加入人類血清白蛋白至其濃度和10%血漿注射劑的人類血清白蛋白濃度相同(0.5%),並無法達到上述效果,顯示10% 血漿注射劑的保護效應可能是白蛋白以外其他的因子(如a和b球蛋白等)造成。此外,過氧化氫和丁硫氨酸-亞碸亞胺也會誘導胚胎幹細胞死亡,而血清或血清替代物可以保護胚胎幹細胞免於受到活性氧的傷害。在未加入過氧化氫及丁硫氨酸-亞碸亞胺的情況下,胚胎培養時加入0.5%胎牛血清或10% 血漿注射劑也可以降低胚胎細胞的活性氧累積,並促進胚胎發育。我們的結果顯示血清和血清替代物可以保護著床前胚胎免於活性氧所導致的細胞凋亡,並因此增進胚胎的發育,而這種保護細胞和促進發育的效應,和白蛋白無關,而可能是由其他因子所造成,因此建議在進行人工生殖技術時,於現行只含白蛋白的胚胎培養液中加入血清替代物,以促進體外胚胎發育,進而提升人工生殖技術的成功率。
Assisted reproductive technology (ART) can help infertile couples have theirown baby. High quality embryo culture is critical to the success in ART. Duringembryo culture in vitro, high oxygen concentration (20%) in the incubator and
procedure-related damage such as light exposure may increase reactive oxygenspecies (ROS) concentration in the embryo. ROS accumulation-induced oxidativestress is related to poor embryo development. Serum and serum substitutes have beenfound to not only increase the percentage of embryo development in vitro to theblastocyst stage but also improve embryo implantation and live birth rates; however,
the underlying mechanisms remain unclear and currently most ART Lab add human serum albumin (HSA), but not serum substitute, to the culture medium. We hypothesized that serum and serum substitutes can reduce oxidative stress during embryo culture in vitro, thereby protecting blastomeres from apoptosis and promoting embryo development. In this study, mouse pre-implantation embryos and embryonic stem (ES) cells were utilized and exposed to hydrogen peroxide (H2O2), a potent exogenous ROS generator, and L-Buthionine-sulfoximine (BSO), a γ-glutamylcysteine synthetase inhibitor that could induce endogenous ROS
accumulation. We found that H2O2 induced blastomere apoptosis and embryo development arrest. The addition of 0.5% fetal bovine serum (FBS) and 10% Plasmanate (a serum substitute) to the culture medium could protect blastomeres from H2O2–induced apoptosis and promote embryo development. By contrast, substitutesation of HSA at the same concentration (0.5%) as that in 10% Plasmanate
was not able to prevent H2O2–induced apoptosis and promote embryo development, suggesting that factors other than HSA (such as a and b globulins) were responsible for the cytoprotective effects of 10% Plasmanate. Furthermore, H2O2 and BSO also induced death in ES cells, and serum and serum substitute could protect ES cells from
ROS-induced damage. In culture conditions without H2O2 and BSO challenge, substitutesation with 0.5% FBS or 10% Plasmanate was also found to reduce ROS accumulation and promote embryo development. Our results demonstrated that
serum and serum substitute could protect pre-implantation embryos from ROSinduced apoptosis and thereby promote embryo development, which was mediated by factors other than HSA. We suggest that serum substitute should be added to currently used culture medium containing HSA only, in order to promote embryo development and increase successful rate in ART.
中文摘要........................................................................................................................6
Abstract………………………………………………………………………………..8
Chapter 1. Introduction………………………………………………………………10
A. The progression of embryo culture medium……………………..............…..10
B. Assisted reproductive technology (ART)………………………………...…..11
C. Effects of serum starvation on cells……………………………………….. ..13
D. Effects of ROS on embryo development……………………………………..14
E. Synthetic serum supplements………………………………………………...16
F. Pre-implantation embryo development……………………………………....17
G. Blastocyst grading……………………………………....................................18
H. Embryonic stem cell as a model for embryo research……………………….19
I. Purpose…………………………………….....................................................20
Chapter 2. Materials & Methods……………………………………………………..21
A. Reagents and antibodies……………………………………………………...21
B. ES cell culture………………………………………………………………..21
C. ES cell treatments…………………………………………………….............22
D. Embryo collection and culture……………………………………………….24
E. Detection of blastomere apoptosis with TUNEL assay...................................25
F. Detection of ROS generation in embryo development……...........................26
G. Total antioxidant capacity measurement……………………………………..26
H. Immunoblotting analysis……………….…………………………………….28
I. Statistical analysis……………………………………………………. . ……29
Chapter 3. Results……………………………………………………………………30
4
A. Effects of exogenous ROS on mouse embryo
development………………………………………………………….............30
B. Effect of serum on ES cell proliferation……………………...........................31
C. Exogenous and endogenous ROS could induce ES cell
death ………………….……………………………………………………...31
D. Serum or serum substitutes could prevent ROS accumulation in preimplantation
embryos………………………………………...........................32
E. Serum or serum substitutes could protect embryos from ROS-induced
apoptosis……………………………………………………………………...33
F. Serum could protect ES cells from exogenous and endogenous ROS-induced
cytotoxicity…………………………………………………….......................34
G. Serum substitutes could protect ES cells from exogenous and endogenous
ROS-induced cytotoxicity……………………………………………………35
H. Serum or serum substitutes could promote embryo
development…………………………………………………….....................35
I. Serum substitutes could enhance total antioxidant
capacity…………………………………………………….............................36
J. Serum and serum substitutes could not enhance MnSOD
activity ……..................... …………………………………………...…........36
Chapter 4. Discussion……………………………………………………………...…37
References……………………………………………………………………………62
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