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)。
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。第一組之老鼠於HCG注射後116-120小時後犧牲。第二組及第三組之老鼠於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。
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)。
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。

Application of Recombinant Leukemia Inhibitory Factor (r-LIF) upon Reproduction
INTRODUCTION: Leukemia inhibitory factor (LIF) is a pleiotropic cytokine of the interleukin-6 family and has different biological actions in various tissue systems. LIF can regulates the growth and differentiation of embryonic stem cells, primordial germ cells, peripheral neurons, osteoblasts, adipocytes, and endothelial cells. It is also crucial for successful implantation of the embryo in mice. The role of LIF in reproduction will be discussed.
ABSTRACT
Objective: To assess the effect of human recombinant leukemia inhibitory factor (LIF) in in the prolonged culture of fresh mouse embryos (Experiment 1) and human cryopreserved-thawing embryos (Experiment 2). The influence of different concentration of LIF in the in-vitro development of mouse embryos was also evaluated (Experiment 3).
Materials and Methods:
Experiment 1: 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 30ml microdroplets of human tubal fluid (HTF) +0.5% human serum albumin (HSA). In Group 3 (HTF + HSA+ 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.
Experiment 2: After thawing, all human embryos were divided into four groups: (1) Human tubal fluid (HTF); (2). HTF + LIF (1000 IU/ml LIF); (3) M3TH medium; (4) M3TH medium + LIF (1000 IU/ml LIF). In the following 5 days post thawing, the embryo development in each groups were compared.
Experiment 3: All 2-4 cell embryos of Female CB6F1 mice were culture in the human tubal fluid (HTF) media which contains 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:
Experiment 1: 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). 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%)(p<.05).
Experiment 2: There were non-different in the early embryo development (2-4 cell to 9-16 cells) between each group. There was non-different between group 1 and 3 (2-4 cell to morula) and group 2 and 4 (2-4 cell to blastocyst). There was lower morula formation in group 1 than other groups and the lower blastocyst formation in group 1 and 3 when compare with group 2 and 4 were noted.
Experiment 3: The percentage of early embryo stage (2-cell embryos to 6-16 cell stages) in each groups were non-significant different. There were higher formation rate of pre-implantation embryos (morula to hatching blastocyst) in group 2, 3, 4 and 5 than those in group 1, 6 and 7.
Conclusion: R-LIF does not provide the obvious stimulation upon the early development of mouse and human embryos. R-LIF has positive effects on pre-implantation blastocyst growth, differentiation and hatching of mouse and human embryos. The r-LIF supplemented HTF could provided a similar culture environment for thawing embryos as M3 TH medium. The lowest concentration of r-LIF which could result in the optimal embryo development is 500 IU/ml.
Keywords: r-LIF, leukemia inhibitory factor, coculture

目 錄
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.5
目錄 ------------------------------------------------------------------------------------- P.6
表目錄 ---------------------------------------------------------------------------------- P.8
圖目錄 ---------------------------------------------------------------------------------- P.9
符號與縮寫 --------------------------------------------------------------------------- P.10
主文 ------------------------------------------------------------------------------------- P.12
前言 -----------------------------------------------------------------------P.12
LIF之發展歷史 ---------------------------------------------------------P.13
LIF的生物活性 ---------------------------------------------------------P.14
LIF在子宮及輸卵管的表現 ---------------------------------------- P.15
LIF對不同時期胚胎之影響 ---------------------------------------- P.17
r-LIF 之發展 -------------------------------------------------------------P.17
研究目的 ---------------------------------------------------------------- P.18
LIF對於人類胚胎體外培養之影響 --------------------------- P.18
材料與方法 -------------------------------------------------------------P.19
r-LIF 之置備 ---------------------------------------------------------P.25
統計 ------------------------------------------------------------------------------------ P.26
結果 ------------------------------------------------------------------------------------ P.27
表格 ------------------------------------------------------------------------------------ P.29
圖片 ------------------------------------------------------------------------------------ P.33
討論 ------------------------------------------------------------------------------------ P.35
細胞素及生長因子 --------------------------------------------------- P.36
LIF之作用機轉 -------------------------------------------------------- P.36
本研究之實驗結果 ---------------------------------------------------- P.37
人工合成之白血病抑制因子(r-LIF)相對於共同培養之優點-- P.37
體外培養之溶液 ------------------------------------------ P.38
人類血清白蛋白之影響 -------------------------------- P.38
不同之r-LIF濃度對於胚胎發育之影響 ------------ P.38
結論 ---------------------------------------------------------- P.40
未來研究、應用方向 ----------------------------------- P.41
參考文獻 ------------------------------------------------------------------------------- P.43
英文摘要 ------------------------------------------------------------------------------- P.50
作者簡歷 ------------------------------------------------------------------------------- P.54
著作權聲明 ---------------------------------------------------------------------------- P.55

