Function Expression Of Hunman MiRNA-30a-5p In The Inducing Has-BMSCs Into Osteoblast

BackgroundStem cells research in tissue engineering as a focus in the field of biomedical research, for the defects and reconstruction of tissues and organs provided a new and very promising paths to clinical application. Adult stem cells (ASC) as an early undifferentiated cells has the potential of strong self-renewal and proliferation and differentiated into osteoblasts, chondrocytes, nerve cells, adipocytes , muscle cells, dermal cells et al. additionally,because it also has many advantages ,such as wide range of sources, easy to in-vitro proliferation and individual treatment to avoid possible rejection that to being studied in-depth. Plenty of studies confirm that bone marrow mesenchymal stem cells(BMSCs)proliferation, differentiation by various cytokines, signaling pathway, intracellular and extracellular micro-environment of the combined effects of various factors. Meanwhile,the study confirmed that a large number of microRNAs (miRNA) is also involved in the regulation of stem cells biological behavior.microRNA is a class of small molecular non-protein coding RNA containing an average of 22nt and mainly regulates the lever of post-transcriptional gene .Recent studies shows that there is a unusually large miRNA family in many species with different types, quantity and time expressions in different species. In addition, in the process of individuals’growth, signal transduction, tissue differentiation and diseases’development miRNA also play an important role in regulation and determines the organism’s physiology, pathology, and behavior changes.Early experiments by our group have used the high-throughput gene microarray detection technology to detected the miRNA,s gene expression profiling in the process of inducing BMSCS into osteoblasts, a series of miRNAs of discrepant expression to be analyzed and selected before and after induced differentiation.this experiment select the miRNA-30a-5p of Significantly increased expression which in the miRNA,s osteogenic gene expression profiling, to verify its biological role in bone formation, and to predict its possible target-genes by bioinformatics methods.ObjectiveTo investigate and verify biological effects of the human bone marrow mesenchymal stem cells(MSCs) in vitro transfection with recombinant miRNA-30a-5p in differentiation into osteoblasts.MedthodsWe select miRNA-30a-5p with significantly hight expression of osteoblast through osteogenic differentiation and miRNA differentially expressed genes microarray from hBMSCs , and then synthesize the recombinant human miRNA-30a-5p .Separate and identify human bone marrow MSCs and induce them into osteoblasts through in vitro transfection of recombinant miRNA-30a-5p. Detect the activity of ALP in osteoblast by alkaline phosphatase (ALP) staining and ALP calcium cobalt quantitative detection, calcium deposition by alizarin red S staining. And investigate the biological functions of the recombinant miRNA-30a-5p in the differentiation of the human mesenchymal stem cells into osteoblasts in comparative observation.Finally,its pos sible target-genes was predicted by bioinformatics methods.ResultsSuccessfully we isolated and cultured MSCs from human bone marrow and inducted the into bone through directional differentiation, The transfection efficiency was 37.32%±2.43%.At different time points(14d、21d) after induction, osteogenetic activity of the cells was detected by ALP staining observation, ALP activity detection and alizarin red staining for calcium nodules,the results show that transfection of artificial re-synthesis miRNA-30a-5p into stem cells significantly enhanced features of stem cells differentiating into bone.Conclusion(1)Multiplicity and supra-purity BMSCs can be separated and cultured in vitro by using Percoll,s density gradient centrifugation combine holo-bone marrow adherent separation, and can be induced to differentiate exclusively into the osteocytic lineages.(2)miRNA-30a-5p has a certain role in promoting orientational differentiation of the MSCs into bone cells. MSCs directely differentiation into osteoblasts was increased by transfected with the recombinant miRNA-30a-5p. That has fundamentally value in elucidating molecular and biochemical mechanisms of osteogenic differentiation and establishing the theoretical basis for cell transplantation of bone defect repair treatment.