Optimization Of Three-dimensional Rabbit Uterine Culture System In Vitro And Embryo Implantation

People learn a little of the development of early embryo, especially the course of development in uterus, while hormone ,nerve, body fluid and endocrine influence the change in vivo. Respecting uterus have an unable to be substituted important contribution and function in the development of embryos and the study of human generation, more and more researchers aware that artificial uterine have important value of science and perspective in the clinical application, establishing tissue engineering artificial uterine has become one of the hot spot in the research sphere of tissue engineering.The study on isolation and culture of rabbit endometrial cells and smooth muscle cells, the results showed that three kinds of cells were successful isolated and purified in vitro; these three kinds of cells acted as seed cells, which were seeded in the mixture of collagen and Matrigel, three dimensional uterine lamellar were built in vitro, which composed of three layers of cells (smooth muscle cell, stromal cell and epithelial cell), through changing the condition of culture , for example: the concentration of estrogen and progestin or added to some growth factors in the culture medium, glandular tube and blood vascular were induced in vitro, three dimensional endometrium was similar to normal structure; Under the effect of estrogen and progestin, three dimensional uterine tissue acted as a model, stromal cells generated decidualization, which imitated normal receptive uterus. Mouse blastocysts and three dimensional uterine tissue coculture, which was used to observe embryos development on the uterine slice, in order to even more learn the early development event of embryo, which is more feasible for deeply learned on early event of embryos development, especially wished that the model could imitate the normal physiological course of embryo early development in the uterine. The mainly results of the research as following:1. The research adopted the way of filtration through copper screen and centrifuge at different speed to isolate endometrial cells, comparing two kinds of ways, the conclusion indicated that the way of centrifuge at different speed was more simple and shortcut, and cells were less contaminated, the number of acquired cells was a great quantity. Two kinds of cells were purified through differential attachment technique, which could acquire more purify endometrial stromal cells and epithelial cells.2. Immunohistochemisty identified the type of cells, mouse antihuman cytokeratin 18 stained, epithelial cells positive, cells show brown, mouse antihuman vimentin stained, stromal cells positive, cells show brown, mouse antihumanα-actin stained, smooth muscle cells positive, cells show brown. The purity of cells was identified by immunofluorescence, the purity of epithelial cells was 97%, the purity of stromal cells was 98%, and the purity of smooth muscle cells was 98%.3. Endometrial cells and smooth muscle cells ahead of 3 generation acted as the seed cells, liquidⅠtype collagen and Matrigel acted as scaffold, under the condition of static state tensile force, uterine tissue were established in vitro. Cultured uterine tissue were taken out of the flask and fixed on 14th days, the structure were observed through H.E staining and immunohistochemistry staining. Conclusion indicated that established uterine tissue emerged evident three layers of structure, the growth of cells showed directionality under condition of static state tensile force, epithelial cells showed some polarity and column aspect. Through scan electron microscope and transmission electron microscope observed cells structure of uterine tissue, the conclusion indicated that the cellular structure integrity, the surface of epithelial cells emerge some microvilli, between cells and cells had tight junction.4. Primary endometrial cells and smooth muscle cells of rabbit acted as seed cells, the mixture of epithelial cells and stromal cells seeded in liquidⅠtype collagen and Matrigel, in this base, the condition of culture was optimized, which precipitated vascular epithelial cells and glandular epithelial cells to emerge shape and construction. First time established uterine tissue was similar to normal uterus in shape and function, which emerged glandular tube and blood vessel, and the surface of tissue emerged big crook, which aggregated some cells. Immunohistochemisty stained respectively with CK7 andⅧ, the conclusion indicated that cells of glandular tube and blood vessel were positive respectively, cells had property of migration and directivity. The emergence of these structures brings hope for the implementation of artificial uterus.5. Through changing the concentration of hormone, three dimensional uterus was induced to being receptive state, that is to say"the window of implantation", H.E staining observed the uterine structure; the stromal cells took place decidualization, cells turned to be dilatants and multinuclear. Blastula and three dimensional uterine tissue cocultured, the result indicated that embryos stick to the three dimensional uterine tissue after being cultured 20h, embryos invaded into three dimensional uterine tissue after being cultured 32h, around of embryos appeared umbilication, embryo implanted three dimensional uterine tissue in vitro.