1. Ichikawa Y. Differentiation of a cell line of myeloid leukemia. Journal of cellular physiology 1969;74,223-234.
2. Smith AG, Heath JK, Donaldson DD, Wong GG, Moreau J, Stahl M and Rogers D. Inhibition of pluripotential embryonic stem cell differentiation by purified polypeptides. Nature, 1988;366(15),688-690.
3. M. Murphy ,K. Reid, D. J. Hilton, and P.F. Bartlett. Generation of sensory neuron is stimulated by leukemia inhibitory factor. Proceedings of the National Academy of Sciences of the United States of American. 1991;88: 3498-3501.
4. Nachtigall MJ, Kliman HJ, Feinberg RF, Olive DL, EnginO, Arici A. The effect of leukemia inhibitory factor (LIF) on trophoblast differentiation: A potential role in human implantation. Journal of Clinical Endocrinology and Metabolism. 1996;81:801-961.
5. Gough NM, Gearing DP, Ling JA, Willson TA. Hilton DJ, Nicola NA, and Donald Metcalf. Molecular cloning and expression of the human homologue of the murine gene encoding myeloid leukemia inhibitory factor. Proceedings of the National Academy of Sciences of the United States of American. 1988;85,2623-2637.
6. Hilton DJ, Nicola NA, Gough NM, and Donald Metcalf. Resolution and purification of three distinct factors produced by Kerbs Ascites cells which have differentiation-inducing activity on murine myeloid leukemic cell lines. The Journal of Biological Chemistry, 1988; 263(19): 9238-9243.
7.Hilton DJ, Niicola NA, Metcalf D. Specific binding of murine leukemia inhibitory factor to normal and leukemic monocytic cells. Proceedings of the National Academy of Sciences of the United States of American. 1988;85:5971-5975.
8. Sutherland GR, Baker E, Hyland VJ, Callen DF, Stahl J, Gough NM. The gene for human leukemia inhibitory factor maps to 22q12. Leukemia 1989;3(1):9-13.
9. Kola I, Davey A, Gough NM. Localization of the murine leukemia inhibitory factor gene near the centromere on chromosome 11. Growth Factors 1990;2:235-240.
10. Wiemer KE, Huffman DI, Masxon WS, Eager S, Muhlberger B, Fiore I, Cuervo M. Embryonic morphology and rate of implantation of juman embryos following coculture on bovine oviductal epithelial cells. Human Reproduction. 1993;8:97-101.
11.Menezo YJR, Guerin JF, Czyba JC. Improvement of juman early embryo development in vitro by coculture on monolayers of Vero cells. Biology of Reproduction. 1990;42:301-306.
12. Watson AH, Hogan A, Hahnel A, Wiemer KE, Schultz GA. Expression of growth factor ligand and receptor genes in the preimplantation bovine embryo. Molecular Reproduction and Development. 1992;31:87-95.
13. Tsai HD, Chang CC, Hsieh YY, Lo HY, Hsu LW, Chang SC. Recombinant human leukemia inhibitory factor enhances the development of preimplantation mouse embryo in vitro. Fertility and Sterility. 1999;71:722-725.
14. Cheng L, Gearing DP, White LS, Compton DL, Schooly K, Donovan PJ. Role of leukemia inhibitory factor and its receptor in mouse primordial germ cell growth. Development 1994;120:3145-3153.
15. Michell MH, Swanson RJ, Hodgen GD, Oehninger S. Enhancement of in vitro murine embryo development by recombinant leukemia inhibitory factor. J Soc Gynecol Invest 1994;1:215-219.
