치아의 치수 줄기 세포와 치주 인대 줄기 세포의 석회화와 분화에 PRP가 미치는 영향
The effect of PRP on the differentiation and minerallzation of DPSCs and PDLSCs
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1 . INTRODUCTION Tissue engineering using stem cell has been studied extensively in attempt to regenerate the lost tooth ancVor damaged periodontal tissue. Although Platelet rich plasma(PRP) isolated from the blood has been applied to the implant surgery, the studies about the direct role of the PRP on the cell are not sufficient. To use PRP in clinic successfully, evaluation about effect of PRP on the cytological lev하 is req버red. n‘ PURPOSE The purpose of this study was to investigate the roles of PRP during the differentiation of dental pulp stem ceJls (DPSCs) and periodontal ligament stem cells (PDLSCs) at cytol맹ic and histologic level. Ill. MATERIALS AND METHOD 1. Cell isolation & primary c띠ture Dental p비，p cells and periodontal ligament cells were isolated from the exf，미 iated third molar of human who dose not have any systernic disease, respectively. The cells were divided into two groups ; PRP was added in one group experimental and was not added in the other group as a control. And the cells were cultured for 15 days. 2. PRP preparation. After taking blood from the 5 rats (male, 6 weeks old), PRP was isolated from the blood with thrombin by the centrifugation method. 3. Immunofluorescence Axioscope multi fluorescence rnicroscopic observation was performed using STRO-1 antibody which is mesenchymal stem ceJl marker to iden디 fy the stem cells in the culture 4. Alizarin-Red S staining At the time of 0, 5, 10 때d 15 days after the cell culture, the Alizarin Red-S stai미 ng was performed to evaluate the rruner때alization. 5. RT-PCR Analysis At the time of 0, 5, 10 없d 15 days after the cell c비ture， total RNA was isolated using Trizol reagent and the first strand cDNA was synthesized. And PCR was performed with the specific primers of DSPP, BSP, ALP, type 1 collagen, osteocalcin, osteonectin and GAPDH to evaluate the differentiation. IV. RESULT 1. Analysis of Immunofluorescence Sever외 stem cells specific to STRO-1 antibody were observed through the FITC in DPSC and PDLSC. 2. Analysis of Alizarin Red-S staining No rnineralization was obseπed before the cultur어 15 days in both control-DPSC group and PRP-DPSCs group. However, at the c띠tured 15 days, mineralization was obseπed in the PRP-PDLSCs group. 3. RT-PCR 1) In PRP-DPSCs group, expression of DSPP were increased compared to those of control group‘ 2) In PRP-PDLSCs group, expression of DSPP, ALP, osteocalcin were almost same cornpared to those of control group V. Conclusion The result may suggests the functional role of PRP in the differentiation 뻐d mineralization of DPSCs by the increase of DSPP.
Abstract
1 . INTRODUCTION Tissue engineering using stem cell has been studied extensively in attempt to regenerate the lost tooth ancVor damaged periodontal tissue. Although Platelet rich plasma(PRP) isolated from the blood has been applied to the implant surgery, the studies about the direct role of the PRP on the cell are not sufficient. To use PRP in clinic successfully, evaluation about effect of PRP on the cytological lev하 is req버red. n‘ PURPOSE The purpose of this study was to investigate the roles of PRP during the differentiation of dental pulp stem ceJls (DPSCs) and periodontal ligament stem cells (PDLSCs) at cytol맹ic and histologic level. Ill. MATERIALS AND METHOD 1. Cell isolation & primary c띠ture Dental p비，p cells and periodontal ligament cells were isolated from the exf，미 iated third molar of human who dose not have any systernic disease, respectively. The cells were divided into two groups ; PRP was added in one group experimental and was not added in the other group as a control. And the cells were cultured for 15 days. 2. PRP preparation. After taking blood from the 5 rats (male, 6 weeks old), PRP was isolated from the blood with thrombin by the centrifugation method. 3. Immunofluorescence Axioscope multi fluorescence rnicroscopic observation was performed using STRO-1 antibody which is mesenchymal stem ceJl marker to iden디 fy the stem cells in the culture 4. Alizarin-Red S staining At the time of 0, 5, 10 때d 15 days after the cell culture, the Alizarin Red-S stai미 ng was performed to evaluate the rruner때alization. 5. RT-PCR Analysis At the time of 0, 5, 10 없d 15 days after the cell c비ture， total RNA was isolated using Trizol reagent and the first strand cDNA was synthesized. And PCR was performed with the specific primers of DSPP, BSP, ALP, type 1 collagen, osteocalcin, osteonectin and GAPDH to evaluate the differentiation. IV. RESULT 1. Analysis of Immunofluorescence Sever외 stem cells specific to STRO-1 antibody were observed through the FITC in DPSC and PDLSC. 2. Analysis of Alizarin Red-S staining No rnineralization was obseπed before the cultur어 15 days in both control-DPSC group and PRP-DPSCs group. However, at the c띠tured 15 days, mineralization was obseπed in the PRP-PDLSCs group. 3. RT-PCR 1) In PRP-DPSCs group, expression of DSPP were increased compared to those of control group‘ 2) In PRP-PDLSCs group, expression of DSPP, ALP, osteocalcin were almost same cornpared to those of control group V. Conclusion The result may suggests the functional role of PRP in the differentiation 뻐d mineralization of DPSCs by the increase of DSPP.