臺灣博碩士論文加值系統

主旨
為評估人工合成之白血病抑制因子(recombinant Leukemia Inhibitory Factor, r-LIF)對於新鮮鼠胚(實驗一)與經冷凍與解凍之人類胚胎之於體外延長培養之影響(實驗二)。並進一步分析不同濃度r-LIF對於鼠胚分裂之影響(實驗三)。比較LIF在不孕症及正常病人之不同子宮內膜位置與月經週期之表現差異(實驗四)。及 以LIF基因植入小鼠之子宮內膜(實驗五)。
實驗方法：
實驗一: (r-LIF對於新鮮鼠胚之影響)
6至8週大之CB6F1雌鼠以10 IU之懷孕的母馬血清雌性激素(pregnant mare’s serum gonadotropin)注射以刺激卵子之發育，以10 IU人類絨毛性腺激素(human chorionic gonadotropin) 於腹膜下注射，然後與BDF1雄鼠進行交配。雌鼠以隨機分配之方法分配。第一組: 體內發育組；第二與第三組: 體外發育組。第一組之小鼠於HCG注射後116-120小時後犧牲。第二組及第三組之小鼠於HCG注射後44-48小時後犧牲並取出胚胎。
第一組之鼠胚屬於囊胚期(blastocyst stage)之胚胎，第二組及第三組之鼠胚屬於2-至4-細胞期之胚胎。第二組之鼠胚培養於培養液(human tubal fluid, HTF)及0.5%之人類血清白蛋白(human serum albumin, HSA)之培養液中，並覆蓋油。第三組之鼠胚培養於HSA及r-LIF (1000 IU/ml) 之培養液中並覆蓋油。各組之不同時期胚胎之數量與比例並進一步予以記錄與比較。
實驗二: (r-LIF對於冷凍與解凍人類胚胎之於體外延長培養之影響)
以捐贈之人類胚胎，經快速冷凍與解凍後，根據不同之培養環境區分為四組: (1). HTF + 0.5% HSA; (2). HTF +0.5% HSA + r-LIF (1000 IU/ml LIF); (3) M3TH medium (Medi-Cult); (4) M3TH medium + r-LIF (1000 IU/ml LIF). 經過五天之長期培養，四組之不同時期胚胎發育予以記錄與比較。
實驗三: (不同濃度r-LIF對於鼠胚分裂之影響)
如同實驗一之鼠胚於HCG注射後46-48小時後犧牲小白鼠並取出胚胎。根據不同之r-LIF濃度，鼠胚以隨機分配之方式分配於七組不同之培養環境之中，包括: (1) HTF + 0.5% HSA; (2) HTF + HSA + 1500 IU/ml r-LIF; (3) HTF + HSA + 1000 IU/ml r-LIF; (4) HTF + HSA + 750 IU/ml r-LIF; (5) HTF + HSA + 500 IU/ml r-LIF; (6) HTF + HSA + 250 IU/ml r-LIF; (7) HTF + HSA + 125 IU/ml r-LIF。七組不同時期之胚胎發育予以進一步記錄與比較。
實驗四: (LIF在不孕症及正常病人之不同子宮內膜位置與月經週期之表現差異)
在不孕症及正常病人分別在濾泡期，排卵期，及黃體期接受子宮內膜細胞之取樣，我們偵測黃體期時之血清黃體素濃度，我們並分析子宮內膜上皮細胞，腺體上皮，實體細胞之LIF immunostaining表現差異，及其與黃體素濃度之關聯性‧
實驗五(LIF基因植入小鼠之子宮內膜)
我們使用Expression plasmids攜帶LIF及luciferase基因施行基因植入，將施行過超級刺激排卵之ICR母鼠與閹割公鼠交配後以LIF-liposome (組1)及luciferase-liposome complexes (組2) 注射入其子宮腔中(第0天)‧以RT-PCR之方式偵測第0天至第4天之子宮內膜LIF及luciferase基因表現，並使用Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)應用於基因植入後之內部控制‧