16. Stewart CL, Kaspar P, Brunet LJ, Bhatt H, Gadi I, Kontgen F, et al. Blastocyst implantation depends on maternal expression of leukemia inhibitory factor. Nature 1992;359:76-79.
17. Moreau JF, Donaldson DD, Bennett F, Giannotti JW, Clark SC, Wong GG. Leukemia inhibitory factor is identical to the myeloid growth factor human interleukin for DA cells. Nature 1988;336(15):690-692.
18. Yamamori T, Fukada K, Aebersold R, Korsching S, Fann MJ, Patterson PH. The cholinergic neuronal differentiation factor from heart cells is identical to leukemia inhibitory factor. Science 1989;246:1412-1416.
19. Metcalf D, Hilton D, Nicola NA. Leukemia inhibitory factor can potentate murine megakaryocyte production in vitro. Blood 1991;77(10):2150-2153.
20. Williams RL, Hilton DJ, Pease S, Willison TA, Steart CL, Gearing DP, Wagner EF, Metcalf D, Nicola NA, Gough NM. Myeloid leukemia inhibitory factor maintains the developmental potential of embryonic stem cells. Nature 1988;336(15):684-687.
21. Conish J, Callon K, King A, Edgar S, Reid IR. The effect of leukemia inhibitory factor on bone in vivo. Endocrinology 1993;132:1359-1366.
22. Bhatt H, Brunet LJ, Stewart CL. Uterine expression of leukemia inhibitory factor coincides with the onset of blastocyst implantation . Proceedings of the National Academy of Sciences of the United States of American 1991;88:11408-11412.
23. Stewart CL. Leukemia inhibitory factor and the regulation of preimplantation development of the mammalian embryo. Molecular Reproduction and Development. 1994;39:233-238.
24. Zeng-ming Yang, Su-ping Le, Dong-bao Chen, and Michael J.K. Harper. Temporal and Sepatial Expression of Leukemia inhibitory factor in rabbit uterus during early pregnancy. Molecular Reproduction and Development. 1994;38:148-152.
25. Charnok-Jones DS, Sharkey AM, Fenwick P, Smith SK. leukemia inhibitory factor mRNA concentration peaks in human endometrium at the time of implantation and the blastocyst contains mRNA for the receptor at the time. Journal of Reproduction and Fertility. 1994;101:421-426.
26. Kojima K, Kanzaki H, Iwai M, Hatayama H, Fujimoto M, Inoue T, Horie K, Nakayama H, Fujita J, Mori T. Expression of leukemia inhibitory factor in human endometrium and placenta. Biology of Reproduction. 1994;50:882-887.
27. Aydin Archi, Oguz Engin, Erkut Attar, and David L. Olive. Modulation of leukemia inhibitory factor gene expression and protein biosynthesis in human endometrium. Journal of Clinical Endocrinology and Metabolism. 1995;80(6):1908-1915.
28. Emily B. Cullinan, Susan J. Abbondanzo, Patrick S. Anderson, Jeffrey W. Pollard, Bruce A. Jessey, and Colin L. Stewart. Leukemia inhibitory factor (LIF) and LIF receptor expression in human endometrium suggests a potential autocrine/paracrine function in regulating embryo implantation. Proceedings of the National Academy of Sciences of the United States of American. 1996;93;3115-3120.
29. Kurzrock R, Estrov Z, Wetzler M, Gutterman JU, Talpaz M. LIF: Not just a leukemia inhibitory factor. Endocrine Reviews. 1991;12:208-217.
30. Fry RC, Batt PA, Fairclough RJ, Parr RA. Human leukemia inhibitory factor improves the viability of ovine embryos. Biology of Reproduction. 1992; 46:470-474.
31. Dunglison GF, Barlow DH, Sargent IL. Leukaemia inhibitory factor significantly enhances the blastocyst formation rates of human embryos cultured in serum-free medium. Human Reproduction. 1996;11:191-196.
32. Jurisicova A, Ben-Chetrit A, Varmuza SL, Casper RF. Recombinant human leukemia inhibitory factor does not enhance in vitro human blastocyst formation. Fertility and Sterility. 1995;64:999-1002.
33. Chang SC, Wang F, Hsu LW. Expression of recombinant human leukemia inhibitory factor (rhLIF) in Escherichia Coli. Medical Journal of China Medical College Hospital. 1997;2:6-11.
