| Objective:Human dental pulp cells (hDPCs) within the cavum medullare and pulp canal of human teeth have been isolated, culture-expanded and characterized. These cells might involve in the regenerative capacity of dental pulp. These cells are also considered to be used in the regeneration of teeth. Those cells from postnatal dental pulp maintaining the characteristics of stem cells, like self-renewal and multipotency, have the ability to differentiate into a variety of cells and tissues. Cultured with odontogenic inductive medium, dental pulp cells could differentiate into odontoblast which is similar to osteoblast-like cells. These cells could express osteogenic markers, such as alkaline phosphatase, type I collagen, bone sialoprotein, osteocalcin, osteopontin and bone morphogenetic proteins and also could form mineralized nodules. In addition, dental pulp cells generate high levels of DSP, which is commonly expressed undetectable levels in osteoblast cells and other cell lines. DSP was considered to be dentin-specific protein. Previous basic research data revealed hDPCs may play an important role in the regeneration of dental tissues. Thus, the postnatal stem cells in the dental pulp are thought to possess great latent energy for differentiating into odontoblast and forming dentin.Our research is to detect the functions of FHL2in human dental pulp cells. FHL2, that is only four and a half Lim protein2, is an emerging protein of279amino acids consisting of four and a half LIM domains and the half LIM domain locate at the N-terminal followed by four full LIM domain. LIM domain is consisted by50-60amino acids forming two adjacent zinc fingers that is a richly cysteine and histidine polypepitide. Based on the number of LIM domains and the presence or absence of additional motifs, LIM proteins are grouped into different classes. FHL2has been shown to be involved in several important cellular processes like transcriptional regulation, DNA replication and signal-transduction pathways. Following the differential usage of different LIM domains, FHL2is able to interact with many different proteins, and involve both in the cytosol and in the nuclei depending on the interaction partner different LIM-domains.Recently results showed that FHL2had been found in human mature teeth. Interestingly, the expressed pattern of FHL2is changed in different tooth development periods. Our data from the gene level analysis suggests that the primary dental pulp cells can express FHL2. The role of FHL2in osteoblast differentiation and mineralization, as well as the development of teeth was similar to bone formation, including the cells differentiation and matrix mineralization and the close correlations biological behavior of FHL2in dental tissue development, leave us with a hypothesis that FHL2possesses some function in the teeth development. To date, only very little is known about the regulation of FHL2for odontoblast in vitro. In this study, we examined the expression of FHL2during the DPSCs differentiation and mineralization and then FHL2was overexpressed stably in DPSCs by using lipofection2000to transfection to demonstrate the possible function of FHL2in odontoblast differentiation and mineralization.Methods and materials:1. Cells isolation and culturePrimary human dental pulp cells are isolated from human third molar dental pulp of the adults. Staplly, the primary DPCs stemmed from dental cavum medull and then used both the enzymatic digestion method and tissue block method to disperse the tissue. To investigate mineralization, cells were cultured in odontoblast induction medium. Transfection assays were made by using lipofectamine2000. Surviving G418treatment was pooled and used for subsequent analysis. Cells transfected pcDNA3empty vector were used as control.2. Alkaline phosphatase (ALPase) activity assayThe primary cells, Cells transfected pcDNA3empty vector and Cells transfected pcDNA3-FHL2-flag were used in this experiment. The primary cells, Cells transfected pcDNA3empty vector were used as controls. The cells were treated with odontoblast induction medium for0,7,14,21,28days. When the time is it, ALPase activity was determined.3. RNA isolation and Quantitative real time PCR:The primary cells, pcDNA3.0empty plasmid and pcDNA3-FHL2-flag infected hDPCs were cultured for0,7,14, and21days respectively. Total RNA was isolated and purified from cells. Reverse transcription was performed and Real-time PCR was carried out by using SYBR Green I Master.4. Alizarin red staining:Cultured cells were washed third with PBS and absolute ethyl alcohol was charged to fix cells and washed third with PBS, then covered with Alizarin red solution. Mineral deposition appeared red after washing wells three times with water.5. Western blot