Molecular Profiling Of Dental Pulp Stem Cells During The Odontoblastic Differentiation Based On Nanoprobes

Dental pulp stem cells(DPSCs)are a population of adult stem cells with high proliferation,self-renewal ability and multi-phase differentiation potential.They can differentiate into specific cell types under certain conditions and play an important role in pulp restoration and tooth regeneration.When the tooth is stimulated by caries,abrasion,trauma and so on,the DPSCs in the pulp are activated to promote the formation of odontoblast-like cells,which can repair the dentin to prevent the progression of the lesion and preserve the pulp vitality.Therefore,understanding the mechanism of dentinal differentiation of DPSCs and discovering the key biological substances involved in the differentiation process is of great significance for the formation of physiological pulp dentin complex and the construction of tissue engineering teeth.Traditional methods used for stem cell differentiation study mainly include immunocytochemistry,gel electrophoresis,Western blotting,and so on.The immunocytochemistry has been widely used for observation the expression of proteins in complex biological processes.It can directly read the change of a single component known.However,this method often suffers from fluorescence quenching or bleaching,resulting in inaccurate results;while western blotting which exploits specific antibody colored cell or biological tissue samples treated by the gel electrophoresis to obtain information about the expression of specific proteins in the cells or tissues analyzed.They both require large amounts of samples and extensive extraction processes for detection.Although,these biotechnologies have many advantages such as strong specificity and high sensitivity,their operational steps are complicated and time-consuming,and are difficult to achieve non-destructive detection of living cells.Moreover,these methods are greatly limited in the multi-components study.Consequently,the development of non-destructive,simple and fast in-situ methods for cell differentiation monitoring and studies is urgently needed.along with the development of nanotechnology,nanomaterials combine with specific biomolecular sensing and performance regulation due to their unique physical and chemical properties,and then develop the wide application of various nanoprobes in biosensing and labeling,and then promote the development of optoelectronic and nanotechnology,especially in surface-enhanced Raman spectroscopy(SERS).In the last few decades,SERS technique as a powerful analytical method,which provides sensitive,fast and non-destructive fingerprint spectrum,has been widely applied for living cell studies,such as monitoring the cell apoptosis process,distribution of pH in cells,cell species identification and so on.However,as far as we known,the study on using SERS method to track the differentiation process of DPSCs and reveal its differentiation mechanism has not been reported.In this study,we applied drug stimulation to accelerate the directional differentiation of DPSCs,and identified key differentiation biomarkers in single-cell level by molecular profiling of DPSCs during cell differentiation using SERS technique.The main content and innovation points are mainly reported in the following aspects:1.In this study,DPSCs were extracted from third molar teeth of healthy adults aged 18-25,which extracted and purified by tissue block using enzymatic digestion.At the same time,immunocytochemistry,fluorescence and flow cytometry were used to determine the extracted cells as dental pulp stem cells.After that,we used differentiation drugs to stimulate of stem cells and established a cell model of DPSCs differentiation to dentin cells.2.Differentiation model was established,and then we prepared gold nanoparticle(AuNPs)targeting to nuclear probe to study the mechanism of tooth differentiation.First of all,we use method of hydrothermal reaction to synthesize AuNPs.The suitable size of gold nanoparticle was then modified by Au-S through the covalent action with mercapto-polyethylene glycol(PEG),cell membrane sequence peptide(RGD),and nuclear localization signal sensing peptide(NLS),respectively.The gold nanoparticle probes(NLS@RGD-PEG-AuNPs,NT-AuNPs)provides more biocompatible and good nuclear targeting.The localization of NT-AuNPs was charactered by bright and dark field imaging,Bio-TEM and fluorescence imaging.The results showed that most of the AuNPs were located into cell nucleus.Moreover,the NT-AuNPs have good biocompatibility.Furthermore,the prepared nuclear probe has no effect for SERS detection of dental pulp stem cells differentiation.3.The SERS nanoprobes were used to investigate the molecular profiling of cell nucleus within dental pulp stem cells during odontogenic differentiation by Surface Enhanced Raman Spectroscopy.In this study,the SERS spectra of cell nucleus within DPSCs during differentiation process were collected by a confocal Raman spectrometer.We have analysed the SERS spectra from label free-single cell SERS spectra,the results of which have indicated that the content of tryptophan were increased significantly at 14 days of differentiation stimulation.Meanwhile,the vibrational mode of the DNA/RNA backbone also changed during differentiation,which indicates that DNA replication or transcription may occur during differentiation.In this thesis,we have employed the drug to stimulate and accelerate directional differentiation of DPSCs.The molecular spectrum of DPSCs during cell differentiation process was identified by SERS technique.It was determined that tryptophan be involved in the process of dentin differentiation,providing a reliable molecular level research method for dental tissue engineering and broadened the field of vision of differentiation theory.