| It was successfully reproted that separation culture of single blastomere was used to produce identical twins or polyembryony. The problem that detection of genetic disease of preimplantation embryo by obtaining single blastomeres from fertilization in vitro through bio-assay is hot topic in human being clinical diagnosis (PGD diagnosis). Research regarding obtaining embryonic stem cells from separation and cultivation of blastomeres using different developmental-stage early embryo in mice was one of recent hot studys on the source of embryonic stem cell. All the above are researchs over the past ten years aimed to find the rapid and new ways of reproduction, to research the prenatal diagnosis means for human beings genetic disease, and to study non-ethically controversially the ES cells reservoir, which was used by the the isolation and culture of the early embryo blastomere. There were many studys on the three aspects, for instance, alive offsprings were obtained from 2-cell and 4-cell stage singal blastomere in goat and cattle, from 4-cell stage in rabbits, and from 8-cell stage in pigs. The successful rate of ES cell restruction by Biopsy blastomere (PGD diagnosis) was rather low, furthermore, not every sister-blastomere could build the ES cell, that leads to whether sister-blastomere has the same capability to develop to the ES cell or has the diffirent fate. Because the selection of blastomere maybe descrease the developmental capability of donor cell, so that it is quite a critical problem to ascertain whether the random selection signal blastomere to create ES cell would break the embryo integrity and embryonal subsequent development. The successful rate of ES cell restruction tended to have negative relationship with cell division times of embryo or the stage of embryonal cells after signal blastomere culture in mice. These results indicated that only one or two blastomeres but not all during the every embryo stage had the capability to develop sucessfully to ES cell. The above reports on the three aspects showed that it had successful potential in every orientation, but its success rate is low, so the question about the success rate focused on the qualiy of the signal blastomere, of which quality resulted from maldistribution of determined factors because of time sequence during the division growth or culture condition that culture media from generations disatisfied higher the non-differentiated enhancement of signal balstomere, the former was hereditary, however, the latter aquired.Trail 1:Optimum and seletion of culture media for 8-cell stage signal blastomere from fertilization in vitroThis study was conducted to research effects on the developmental rate of 2-cell,4-cell, 8-cell stage and morula and blastula of different concentration of vitamine E, cysteine and L-glutamine supplementation to the CZB'culture solution that was made by the author's lab as the basical culture solution, which was on the basis of analyzing and selecting culture solution for early embryo from fertilization in vitro in mice. The trial was aimed to pave the way for selecting culture media of 8-cell embryon signal blastomere from fertilization in vitro in mice, which was on the basis of improved culture system for early embryo from fertilization in vitro in mice. Results indicate:the antioxidative incubation treatment of 0.15 mg·ml-1 L-glutamine,0.05 mg·ml-1 cysteine and 100μmol·L-1 vitamine E increased obiviously the developmental rate of 2-cell stage (68.68%,61.35%,67.88%VS 53.14%),4-cell stage(51.77%,49.72%,50.71%VS 27.83%),8-cellstage(42.18%,41.32%,40.52% VS 17.70%) and morula and blastula (33.75%,32.37%,30.89% VS 12.70%). Results showed:the effectiveness of antioxidative incubation which was used the CZB'culture solution from the author's own laboratory supplemented with the appropriate content of antioxidant was acceptable, so that this antioxidative culture media could be utilized as basic solution for 8-cell stage signal blastomere from fertilizertion in vitro.Trial 2:a study on the cultivation method of 8-cell signal blastomere from fertilization in vitro embryoThis study was based on results of trial 1, the trial seleceted antioxidative early embryonal culture media as culture solution for 8-cell embryonal signal blastomere through observing the effect of the cell co-culture, artificial zona pellucida and the blastomere cultivation density on the cultivation, furthermore, and studied the cultivation method of signal blastomere in vitro. Results indicated:MEF 3rd generation co-culture and endometrial cell co-culture group increased the developmantal ability and the blastula rate of 8-cell signal blastomere compared the control group (15.90%,6.06% VS 3.48%); the blastula developmental rate cultivated by putting the signal blastomere into the artificial zona pellucida was not obivious (16.15% VS 15.87%), however, the multilocular blastula rate of culture treated with zona pellucida was significantly lower than that without zona pellucida (1.09% VS 4.63%); the concentration of 10 per 20μl in the incubation microdrop could enhance differently the blastula rate (18.44% VS 14.33%). Results showed that the selection of MEF 3rd generation co-culture buried with the zona pellucida in the concentration of 10 per 20μl could significantly enhance the in vitro developmental ability of blastomere and the appearance rate of the blastula.Trial 3:the segregation, cultivation and identification for blastula-like stem cell from 8-cell embryonal signal blastomereAfrer obtaining mice blastula developed from the signal blastomere, the proliferative ICM colony was picked by microscopicall operation means from co-culture system and purified by vicariance, segregation and cultivation, and through morpholoical identification, AKP staining and cell differentiation in vitro to identify the ES from mice 8-cell stage embryonic signal blastomere by fertilization in vitro. Results indicate:the ES-like cells colony had a clear borderline between the colony and the feeder layer and had a smooth surface, the cells were condensing and the colony was typically nest-like, the volume of cells were small but the cell nuclei were large under microscope, which was consistent with the morphological characteristic of ES cells; It was detected by the AKP that the ES-like cells had been dyed red and the surrounding breeding layer was not stained; ES-like cells began to differentiate after being sought out from the ES-like cell colony and being inoculated into the ES cells culture solution without LIF, then they began to differentiate. Results showed the cell colony of 8-cell stage embryonic signal blastomere from fertilization in vitro could be judged as the ES-like cells through preliminary identification.Conclution; the antioxidative culture media from the author's laborotary could improve the cultivation effectiveness of embryo from fertilizatio in vitro, which was on basis of analyzing and selecting some general culture medias of embryo from fertilizatio in vitro in mice; The application of MEF 3rd generation co-culture system that put the signal blastomere into the artificial zona pellucida at the concentration of 10 per 20μl could significantly enhance the developmental rate of 8-cell fertilization in vitro signal blastomere to blastula in mice, which used this culture media as the basic culture solution for 8-cell fertilization in vitro signal blastomere; the cell colony picked from the colony of mice 8-cell stage embryonic signal blastomere by fertilization in vitro, which was characteristic on the clear boundary, smooth surface, compact cells and nest-like, could be preliminarily identified as the ES-like cells through the morpholigical identification, AKP steining and differentiation in vitro.
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