腹腔巨噬细胞对小鼠卵受精率和囊胚形成率的影响

发布时间：2017-12-31 10:17

本文关键词：腹腔巨噬细胞对小鼠卵受精率和囊胚形成率的影响　出处：《中国协和医科大学》2006年硕士论文　论文类型：学位论文

【摘要】：腹腔巨噬细胞对小鼠卵受精率和囊胚形成率的影响近年来子宫内膜异位症(endometriosis，EM)发病率不断上升，约5％～15％的育龄期妇女受其影响，其与不育症的关系日益成为研究的热点，但其病因与发病机制尚未完全阐明，EM患者分泌的腹腔液增多，腹腔液中巨噬细胞数量增多、功能增强，腹腔液和血清中山巨噬细胞分泌的细胞因子也明显增多，且与病变程度成正比。这些与不育是否有关、关系如何，目前尚不十分清楚。国内外的研究对巨噬细胞对囊胚形成的作用意见不一致。本研究拟通过建立小鼠体外受精模型，观察巨噬细胞对小鼠卵子受精率和囊胚形成率的影响。研究目的一．建立小鼠体外受精模型二．巨噬细胞对小鼠卵子受精率和囊胚形成率的影响 1．分离、培养及鉴定小鼠腹腔巨噬细胞； 2．昆明小鼠精子提取； 3．昆明小鼠的药物诱导排卵； 4．昆明小鼠的取卵； 5．小鼠卵子与巨噬细胞体外共培养，观察其对小鼠卵受精率和囊胚形成率的影响。研究方法 1．观察小鼠不同周龄(＜8周，8周～10周，10～14周和＞14周)，不同体重(32克，32克～40克和)40克)，以及促排卵药物不同剂量(5IU、10IU和15IU)和不同状态(冷冻还是新鲜)下，，所取出鼠卵的数量和质量，确定最佳的小鼠状态和药物剂量建立昆明小鼠体外诱导排卵模型。 2．利用冰盐水对小鼠进行腹腔内注射，诱导腹腔巨噬细胞产生，经分离纯化，通过鉴定证明收获细胞约95％为巨噬细胞备用。 3．通过设立实验组和对照组，取卵后随机把卵分成三组：①对照组，进行小鼠卵子单独培养，培养过程中不与巨噬细胞共培养；②实验组1：取卵后即与巨噬细胞进行共培养，观察巨噬细胞作为共培养体系对小鼠卵子受精率和囊胚形成过程的影响；③实验组2：在受精50小时～55小时后，即在8细胞形成前后与巨噬细胞共培养进行共培养，观察巨噬细胞作为共培养体系对小鼠受精卵囊胚形成率的影响。研究结果 1．建立的标准体外受精模型，以昆明小鼠为实验材料，小鼠最佳周龄是10～14周，体重为32克～40克，所用促排卵药物以HMG10IU和HCG10IU为最佳，取卵数量最多，质量最好。 2．腹腔诱导的巨噬细胞经过染色证明收获细胞约95％为巨噬细胞，且一只小鼠腹腔收获的细胞数目，如果在1个3653培养皿内池中1ml培养液里约为10~6。 3．与未加巨噬细胞的对照组(受精率为91.0％)和实验组2(受精率为93.7％)比较，受精时即与巨噬细胞共培养的实验组1受精率为72.0％，明显降低，差异有统计学意义(p＜0.001)。在受精后与巨噬细胞共培养的实验组2受精卵的囊胚形成率为78.2％明显高于对照组的受精卵的囊胚形成率60.3％，差异有统计学意义(p＜0.001)。 4．受精后与巨噬细胞共培养的实验组2的卵子囊胚形成率73.2％高于对照组的卵子囊胚形成率54.8％，差异具有显著的统计学意义；高于实验组1的卵子囊胚形成率50.0％，具有显著的统计学差异。 5．任何一个时间点观察都发现加巨噬细胞的实验组较对照组里的细胞分裂要快，提前约7～8小时，细胞分裂质量好，碎片少，囊胚形成的时间提前。结论 1．昆明小鼠的体外受精模型以10周～14周，32克～40克昆明小鼠为最佳，所用促排卵药物以HMG10IU和HCG10IU为最佳，取出的卵子数量最多质量最好。 2．受精阶段与巨噬细胞共培养，可降低小鼠卵子的受精率。 3．与巨噬细胞共培养对受精卵的卵裂、胚胎的生长发育和囊胚形成有明显的促进作用。 4．在8细胞形成前后开始添加巨噬细胞共培养，能最大程度增加囊胚形成率。
[Abstract]:Effects of peritoneal macrophages on the fertilization rate and blastocyst formation rate of mouse eggs
In recent years, endometriosis (endometriosis, EM) the rising incidence of about 5% to 15% of women of childbearing age affected, its relationship with infertility has become a research hotspot, but its etiology and pathogenesis has not been fully elucidated, peritoneal fluid secretion in patients with EM increased, the number of macrophages in the peritoneal fluid. And enhance the function of cytokines in peritoneal fluid and serum Zhongshan macrophages also increased significantly, and the lesion is proportional to the degree. These are associated with infertility, how the relationship, is currently unclear. Research at home and abroad on the effect of macrophages on blastocyst formation views are not consistent. The purpose of this study was to establish in vitro fertilization of mouse model to observe the rate of macrophage, influence on the formation of the fertilization rate of eggs and mouse blastocysts.
research objective
1. Establishment of a model of in vitro fertilization in mice
Two. The effect of macrophage on the rate of fertilization and the rate of blastocyst formation in mice
1., the mouse peritoneal macrophages were cultured and identified.
2. sperm extraction from Kunming mice;
3. the drug induced ovulation in Kunming mice.
4. the eggs of Kunming mice were taken.
5. mouse eggs and macrophages were co cultured in vitro, and the effects on the fertilization rate and the blastocyst formation rate of mouse eggs were observed.
research method
1. mice of different ages (less than 8 weeks, 8 weeks to 10 weeks, 14 weeks and 10 to > 14 weeks), different weight (32 grams, 32 grams to 40 grams and 40 grams)), and ovulation induction with different doses (5IU, 10IU and 15IU) and different state (frozen or fresh), then the quantity and quality of egg, determine the ovulation inducing mouse model and the optimal dose of drugs to establish Kunming mice in vitro.
