Isolation And Identification Of Bone Marrow Mesenchymal Stem Cells From Takin

Budorcas taxicolor is a rare mammal and is listed as a "vulnerable species" by the World Conservation Union.In recent years,the number of Budorcas taxicolor in China has been declining due to habitat destruction,illegal poaching and food shortage caused by continuous interference of human activities.Therefore,all localities have taken active protection measures to preserve their high-quality genetic resources.At present,it is mainly focused on the macro aspects of in-situ protection such as the establishment of Budorcas taxicolor ecological habitat and ex-situ protection through domestication;There are relatively few studies on the protection of Budorcas taxicolor species diversity through cell cloning and culture,whole genome determination and analysis and other cellular and molecular micro means.Therefore,in this study,stromal cells were isolated from Budorcas taxicolor bone marrow,and the characteristics of cultured cells were identified at the cellular and molecular levels.(1)Extraction and culture of bone marrow stromal cells from takin.Bone marrow was taken from male takin that died unexpectedly in the wild and transported to the laboratory aseptically for isolation and culture of bone marrow stromal cells.The next day,under the inverted microscope,it was observed that this kind of cells,like fibroblasts,showed a long spindle shape and adhered to the wall,When the cell density reached about 80%,the cells began to subculture.When subcultured to P5,the cells remained active.The karyotype of bone marrow stromal cells of takin was analyzed.After measuring the long and short arms of chromosomes and calculating the ratio between them,it was found that takin had 52 chromosomes,including 25 pairs of autosomes and 1 pair of sex chromosomes(XY).Its chromosome structure remained normal without chromosome structure and number alienation.Flow cytometry was used to identify the associated surface antigen of takin bone marrow stromal cells.It was found that CD105(97.1%),CD73(95.9%),CD90(97.6%),CD166(94.7%)and CD44(97.2%)were positive;At the same time,CD14(3.1%),CD19(2.4%),CD34(1.4%),CD45(1.8%)and HLA-DR(3.7%)were negative,which comply with the criteria for the identification of mesenchymal stem cells.Meanwhile,it was found that the expression of CD166 was close to but less than 95%,The expressions of CD14,CD19 and HLA-DR were close to but not less than 2%.(2)Differentiation potential of takin bone marrow stromal cells.Three line differentiation induction and staining identification of the isolated cells were carried out with the corresponding differentiation medium.After 21 days of culture,the cell morphology in osteogenic,adipogenic and chondrogenic differentiation medium changed significantly,the osteoblast nucleus became larger,located on one side of the cell,the nucleolus was obvious,the cells accumulated calcium and gradually calcified,which could be stained red by Alizarin Red staining solution.The morphology of cells in the adipocyte induction medium gradually changed from the original long spindle shape to round,and the volume gradually increased.Small lipid droplets began to accumulate in the cytoplasm.Finally,these small lipid droplets gathered together to become large lipid droplets,which were dyed orange by oil Red O.Chondroblasts gradually increase in volume and show irregular multilateral shape in morphology,which can be dyed blue by alcian blue dye.Combined with the above two points,it can be preliminarily proved that the isolated and cultured cells are mesenchymal stem cells from three aspects: cell morphology,Surface specific markers and cell directional differentiation.(3)Transcriptome Sequencing of takin bone marrow stromal cells.To further verify whether the cultured cell types express genes for stem cell characteristics.Firstly,the c DNA library was constructed and sequenced using Illumina to obtain a 150 bp double ended sequence.After filtering,the higher the quality of the original sequence and the proportion of the measured sequence,the better the quality of the original sequence and the total number of filtered sequence.By removing low-quality sequences and sequences with joints,high-quality sequences(clean reads)are finally obtained for subsequent analysis.In the obtained sequences,the high-quality sequence of overlap is further spliced into Uni Gene through Trinity software,and the redundant fragments are eliminated to finally obtain Uni Gene with a length of 150-270 bp.Compare the length distribution trend of long-chain noncoding RNA and m RNA to judge the consistency between them.Stem cells express specific genes CD105,CD73,CD90,CD166 and CD44.Finally,FPKM is used to quantify the gene expression.In conclusion,takin bone marrow stromal cells were successfully isolated and cultured,characterized and determined at the molecular,cellular and protein levels,which comply with the identification criteria of mesenchymal stem cells in this study.It not only enriches the database of takin bone marrow mesenchymal stem cells,but also lays a foundation for the research and preservation of takin bone marrow mesenchymal stem cells in the future.