| Chronic periodontitis is a common disease of periodontal tissue loss.Patients with periodontitis often have teeth displacement,gap scattered in anterior teeth,which affects the smile.Patients with periodontitis often seek orthodontic treatment to restore smiling aesthetics.However,the response of orthodontic treatment between healthy periodontal tissue and inflammatory periodontal tissue is very different.The "light force" relative to healthy tissue may become "heavy force" for periodontal tissue in patients with periodontitis.In some severe cases,it may even leading loss of periodontal attachment and alveolar bone absorption,which affects the health of the teeth and the stability of the occlusal function.This different response of orthodontic force may closely relate to the functional roles of periodontal ligament stem cells?PDLSCs?.PDLSCs have high proliferation capacity,self-renewal and multi-directional differentiation power,which can influence the reconstruction of periodontal tissues under the guidance of mechanical signals.It is an important cell to study the process of orthodontic force acting on periodontal tissues.Understanding the response of PDLSCs under different mechanical loads in the inflammatory microenvironment can help us understand the causes and mechanisms of orthodontic responses between healthy periodontal tissues and periodontal tissues in patients with periodontitis,and rationally apply the corrective force in clinical treatment.In our early study,we applied different levels of static mechanical strain?SMS?to PDLSCs.It was found that 8% SMS loading of PDLSCs derived from periodontal tissues of periodontitis patients?PPDLSCs?had the strongest osteogenic differentiation,but Osteogenic differentiation of PPDLSCs was inhibited when 12% SMS was loaded,which was significantly different from the osteogenic differentiation of HPDLSCs?PDLSCs obtained from healthy periodontal tissues,HPDLSCs?at 12% SMS loading.The reason of differences between PPDLSCs and HPDLSCs in osteogenic differentiation under the same level of SMS loading is the focus of our follow-up study.Long non-coding RNA?LncRNA?is an RNA with no coding ability and a length of more than 200 nt,which can play an important regulatory role in cell proliferation,differentiation and development.Studies have shown that there are a large number of differentially expressed lncRNA between inflammatory periodontal tissues and healthy periodontal tissues,suggesting that it may affect the cell regeneration and osteogenic differentiation of PDLSCs in the inflammatory microenvironment.Therefore,we hypothesize that lncRNA is also likely to be involved in the regulation of osteogenic differentiation of PDLSCs under mechanical stress loading,which provides a research direction for exploring the osteogenic differentiation of PDLSCs under mechanical stimulation.Based on this,this study designed the gene microarray study of PPDLSCs loaded with 12% SMS,and screened a large number of lncRNA differentially expressed before and after loading of PPDLSCs.After verifying the regulatory function of lncRNA during osteogenic differentiation,It was found that LINC00638 can compete with FGFR1 gene for miR-424-5p,which is actively involved in regulating the osteogenic differentiation of PPDLSCs after SMS loading,and provides a theoretical reference for the regulation of osteogenic differentiation of PDLSCs mediated by mechanical stimulation.This topic mainly includes three parts: Part ? The study of the response of HPDLSCs and PPDLSCs to different magnitudes of SMS Objective: 1.Culture and identify HPDLSCs and PPDLSCs in vitro;2.To study the effects of different levels of SMS loading on osteogenic differentiation of HPDLSCs and PPDLSCs;3.Determine the target strength of SMS loading.Methods: 1.HPDLSCs and PPDLSCs were obtained by low density method;cell properties were identified by flow cytometry;colony formation and CCK-8 were used to detect the proliferation of cells in each group,and growth curves were drawn;alizarin red staining and oil O were induced to identify the multi-directional differentiation potential of cells;2.Fx-4000 T system was used to construct different levels of SMS loading of cell model;CCK-8 was used to detect the proliferation of HPDLSCs and PPDLSCs after different levels of SMS loading;PCR and alizarin red staining were used to detect the osteogenic differentiation of HPDLSCs and PPDLSCs after SMS loading.Results 1.HPDLSCs and PPDLSCs were successfully obtained in vitro;the two groups of cells positively expressed the surface markers of mesenchymal stem cells,Stro-1,CD146,and negatively expressed the hematopoietic markers CD31 and CD14.The results indicated that both HPDLSCs and PPDLSCs were mesenchymal stem cells.The results of crystal violet staining showed that the clone formation rate of PPDLSCs was significantly higher than that of HPDLSCs.CCK-8 assay showed that the overall growth rate of PPDLSCs was greater than HPDLSCs.2.The results of alizarin red staining showed that compared with HPDLSCs,the mineralized nodules formed by PPDLSCs were smaller and more dispersed,and the coloration was lighter than HPDLSCs.The results of oil red O staining showed that the fatogenic differentiation ability of PPDLSCs was weaker than that of HPDLSCs.3.The SMS cell loading model was successfully established using the FX-4000 T system.The CCK-8 results showed that the proliferation