Isolation,Culture And Identification Of Mesenchymal Stem Cells From Bone Marrow Of Forest Musk Deer

China has the most abundant musk deer resources in the world,and the musk deer resources have high medicinal and economic value.Forest musk deer is one of the endangered species in China,which is listed as the first-level protected animal in China.The state has invested a lot of funds to establish nature reserves for on-site protection of forest musk deer,or to carry out off-site protection of forest musk deer through domestication.In addition to these macro conservation methods,more and more scientists have been protecting the genetic resources and ecological diversity of musk deer at the cellular level in recent years.At present,the conservation of forest musk deer germplasm resources is mainly focused on macro conservation,and there is little protection of forest musk deer germplasm resources through cell level microscopic research,which is one of the important means to protect endangered species.Between bone marrow is a kind of pluripotent stem cells,mesenchymal stem cells have multiple differentiation potential,can release a variety of factors,and to participate in antiapoptotic signal expression,cell growth and wound healing process and promoting endogenous regenerative potential,also have immunomodulatory effect,its various key signaling pathways involved in cell proliferation,differentiation and embryonic development,is an ideal seed cells.In this study,the cell level of forest musk deer was studied from three aspects: cell isolation and culture,transcriptome sequencing and cell differentiation.(1)Isolation,culture and karyotype analysis of interstitial cells from bone marrow of musk deer.Bone marrow was isolated from the bone marrow tissue of adult male musk deer within 48 h of death,and a large number of primary cells were cultured in vitro.After 48 h of culture,the cells could stick to the wall and grow.At first,the cell morphology is different,some cells are fusiform,some cells are round;On the third day of culture,bone marrow stromal cells had become colonies,and on the seventh day of culture,the cells covered the dish bottom,and the cell morphology was mainly shuttle type.When the cells were transferred to P3,the morphology was gradually uniform and the cells maintained proliferation activity.Karyotype analysis of isolated cultured cells showed that musk deer had58 chromosomes,including 56 autosomes and 2 sex chromosomes.Fifty-six chromosomes were successfully paired,except for the longest chromosome,which was X,and the shortest chromosome,Y.The results showed that the mesenchymal cells of musk deer bone marrow were obtained successfully,and the chromosome structure did not change abnormally in the process of in vitro culture.(2)Transcriptome sequencing of bone marrow stromal cells of musk deer.Using the second-generation sequencing technology,78.45% of the raw data(reads)obtained from the sequenced samples could be mapped to the genome.High quality reads were assembled using the Trinity program,and most of the unigenes in the transcript were 150-270 bp in length.The fluctuation of the first 6 bp base proportion was normal.The species distribution of the samples showed no contamination.In the KOG function classification diagram of consensus sequences,signal transduction mechanism,protein post-translational modification,turnover and chaperoning,and transcription constitute the largest functional group.In the results of GO annotation of Unigene sequence genes,most GO terms were classified as biological processes.In the category of biological processes,single organic process,cellular process,metabolic process and biological regulation were the most abundant.Among KEGG annotation results of Unigene sequence genes,the largest unigenes related subgroup is signal transduction,followed by immune system.(3)Bone marrow stromal cells of musk deer differentiated into osteoblasts and adipocytes.After the cells were treated with osteogenic differentiation medium,the cell growth slowed down gradually until it stopped growing.After 21 days of culture in the medium,the cells showed polygonal shape,and the deposition of extracellular matrix was observed after alizarin red staining.The results showed that the isolated cells could differentiate into osteoblasts.After 21 days of treatment in lipogenic differentiation medium,cell morphology changed,cell volume became larger,and lipid bubbles in cytoplasm increased.The results showed that the isolated cells could differentiate into adipocytes.Gene expression levels in osteoblasts and adipocytes were compared with isolated cells,the isolated cells expressed CD29 and CD59,and the expression of CD29 and CD59 was higher in osteoblasts and adipocytes after differentiation,but the difference was not significant(P>0.05).The isolated cells expressed CD105 and CD44,which were higher in osteoblasts and adipocytes(P<0.05).OPN expression was higher in osteoblasts and lower in adipocytes(P<0.05).RUNX2 is highly expressed in osteoblasts and adipocytes(P<0.05),especially in osteoblasts(P<0.05).ADIPOQ and PPAR were highly expressed in osteoblasts and adipocytes(P<0.05),PPAR was highly expressed in adipocytes(P<0.05).The results showed that the differentiation of bone marrow stromal cells into adipocytes and osteoblasts resulted in the change of expression profile.Conclusion: The karyotype analysis showed that the cells maintained normal karyotype,and the bone marrow mesenchymal cells isolated by us were bone marrow mesenchymal stem cells after partial marker gene detection and differentiation of cells.This study lays a foundation for the follow-up research.