結果:
實驗一: (r-LIF對於新鮮鼠胚之影響)
比較第二組及第三組之鼠胚發育，發現二組之4-至8-細胞期及桑椹期之鼠胚發育比率類似(4-至8-細胞期: 87.3% vs. 91.0%, P <.05; 桑椹期: 74.6% vs. 87.1%, P <.05)。進一步比較第二組及第三組之囊胚期，成熟囊胚期，孵化囊胚期之比例，發現第二組(48.1%, 31.7%, 18.5%, P <.05)之胚胎分裂與發育均較第三組(83.6%, 53.7%, 37.8%, P <.05)為差。
實驗二: ( r-LIF對於冷凍與解凍人類胚胎之於體外延長培養之影響)
比較四組之胚胎之早期發育於2~16細胞期，並無統計學上之差異。進一步比較第一組及第三組其2~桑椹期，並無統計學上之差異；比較第二組及第四組，其2~囊胚期，亦無統計學上之差異。第一組之胚胎發育成桑椹期之比例較第二，三，四組之比例為低，第一與第三組之胚胎發育成囊胚期之比例較第二與第四組之比例為低。
實驗三: (不同濃度r-LIF對於鼠胚分裂之影響)
七組之胚胎之早期發育於2-16細胞期，並無統計學上之差異。進一步比較胚胎植入前之發育(桑椹至孵化囊胚期)，第2, 3, 4, 5組之胚胎(HTF + r-LIF 1500, 1000, 750, 500 IU/ml) 比第1, 6 ,7組(HTF only, HTF + r-LIF 250, 125 IU/ml)之比例為高(P <.05)。
實驗四: (LIF在不孕症及正常病人之不同子宮內膜位置與月經週期之表現差異)
LIF在正常之女性之子宮內膜之表現較不孕症之女性為高，LIF在子宮內膜之表現最高為上皮細胞，其次為腺體上皮，最低為子宮內膜實體細胞，子宮內膜上皮及腺體細胞之表現最低在濾泡期，其次為排卵期，最高為黃體期；子宮內膜實體細胞之LIF表現並無週期性差異；LIF表現與黃體素之濃度並無顯著關聯性‧
實驗五(LIF基因植入小鼠之子宮內膜)
LIF mRNA及luciferase之活性表現最高出現在基因植入後第三天，在第一組中LIF/GADPH在第一至第四天之比例分別為0.414, 1.096, 1.162, 及0.782‧在第一組中LIF/GADPH在第一至第四天之比例分別為0.24, 0.22, 0.35, 及0.32‧
結 論
對於早期之鼠胚與經冷凍與解凍之人類胚胎體外培養，r-LIF並無法提供明顯胚胎發育之幫助。對於較晚期植入前之胚胎(pri-implantative stage；桑椹至孵化囊胚期)，r-LIF能明顯提升胚胎之生長、分化與孵化。於HTF溶液之中加以r-LIF能提供類似M3 TH溶液之培養環境。500 IU/ml.為r-LIF理想之最低濃度。
子宮內膜細胞之LIF表現於人類之不孕症發生有關，LIF表現亦與人類子宮內膜之細胞位置與月經週期有關，較強之LIF表現於黃體期時之人類子宮內膜表皮細胞‧小鼠之子宮內膜可以使用liposome-DNA之混合液予以基因轉殖入，未來將可能應用此方式於人類之基因治療‧

Part 1. Recombinant Leukemia Inhibitory Factor (r-LIF) Enhance Pre-implantation Mouse Embryo Development In Vitro
Objective: To assess the effect of human recombinant leukemia inhibitory factor (r-LIF) in mouse embryos.