2., intraperitoneal injection of ice saline into mice was used to induce peritoneal macrophages production. After isolation and purification, 95% of the harvested cells were identified as macrophages.
3. through the establishment of experimental group and control group randomly after oocyte eggs into three groups: the control group, the mouse eggs cultured alone, and not in the training process of macrophage co culture; the experimental group: 1 oocytes after co cultured with macrophages, macrophage was observed as system on formation process of fertilization rate the egg and mouse blastocysts were cultured; the experimental group: 2 in 50 hours to 55 hours after fertilization, namely before and after the formation of macrophage in 8 cells were co cultured macrophages were cultured as the rate of observation system influence on the formation of mouse embryo blastocysts were cultured.
Research results
1. the standard in vitro fertilization model established by Kunming mice was 10~14 weeks and the body weight was 32 grams to 40 grams. The best ovulation agents were HMG10IU and HCG10IU. The number of eggs was the largest and the quality was the best.
2., peritoneal macrophages were stained by macrophages. About 95% of the harvested cells were macrophages, and the number of cells harvested from abdominal cavity in one mouse was 10~6. in the 1 culture medium of 3653 1ml.
The control group 3. with macrophages (the fertilization rate was 91% (2) and the experimental group fertilization rate was 93.7%), fertilization with macrophages co cultured in experimental group 1 fertilization rate of 72%, significantly decreased, the difference was statistically significant (P < 0.001). Co cultured with macrophages after fertilization in the experiment group 2 fertilized blastocyst formation rate was 78.2% higher than the control group obviously fertilized blastocyst formation rate was 60.3%, the difference was statistically significant (P < 0.001).
4. after fertilization, the percentage of blastocyst formation in 2 of the experimental group co cultured with macrophages was 73.2% higher than that of the control group. The difference in egg blastocyst formation rate was 54.8%. The difference was statistically significant. The ovum blastocyst formation rate was 1 higher than that in the experimental group 50%, with significant statistical difference.
5. at any time point, we found that the macrophages in the experimental group were faster than those in the control group, and about 7~8 hours earlier. The cell division quality was good, the fragments were few, and the time of blastocyst formation was ahead.
conclusion
1. the Kunming mouse's in vitro fertilization model is 10 weeks to 14 weeks, 32 grams to 40 grams of Kunming mice is the best, the best ovulation drugs are HMG10IU and HCG10IU, and the number of eggs taken is the best.
The 2. fertilization stage and macrophage co culture can reduce the fertilization rate of mouse eggs.
3. with macrophage co culture, the cleavage of the fertilized egg, the growth and development of the embryo and the formation of blastocyst were obviously promoted.
4. the macrophage co culture was added before and after the formation of 8 cells, and the rate of blastocyst formation could be increased to the maximum.

【学位授予单位】：中国协和医科大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R711.71;R711.6;R321

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