The Effect Of Reversine On The Reprogramming Capability Of Sika Deer(Cervus Nippon) Fibroblast And Interspecific Nuclear Transfered Embryos

The sika deer (Cervus nippon) was listed as the national level I protected wild animals of China, and endangered rating in China Red Data Book of Endangered Animals.If the wild sika deers have not been preserved in any way before extinction, not only the genetic resources will lost evermore, but also the research on biological mechanisms of various unknown cells and molecules of the extinct spaces will not be realized as well as the wish to regenerate the animals through somatic cell cloning. Therefore, there is a very urgent need to commence conservation of endangered species. Moreover, with the future development of science and technology, the roles of cell lines will become increasingly prominent and they may be useful in currently unforeseen applications.Then we should find a way to dig the potentiality of the somatic cell, we wish this species will regenerate by this way.From the cloned frogs to induced pluripotent stem cell(iPS), the researchers are struggling in the problems of safety and effectivity in process of reprogramming, they try to add different kinds of small compounds to figure it out, reversine is a small compound, the mouse muscle cells cells were transformed as a stem-like cell to an original state after treated with reversine, moreover, the development of nuclear transfer embryos could increased by using this small compound.Therefore, in the present study, the sika deer’s ear marginal was used as the research material, the primary cells were cultured by using the method of tissue piece, then the cells was passaged by Trypsin digesting. According to the cell growth state, the cell growth curve was described. The cells were frozen when100%contact inhibiting. Count the number of cryopreserved cell, then recovery the cells and detected cell viability of the recovery. The fibroblast karyotype was analyzed by Giemsa staining. The results showed that, tissues from sika deer’s ear marginal were isolated to culture fibroblasts and to develop a fibroblast cell line, the number of sample is137, the cell number of each sample is (1-3)×106/ml.Chromosome analysis showed that the chromosome number is2n=66,we can definite the sample was from the sika deer.After the fibroblast cell bank was established successfully, the present research was began to focus on the totipotency of somatic cell.5μM reversine were used to treat the recovered fibroblast for4d. At first, the cell morphologic changed were observed before and after treatment, then the flow cytometry were use to detect the cell cycle distribution and apoptosis, then the indirect immunofluorescence assay was used to detected the reversine treated cells proliferation and cytoskeletal changes. In order to reflect the dedifferentiate ability of reversine treated adult cells, we induced these cells to the osteogenic, adipogenic and liver cells before and after reversine treatment. Then the confocal microscopy and flow cytometry were used to analysis the histone modifications of the dedifferentiation cells. The results showed the reversine-treated fibroblast become the multinucleated cells by decreased the expression of the BrdU,and this kind of cell could be induced to differentiate into osteogenic, adipogenic and liver cells, then immunohistochemistry and RT-PCR were used to detect. After confocal qualitative and flow cytometry quantitative detection, we found the level of acetylation was incresed after reversine treatment, the level of methylation and phosphorylation was decresed, which indicated that reversine could induce the Sika Deer fibroblast into more multipotent progenitor-type cells by dedifferentiation.The most useful way to regenerate the endangered animals is to built the interspecies somatic cell nuclear transfer(ISCNT) embryos. In this study, the sika deer fibroblasts were used as the donor cells, the bovine MⅡ stage of oocytes were used as the receptor to built the ISCNT embryos.Then we compare the developmental level of ISCNT embryos, in vitro fertilization (IVF)embryos and normal NT embryo. In order to improve the developmental level of reconstructed embryo, we use reversine to deal with these NT embryos, then the indirect immunofluorescence assay was used to detect the expressed characteristics of the linker histone. The results showed that the cattle-deer ISCNT embryos was successfully constructed in this study, and ISCNT embryonic blastocyst were obtained successfully. Then compare the developmental time of cattle-deer species ISCNT embryo was not hysteresis to the normal NT embryos, but the efficiency of development to the blastocyst stage was much lower than the normal NT embryos (16±8.8%vs7±3.9%>P<0.05). It’s surprise that, the cattle-cattle normal NT embryos and cattle-deer interspecies NT embryos’ developmental level has been improved significantly after reversine treatment(16±8.8%vs28.2±5.9%, P<0.05;3.7±3.9%vs16.1±5.3, P<0.05);After analyze the expressed pattern between oocyte-specific linker histone Hlfoo and the somatic cell types H1protein, a shift trend were existed. The shift trend expressed pattern of H1Foo and H1was started in the16-cell stage or even later in the normal fertilized embryos,however, this kind of shift trend expression in normal NT embryos was happened in2-cell stage, which explains the phenomenon of incomplete reprogramming in the NT embryos. After reversine treatment, the expressed patterns of linker histone in NT embryos was corrected similar with the expression pattern of IVF embryo.