| Summary: | 碩士 === 中國醫藥學院 === 醫學研究所 === 89 === 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 30l 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.
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