analysis:Cellular protein was isolated by cellular protein extraction solution. Protein concentrations were measured as above mentioned approach. And as the previous method, doing the following experiment.Results:1. Expression pattern of FHL2in hDPCsTo observe the presence of FHL2in hDPCs, we measured the mRNA and protein level of FHL2by semi-quantitative real time PCR and Western blot analysis. The mRNA and protein express level of FHL2witnessed obvious increase during the stage of odontoblast differentiation. GAPDH mRNA and protein were used as control.2. Transfection of FHL2into hDPCsStable cell populations of hDPCs/FHL2and hDPCs/pcDNA3.0were generated. Cells that transfected of pcDNA3-FHL2-flag can express both FHL2and flag. Flag expression from the hDPCs/FHL2cells enables examinations of the transfection consequence. The mRNA and protein express levels of FHL2in hDPCs/FHL2are higher than the hDPCs/wt and hDPCs/pcDNA3.0. Flag can express in the hDPCs/FHL2and we cannot detect the expression of flag in the hDPCs/wt and hDPCs/pcDNA3.0. These results showed that FHL2was stably overexpressed in hDPCs/FHL2.3. ALP activityTo investigate the expression pattern of ALP and the impacts of FHL2on expression of ALP, we detected the ALP activity in the primary cells, hDPCs/pcDNA cells and the hDPCs/FHL2cells for0,7,14,21,28days. At day0and day7, ALP activity was low in all studied groups. However, we observed up-regulation of ALP in the hDPCs/FHL2.At14days post-induction, a dramatic increase was observed in the ALP activity in all groups; on day14, the ALP activity also markedly increase in the hDPCs/FHL2compared with the two control groups. ALP gradually increased during the differentiation induction by mineral medium, reaching the peak on day21and then begined to attenuate at28days. ALP activity in the hDPCs/FHL2was significantly elevated. FHL2overexpression stimulated odontoblast differentiation as shown by increasing ALP activity.4. Cellular mineralizationTo further verify the functional impact of FHL2on formation of the mineralized nodules, Alizarin red staining were performed. The result clearly revealed FHL2overexpressing cells were stronger positive staining in red. Correspondingly, only some parts of the cellular construct were weakly stained in the hDPCs/wt and hDPCs/pcDNA3.0during the stages of odontoblast differentiation.5. Gene expressions by Real time PCRBased on promising results shown by Purmorphamine in increasing ALP activity, we evaluated the relative mRNA levels with RT followed by real time PCR confirmed the upregulation of ALP, BSP, OPN and DSPP at0,7,14and21days post-induction. The expression levels of mRNA for ALP, BSP, OPN and DSPP were compared on the hDPSCs/wt, hDPCs/pcDNA cells and the hDPCs/FHL2cells after0,7,14, and21days of culture. The mRNA level of the hDPSCs/wt at0days was set to baseline (=1.0). ALP was expressed actively during the early stages of odontoblast differentiation and reached a peak at day21. Expression of DSPP was relatively low at day0, but it was expressed in greater levels with time up to21days. Other genes coding proteins related to the dentin matrix were quite inactive during the first14days. However, they were highly upregulated at21days and this upregulation was more conspicuous on the hDPCs/FHL2cells than hDPSCs/wt and hDPCs/pcDNA cells.6. Protein analysis by Western blotTo detect the secretion of OPN, BSP, OCN and DSPP, which were important proteins of odontoblast or dentin matrix, western blot analysis was conducted on the21st day. The data clearly revealed the expression of OPN, BSP, OCN and DSPP in the hDPCs/FHL2, but these proteins were weakly expressed in hDPSCs/wt and hDPCs/pcDNA cells.Conclusions:The main findings of this study are as follows:1. The primary DPCs were stemed from dental cavum medull and then used both the enzymatic digestion method and tissue block method.2. The plasmid of pcDNA3-FHL2-Flag could express stably in human dental pulp cells. And the expression of FHL2gradually increases during the odontoblast differentiation and mineralization. The changed expression of FHL2demonstrate FHL2may be involved in the development of teeth.3. The potential functional roles were investigated by infected FHL2. hDPCs/FHL2cells were shown to activelyles of FHL2in human dental pulp cells and their odontoblast differentiation w biological behaviors with respect the hDPCs/wt cells and hDPCs/pcDNA, as determined by the expression of dentin formation associated genes, matrix synthesis and mineralization. Considering these findings as a whole, our observations confirm that the expression of FHL2in the mature dental pulp can promote the dental pulp stem cells to differentiate into odontoblast and stimulated odontoblast. |
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