Materials and Methods: Female CB6F1 mice that were between 6 and 8 weeks old were superovulated by 10 IU pregnant mare‘s serum gonadotropin (PMSG) and 10 IU HCG intra-peritoneally; then mated with BDF1 male mice. Mice were divided randomly into three Groups, which included 1 Group of in vivo (Group 1) and 2 Groups of in vitro study (Group 2, 3). In Group 1 (control Group), mice were killed after HCG injection 116-120 hours. In Group 2 and 3, mice were killed after HCG injection 44-48 hours. The 2-cell embryos (Group 2, 3) and blastocyst stage embryos (Group 1) were washed by flush medium from the ampulla after the excision of the oviduct. In Group 2 (HTF + HSA), embryos were co-cultured with 30l microdroplets of human tubal fluid (HTF) +0.5% human serum albumin (HSA). In Group 3 (HTF + r-LIF): mouse embryos were co-cultured with HTF and r-LIF (1000 IU/ml) under paraffin oil. The embryonic numbers in different stage including 4-8 cell, morula, blastocyst, expanded blastocyst, and hatching blastocyst were recorded and compared.
Results: Similar embryos development to 4-8 cell and morula stages were noted between Group 2 and 3 (87.3% vs. 91.0%; 74.6% vs. 87.1%, respectively). However, further embryo development in blastocyst, expanded, and hatching blastocyst in Group 2 (48.1%, 31.7%, 18.5%) were lower than that of Group 3 (83.6%, 53.7%, 37.8%).
Conclusion : R-LIF does not provide the obvious stimulation upon the early development of mice embryo. However, r-LIF has positive effects on pre-implantation blastocyst growth, differentiation and hatching.
Part 2. The effect of different concentrations of Recombinant leukemia inhibitory factor (LIF) on different development stage of mouse embryo in vitro
Purpose: To assess the influence of different concentrations of recombinant human leukemia inhibitory factor (LIF) on the in-vitro development of mouse embryos.
Materials and methods: The 2-4 cell embryos of CB6F1 mice were culture in the human tubal fluid (HTF) media containing different concentration of LIF. Mouse embryos were divided into 7 groups: (1) HTF; (2) 1500 IU/ml LIF; (3) 1000 IU/ml LIF; (4) 750 IU/ml LIF; (5) 500 IU/ml LIF; (6) 250 IU/ml LIF; (7) 125 IU/ml LIF. The embryonic numbers of different stages including 5-8 cell, 9-16 cell, morula, blastocyst and hatching blastocyst were recorded.
Results: The percentage of early embryo stage (2-cell embryos to 6-16 cell stages) in all groups were non-significantly different. There were higher formation rates of pre-implantation embryos (morula to hatching blastocyst) in groups 2, 3, 4 and 5 than in groups 1, 6 and 7.
Conclusion : LIF has positive effects on pre-implantation embryo development and has non-significant influence upon the early embryo development. The lowest concentration of LIF which could provide the optimal embryo development is 500 IU/ml.
Part 3. Prolonged culture of human cryopreserved embryos with recombinant human leukemic inhibitory factor (rhLIF)
Purpose: To evaluate the efficiency of recombinant human leukemic inhibitory factor (LIF) in the prolonged culture of human cryopreserved-thawing embryos.
Patients and methods: After thawing, all embryos were divided into four groups: (1) Human tubal fluid (HTF); (2). HTF + LIF; (3) M3TH medium; (4) M3TH medium + LIF. In the following prolong culture, the embryo development in each groups were compared.
Result(s): About the embryo development from 2-4 cell to 9-16 cell stage, there were non-different between each group. There was lower morula formation rate in group 1 (6.9%) than those in other groups (23.2%, 19.7%, 23.1%). The lower blastocyst formation in group 1 and 3 (0%, 0%) than those in group 2 and 4 (11.0%, 12.8%) were noted.
Conclusion(s): LIF is beneficial for pre-implantation embryos. LIF does not influence the early embryo development. The LIF-supplemented HTF provided a similar culture environment for thawing embryos as M3 TH medium.
Part 4. Leukemia Inhibitory Factor (LIF) expression in different endometrial location between fertile and infertile women throughout different menstrual phases
Purpose: To demonstrate the leukemia inhibitory factor (LIF) expression in different endometrial locations between fertile and infertile women throughout different menstrual phases. The relationship between progesterone level and LIF expression were evaluated.
