MiR-146a-5p Promotes The Osteoblastic Differentiation Of Periodontal Ligament Stem Cells Induced By APTG-CM

Alveolar resorption due to poor oral hygiene is very common during orthodontics treatment. Alveolar bone and teeth is closely linked with the aid of the periodontal ligament and it plays an important role in maintaining the stability of teeth and other physiological function. Once alveolar bone loss occurred is irreversible because osteoblast is equal to osteoclast after maxillofacial development complete. Researches have demonstrated that characters of self-renewing and multipotential differentiation of human periodontal ligament stem cells(h PDLSCs) makes it be the reliable cell-sources of tissue engineering. Studies have shown that h PDLSCs could differentiate into osteoblast, cementoblast and fibroblast by means of different condition to fix periodontal defects. Micro RNAs are approximately 22-nucleotide-long small non-protein-coding RNA molecules that negatively regulate gene expression at the post-translational level by acting with RNA-induced silencing complex(RISC). Several researches have demonstrated that micro RNAs play an important role in regulating the self-renewing and multipotential differentiation characters of stem cells. Though h PDLSCs possess multi-directional differentiation potential, whether hsa-mi R-146 a plays roles in regulating PDLSCs differentiate into osteoblast is not yet known.ObjectThe aim of this study is to demonstrate the effect of mi R46a-5p on the differentiation of PDLSCs into osteoblast induced by APTG-CM. Material and Methodsfirst, lentivirus vectors were structured to up/down regulate mi R146a-5pspecifically and been transfected into PDLSCs. Then ALP activity and ALP, BSP, OCN expression level were detected after PDLSCs treated by APTG-CM for 7, 14,21 day respectively.1. PDLSCs culture and screen; 2. APTG-CM preparation; 3. Verification of mi R146a-5p expression by RT-QPCR; 4. Lentivirus transfection; 5. ALP activity test; 6. ALP, BSP, OCN expression level test.RESULTS1. The positive immunomagetic sorted cells exhibited several characteristics of PDLSCs, as indicated by positive immunocytochemical staining of STRO-1 and CD146. 2. RT-QPCR showed that mi R146a-5p expression rised during differentiation into osteoblast from PDLSCs. 3. Transfection efficiency test showed that mi R146a-5p increased/decreased after treated by LV-has-mi R146a-5p/LV-has-mi R146a-5pinhibitor respectively. 4. ALP activity test showed that ALP activity increased obviously in PDLSCs after treated by LV-has-mi R146a-5p while decreased by LV-has-mi R146a-5pinhibitorcompared with their negative control, respectively. 5. Osteogenic genes such as ALP, BSP, OCN increased obviously in PDLSCs after treated by LV-has-mi R146a-5p while decreased by LV-has-mi R146a-5pinhibitorcompared with their negative control, respectively.Conclusionmi R-146a-5p promotes the osteoblastic differentiation of periodontal ligament stem cells induced by APTG-CM.