Plastrum Testudinis Between The Active Ingredient And Promote Mesenchymal Stem Cells Into MiRNA-VDR Network Mechanisms Osteoblasts

Research purposes:Bone marrow mesenchymal stem cells (Bone mesenchymal stem cells, BMSCs) is currently the most studied, one of the largest potential applications of stem cells, is ideal model for studying the mechanism of proliferation and differentiation. However, bone marrow BMSCs content is extremely low, long-term proliferation of weak amplification slow, fat gradually aging, unable to meet the large number of clinical needs. Numerous in vitro transfection and in vivo studies indicate that transplantation, BMSCs transplantation proliferation activity is not enough, less the amount of survival, severely limits the application of BMSCs. BMSCs and osteogenic activity is weak, slow process of ossification, therefore, how to improve the osteogenic potential of BMSCs is focus in recent years.Kidney Chinese medicine extract Plastrum testudinis and an active ingredient can induce stem cells to differentiate into osteoblasts. In recent years, our group focuses on the directed differentiation of various types of Plastrum testudinis kidney stem cells. He accumulated a wealth of previous research work basis. Previous studies found that Plastrum testudinis extract to promote osteogenic differentiation of BMSCs may be small molecules within their fatty acids, fatty acid esters, steroids directly into the cell membrane inside the nuclear receptor. Plastrum testudinis extract may be directed osteogenic differentiation of BMSCs upregulated during VDR expression, which may reveal the mechanism of Plastrum testudinis promote osteogenic differentiation of BMSCs oriented in a certain extent. Therefore, this study on the basis of previous work on in-depth study to explore the molecular mechanism of Plastrum testudinis and its application targeting VDR pathway on bone marrow mesenchymal stem cell osteogenic differentiation of.Research methods:1. lncRNA microarray Plastrum testudinis active ingredient differentially expressed, the use of bioinformatics, lncRNA predictive analysis of differentially expressed genes co-expressed, predictive analysis of their co-expression of target genes, the use of biological function cluster analysis Learn their participation in osteogenic differentiation of the relevant circumstances, the difference lncRNA mechanisms involved in the regulation of gene construct networking TF-lncRNA- target gene by trans and cis three predictive analysis.2. Constructed VDRE driven luciferase (Luc) reporter gene system, identified the VDR nuclear receptor target genes of osteogenic differentiation Plastrum testudinis active ingredient response element, to find out the pro-osteogenic differentiation of BMSCs best Plastrum testudinis extract (PTE) component.3. Transfected vitamin D receptor (VDR) and its functional area truncated body to bone marrow mesenchymal stem cells (BMSCs), a nuclear receptor which was observed Ribbon Plastrum testudinis active ingredient in osteogenic differentiation plays a role.4. Plastrum testudinis and its application intervention bone marrow mesenchymal stem cells (BMSCs), normal BMSCs as controls, verified by miRNA gene microarray and qRT-PCR, miRNA discovery information aspects of osteogenic differentiation.5. The combination of front miRNA microarray and qRT-PCR information, selected miRNA-351, its target gene VDR mRNA, the introduction of special rno-miR-351 mimic the predicted target genes VDR 3’UTR dual luciferase assay to verify miRNA inhibits candidate target gene translation level.Research result:1. By analyzing the results of gene chip lncRNA found lncRNA Plastrum testudinis and mRNA differentially expressed an active ingredient.2.Pathway and cluster analysis to determine the biological function of molecules, and further analysis of the active ingredient lncRNA Plastrum testudinis differentially expressed relationship between TF and cis regulation.3. Trans analysis, constructed lncRNA-mRNA- ternary transcription factor regulatory networks.4. Aging Expression preliminary conclusions:the components are promoted to varying degrees. Structural analogues of different doses of the drug the best point in time, there are different levels of promotion, highest 30μg/mL expression efficiency of VDRE.5.S9 role binding domain (pCMV-Myc-VDR-C) in VDR ligands promote osteogenic differentiation of BMSCs.6. After cDNA microarray data analysis, we found that after 2B, S9, K9 intervention, miRNA-330-3p, miRNA-200a-5p, miRNA-296-3p upregulated, while miRNA-615, miRNA-351-3p, miRNA-129-1-3p, miRNA-466b-2-3p, miRNA-466b-1-3p downregulation.7. miRNA-351 mimic can effectively inhibit the expression of VDR, and in transfected miRNA-351 inhibitor can promote the expression of VDR protein, miRNA-351 and VDR corresponding binding sites effectively at the level of translation inhibit the expression of VDR.Analysis conclusion:Plastrum testudinis and an active ingredient may be targeted VDR promote BMSCs osteogenic differentiation, while inhibiting miRNA-351 expression can also enhance the VDR translation.