Patients and methods: Idiopathic infertile and normal fertile women accepted the endometrial biopsies in follicular, periovulatory, and luteal phases. The luteal progesterone level was measured. Endometrial LIF immunostaining of luminal epithelium, glandular epithelium, and stroma were detected. The relationship between luteal LIF expression and progesterone level was evaluated.
Results: Significant LIF expression was noted in the endometrium of fertile women than that of infertile women. The LIF expression was highest in the luminal epithelium, moderate in the glandular epithelium, and lowest in the stroma. The luminal and glandular epithelial staining were lowest in follicular phase, moderate in periovulatory phase, and strongest in luteal phase. The stromal LIF presented with a non-cyclical manner. The LIF expression is not related with the progesterone level.
Conclusion: Endometrial LIF expression is related with human fertility. Endometrial LIF expression is dependent on cellular localizations and menstrual stages. Stronger LIF expression presents in the endometrial epithelium during luteal phase.
Part 5. In-vivo gene transfer of leukemia inhibitory factor into mouse endometrium
Objectives: Leukemia inhibitory factor (LIF) is important for embryogenesis and implantation. We aimed to transfect LIF gene into the mouse endometrium.
Patients and methods: Expression plasmids carried LIF and luciferase genes for transfer. After superovulation, 100 ICR mice were mated with vasectomized mice. Then LIF-liposome (Group 1) and luciferase-liposome complexes (Group 2) were injected into their uterine lumen (day-0). Endometrial LIF and luciferase expressions were detected by reverse RT-PCR on day-0 to 4 post gene transfer. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control to normalize the gene transfection.
Results: LIF mRNA and luciferase activities reached the peak expression on day-3. In Group 1, the ratios of LIF/GADPH on day-1 to 4 were 0.414, 1.096, 1.162, and 0.782. In Group 2, LIF/GADPH on day-1 to 4 were 0.24, 0.22, 0.35, and 0.32
Conclusions: Mouse endometrium could be effectively transfected with liposome-DNA mixtures. Endometrial LIF transfer via liposome may be effective in human.

誌謝 ----------------------------------------------------- P.1
中文摘要 --------------------------------------------------P.2
主旨----------------------------------------------------P.2
實驗方法----------------------------------------------- P.2
結果--------------------------------------------------- P.4
結 論-------------------------------------------------- P.6
目錄------------------------------------------------------ P.7
表目錄---------------------------------------------------- P.9
符號與縮寫------------------------------------------------ P.10
主文 ----------------------------------------------------- P.11
前言----------------------------------------------------P.11
LIF之發展歷史-------------------------------------------P.12
LIF的生物活性-------------------------------------------P.13
LIF在子宮及輸卵管的表現-------------------------------- P.14
LIF對不同時期胚胎之影響-------------------------------- P.16
r-LIF 之發展--------------------------------------------P.16
研究目的----------------------------------------------- P.17
材料與方法----------------------------------------------P.18
Medium 之製備-------------------------------------------P.24
r-LIF 之製備--------------------------------------------P.25
統計------------------------------------------------------ P.25
結果------------------------------------------------------ P.26
表格------------------------------------P.27, 29, 31, 33, 35-38
討論--------------------------------- ----------------- P.39
LIF之作用機轉-------------------------------------------P.40
LIF對於人胚與鼠胚之影響---------------------------------P.41
LIF對於胚胎共同培養之優點-------------------------------P.41
體外培養溶液之影響--------------------------------------P.42
人類血清白蛋白(human serum albumin, HSA)之影響----------P.42
不同LIF濃度對於胚胎發育之影響---------------------------P.42
LIF不同子宮內膜位置與月經週期之表現---------------------P.43
LIF之基因轉殖-----------------------------------------P.43 結論與未來研究、應用方向---------------------------------- P.45
參考文獻 ---------------------------------------------- P.47
英文摘要 ---------------------------------------------- P.55
作者簡歷 ------------------------------------------------- P.59
著作權聲明 ----------------------------------------------- P.55

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