34. Lavranos TC, Rathjen PD, Seamark RF. Trophic effects of myeloid leukaemia inhibitory factor (LIF) on mouse embryos. Journal of Reproduction and Fertility. 1995;105:331-338.
35. Eckert J, Tao T, Niemann H. Ratio of inner cell mass and trophoblastic cells in blastocysts derived from porcine 4- and 8-cell embryos and isolated blastomeres cultured in vitro in the presence or absence of protein and human leukemia inhibitory factor. Biology of Reproduction. 1997;57:552-560.
36. Tasi HD, Chen CM , Lo HY, Chang CC. Subcutaneous low does Leuprolide Acetate Depot Versus Leuprolide Acetate for Women Undergoing Ovarian Stimulation for in-Vitro Fertilization .Human Reproduction. 1995;10:2909-2912.
37. Hsieh YY, Tsai HD, Chang CC, Chang CC, Lo HY, Andrew Lai CH. Ultra-rapid cryopreservation of human embryos---1582 embryo experience. Fertility and Sterility 1999, in press.
38. Lai AC, Lin BP, Chang CC, Tsai HD, Hwang VW, Lo HY. Pregnancies after transfer of ultrarapidly frozen human embryos. Journal of Assisted Reproduction and Genetics. 1996;13: 625-628.
39. Edward RG. New concepts in embryos growth and implantation. Human Reproduction 1998;13:supplement: 271-282.
40. Harvey MB, Leco KJ, Arcellana-Panlilio MY, Zhang X, Edward DR, Schultz GA. Proteinase expression in early mouse embryos is regulated by leukemia inhibitory factor and epidermal growth factor. Development 1955;121:1005-1014.
41. Charnok-Jones DS, Sharkey AM, Fenwick P, Smith SK. leukemia inhibitory factor mRNA concentration peaks in juman endometrium at the time of implantation and the blastocyst contains mRNA for the receptor at the time. Journal of Reproduction and Fertility 1994;101:421-426.
42. Virginia R. Coculture cells that express leukemia inhibitory factor (LIF) enhances mouse blastocyst development in vitro. Journal of Assisted Reproduction and Genetics 1995;12:153-156.
43. Gardner DK, Lane M. Alleviation of the 2-cell block and development to the blastocyst of CF1 mouse embryos: role of amino acids, EDTA, and physical factors. Human Reproduction 1996;11:2703-2712.
44. Lane M, Gardner DK. Differential regulation of mouse embryo development and viability by amino acids. Journal of Reproduction and Fertility 1997;109:153-164.
45. Bolton VN, Hawes SM, Taylor CT, et al. Development of spare human preimplantation embryos in vitro: An analysis of the correlation among gross morphology, cleavage rates, and development to the blastocyst. Journal of In Vitro Fertility and Embryo Transfer 1989;6:30-35.
46. Quinn P, Kerlin JF, Warnes GM. Improved pregnancy rate in human in vitro fertilization with the use of a medium based on the composition of human tubal fluid. Fertility and Sterility 1985;44:493-498.
47. Jones GM, Trounson AO, Gardner DK, Kausche A, Lolatgis N, Wood C. Evolution of a culture protocol for successful blastocyst development and pregnancy. Human Reproduction 1998;13:169-177.
48. Dale PO, Kjekshus E, Tanbo T, Holst N, Abyholm T. Clinical outcome of day 3 versus day 2 embryo transfer using serum-free culture media. Abstract of the 10th Annual Meeting of the ESHRE, Brussels 1994 (No. 068).
49. Forsdahl F, Bertheussen K, Bungum LJ, Bungum MH, Willumsen J, Maltau JM. A study of extended culture time with embryo replacement of the morula and blastocyst stage. Abstract of the 10th Annual Meeting of ESHRE, Brussels 1994 (No. 375).
50. Gardner DK, Vella P, Lane M, Wagley L, Schlenker T, Schoolcraft WB. Culture and transfer of human blastocysts increases implantation rates and reduces the need for multiple embryo transfers. Fertility and Sterility 1998;69:84-88.
51. M Iean, S Mirallie, P Barriere, A Godard, A Mensier, P Lopes, Aydin Arici. Leukemia inhibitory factor expression in human follicular fluid. Human Reproduction 1999;14:571-572.