Inter-Species Nuclear Transplantation Between Human And Bovine

"Therapeutic cloning" is based on the technology of somatic cell nuclear transfer and human ES cell. It uses patients' somatic cells as donor and the enucleated oocyte asreceptor. After the reconstructed embryos are cultured to blastocysts, cells isolated from the inner cell mass are used to establish new embryonic stem cell lines. The ES cell lines containing the patient's genetic material are used to cure the patient himself. Now "therapeutic cloning"has been research hotspot. Because of the restriction of law and other factors, it is difficult to gain enough human oocytes for the research. As the technology of inter-species nuclear transfer appeared, it is possible to solve these problems. This study focused on whether human fetal fibroblast cells can be reprogrammed in bovine oocytes and the ability of in vitro development of human-bovine interspecies reconstructed embryos, meanwhile the fusion condition and culture system of the reconstructed embryos were reserached.1. Culture of the human fetal fibroblast cells in vitro(1) Proliferation-promoting rule of the human fetal fibroblast cells in 6^th passage were studied by drawing the growth curve. The results indicated that the growth was very prosperous. Latent phase is 1-2 day. Logarithmic growth phase is 3-6 day. Plateau phase is 7-8 day.(2) The human fetal fibroblast cells were cryo-preserved with DMSO as cryoprotectants in manual way. The results indicated that this cryo-preservation method is an effective way for the human fetal fibroblast cells.(3) The human fetal fibroblast cells in 10^th passage were observed by the karyotype analyzed. The results indicated that 75% of the cells maintained diploid karyotype in 10th passages. Cell aging phenomenon was not oberved.(4) The human fetal fibroblast cell cycles were detected by flow cytometry after cells in 8^th and 20^th passage were treated with serum starvation and general culture. The results indicated that the human fetal fibroblast cells could be synchronized to G₀/G₁ by both treatments. After the 20^th passage cells were treated by serum starvation, the cell percentage of G₀/G₁ was significant higher than that of the other three groups.2. Reconstruction of human-bovine embryos by interspecies nuclear transfer(1) The results indicated that the human fetal fibroblast cells could be reprogrammed in bovine oocytes and reconstructed embryos could develop to blastocyst stage in vitro.(2) When the donor cells were in different cell cycles and different passages, the fusion rates, cleavage rates and blastocyst rates of reconstructed embryos were compared. The results indicated that starvation treatment might not be the essential factor for low passage donor cells. When donor cells were in high passages, starvation treatment was the important way to increase fusion rates and development rates significantly and improve the efficiency of interspecies nuclear transfer.(3) The effects of donor cell preparation methods (directly digested and stored at 4 0^C) on reconstructed embryos were investigated. The results indicated that both of two methods could be used in human-bovine nuclear transfer. There was not significantly difference between two methods. Refrigeration is a convenient and efficient donor cell preparation method.(4) Human-bovine reconstructed embryos were fusd at three parameters, pulse-strength 160V/mm, pulse-interval 1s; 180V/mm, pulse-interval 1s; 200V/mm, pulse-interval 0.1s. The results indicated that the best fusion rates(61.90%) were obtained by pulse-strength 200V/mm, pulse-width 100μs, pulse-interval 0.1s.(5) Human-bovine reconstructed embryos were cultured in Queen's and SOFaa medium respectively. The results indicated that both of two mediums could sustain development of human-bovine reconstructed embryos in vitro. There was not significantly difference in cleavage rates and morula-blastocyst rates.(6) The reconstructed embryos were analysised in number and shape of chromosomes, and the results indicated the nuclears of embryos were derived from human somatic cells.