| Osteoporosis is a chronic inflammatory related disorder， characterized bydisequilibrium of osteogenesis and bone resorption. Currently, osteoporosis is viewed as aheterogeneoue condition which can occur in many factors such as aging, estrogendeficiency and inflammation disease. The bone marrow-derived mesenchymal stem cells(BMMSCs) are known as progenitor cells of osteoblasts and osteocytes in bone formation.Studies have shown the differentiation of BMMSCs into osteoblasts play an important rolein osteogenesis. Therefore, investigating the biological behaviour changes in disease andthe molecular mechanisms that regulate bone marrow mesenchymal stem cells (BMMSCs)differentiation into osteogeniclineage as well as improving the osteogenic capability isimportant for the development of therapies for treatment of osteoporosis.NDRG2belongs to the N-Myc downstream-regulated gene family which play animportant role in regulating a variety of physiological and pathological processes,including cell differentiation, proliferation, and cancer. Especially in the process of cancers, more and more researchers keep a watchful eye on it and intend to determine it asa prognostic maker. In previous studies, NDRG2has been reported to regulate foetalosteogenesis. We have screened its expression in different tissues and cells. There isdifferent expression in normal and patients’ cells, osteoblasts and osteoclasts. However, it isnot clear whether or not NDRG2can regulate BMMSCs differentiation and how to do that.Studying the regulatory role of NDRG2in BMMSCs differentiation as well as abnormalexpression and function of NDRG2in osteoporosis would help us to understand themechanism of osteogenic differentiation and develop promising stragegies to treatosteoporosis.Objectives:1. To investigate the expression of NDRG2during osteogenic and adipogenicdifferentiation of mBMMSCs.2. To investigate the mechanism of NDRG2during differentiation in vitro.3. To investigate the expression and molecular mechanism of NDRG2in BMMSCsderived from osteoporosis mice.Methods:1. We use density gradient centrifugation and to isolate, purify and culture mice Bonemarrow-derived mesenchymal stem cells. Phenotypes of mBMMSCs were analyzed byflow cytometry analysis. The multi-differentiation potential of mBMMSCs wasassayed by osteogenic and adiopogenic induction. ALP, Alizarin Red S, and Oil red Ostaining was used to determine the differentiation ability. And osteogenic andadipogenic specific genes were determined by real time RT-PCR.2. We analysed the NDRG2expression after osteogenic and adipogenic induction byRT-PCR. NDRG2was up-regulated and down-regulated through lentiviraltransfection. ALP, Alizarin red S and Oil red O staining were used to determine thechange of osteogenesis and adipogenesis. RT-PCR and Western Blot were used toanalyses marker genes expression. 3. In order to investigate the mechanism of osteoporosis in mice models, we isolate andculture bone marrow-derived mesenchymal stem cells from normal and osteoporoticmice. Comparing their differentiation ability and the expression of NDRG2. We addsome inflammatory factors to imitate inflammatory environments as well as additionof depressor to find out the relative signal pathways. We also construct lentiviralvector of NDRG2to regulate NDRG2in mBMMSCs. ALP, Alizarin red stainingand RT-PCR and Western blot analysis were used to determine the osteogenic andadipogenic potential of mBMMSCs after regulation of NDRG2. Based on the above,we further investigate the influencing factors during the mechanism.Results:1. Cultured mBMMSCs expressed surface markers of mesenchymal rather than theexpression of hematopoietic markers. Osteogenic and adipogenic induction showedthat mBMMSCs could differentiate into multiple mesoderm-type lineages.2. NDRG2was increased during osteogenic and adipogenic differentation of mBMMSCs.NDRG2was over/lower-expression after lentiviral transfection. Up-regulatingNDRRG2could inhibit mBMMSCs osteogenic differentiation and promoteadipogenic differertiation in vitro. On the contrary, reducing the expression of NDRG2could promote the osteogenic differentiation of mBMMSCs and inhibit adipogenic.3. The osteogenic differentiation was impaired in mBMMSCs derived from osteoporosis.The adipogenic differentiation was enhanced in senile osteoporosis. There weredifferent NDRG2expression between normal and osteoporotic cells. NDRG2take partin the process of osteoporosis. Furthermore, TNF-α could inhibit mBMMSCsdifferentiation, impress the expression of NDRG2through NF-κB pathway ratherthan WNT pathway. Down-regulated NDRG2could restore the impaired osteogenicpotential in osteoporosis.Conclusion:1. NDRG2existed in bone tissues and regulated mBMMSCs differentiation in vitro.. 2. NDRG2could be response to TNF-α through NF-κB pathway.3. There were different expression of NDRG2in osteoporotic cells. NDRG2play a rolein postmenopausal osteoporosis mice through NF-κB pathway rather than WNTpathway. |
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