activity of PPDLSCs was activated after 8% SMS loading,but the proliferation of PPDLSCs was inhibited after 12% SMS loading;8% SMS and 12%SMS can both promote the proliferation of HPDLSCs,and the 12% SMS group has the highest degree of proliferation.4.Quantitative analysis of PCR and mineralized nodules showed that 12% of SMS could promote osteogenic differentiation of HPDLSCs,but did not promote osteogenic differentiation of PPDLSCs.Conclusions 1.HPDLSCs and PPDLSCs obtained in vitro has the potential of proliferation and multi-directional differentiation;the inflammatory microenvironment affects the osteogenesis and adipogenic differentiation of PPDLSCs.2.PPDLSCs and HPDLSCs respond differently to SMS loading.12% SMS is the most obvious difference mechanical loading in HPDLSCs and PPDLSCs.Part? The study and bioinformatics analysis of lncRNA microarray expression profiles in PPDLSCs under 12% SMS loading Objective 1.Construction of lncRNA gene expression profiles of PPDLSCs after SMS loading.2.Bioinformatics analysis of differentially expressed lncRNA.Methods 1.After loading the 12% SMS in PPDLSCs,TRIzol extracts total cellular RNA,then amplifies each sample and transcribes it into fluorescent cRNA along the length of the entire transcript.The cRNA was purified using the RNeasy Mini Kit,and the concentration and activity of the labeled cRNA were determined using NanoDrop ND-1000.Cleaning,immobilizing and scanning hybridization arrays using an Agilent DNA microarray scanner.2.Perform image data analysis using Nimble Scan software and perform hierarchical clustering using Agilent Gene Spring GX software?11.5.1 version?.And data grouping according to the up-and down-regulation of the differential expression of lncRNA and mRNA;3.Using KEGG analysis,GO analysis,CNC analysis to predict the molecular pathways that lncRNA may be involved in regulation,biological regulation function.4.qPCR technology was used to verify differentially expressed lncRNA in the chip.Results 1.Scatter plot,volcano map and Heatmap showed that there were a large number of differentially expressed lncRNA and mRNA in PPDLSCs before and after loading.Among them,4471 lncRNA expression was up-regulated,6444 lncRNA expression was down-regulated,and 7077 mRNA expression was up-regulated.,5816 mRNA expression levels were down-regulated.2.In the mRNA with up-regulated expression,the KEGG results showed the top ten pathways were mainly prostate tumor,tumor proteoglycan synthesis pathway,actin cytoskeletal pathway,MAPK pathway,hormone.Secretory pathways,cancer-mediated pathways,insulin resistance pathways,viral oncogenic pathways,long-term potentiation pathways,epidermal growth factor receptor pathways.Among the mRNAs whose expression levels are down-regulated,the top ten pathways are: cell cycle-mediated,DNA replication,gene homologous recombination pathway,DNA repair,FA-BRCA network,NOD-like receptor signaling pathway,splice,non-Homologous terminal junction pathway,nucleotide excision repair,base excision repair pathway.3.In molecular function,GO analysis results showed the differentially expressed mRNA were mainly involved in protein synthesis,growth factor binding,ATPase activity activation,damage DNA repair,etc.;In biological process: mainly involved in signal pathway regulation,growth and development,Multicellular tissue development,cell cycle,molecular differentiation,etc.;In cell composition: mainly involved in cell binding,intracellular junction,nuclear division,nucleolar formation,etc.4.CNC analysis showed that there are multiple molecules involved in the osteogenesis regulatory pathway may related to some differentially expressed lncRNA in our chip,such as FGFR1,FPR2,DAPK1,etc.5.The trend of 7 lncRNAs such as ENST00000550947 is basically similar to the trend of chip results,only slightly different in the expression change.The PCR results of TCONS00008000 were inconsistent with the chip results,and the difference was not statistically significant.Conclusion PPDLSCs produced a large number of differentially expressed lncRNAs and mRNAs after 12% SMS loading,some of which were associated with osteogenesis regulatory pathways such as MAPK signal pathway.Part ? The study of LINC00638 regulates osteogenic differentiation in PPDLSCs under SMS loading Objective: To investigate the effect of LINC00638 on the osteogenic differentiation of PPDLSCs under the 12% SMS,and explore the mechanism.Methods: 1.Screening target lncRNA by observing the expression of selected lncRNA during osteogenesis regulation.2.Lentivirus stably transfected cells,overexpressed and silenced the gene expression of LINC00638,and detected the changes of osteogenic differentiation of PPDLSCs under 12% SMS by using PCR,ALP staining,alizarin red staining analysis.3.Silencing LINC00638 and detecting the osteogenic differentiation ability of PPDLSCs under 8% SMS.4.Bioinformatics predicts the possible mode of action of LINC00638.5.Detected the expression level of LINC00638 and the expression level of miR-424-5p by PCR after silencing LINC00638 and miR-424-5p;ceRNA analysis of its binding site.6.RT-PCR and Western Blot were used to find the expression of FGFR1 whicn was observed after overexpression and silencing of LINC00638.Results 1.LINC00638 was our target lncRNA;after transfecting overexpressing LINC00638 lentivirus,the expression level of LINC00638 increased by 3.25 times.After transfection of silencing LINC00638 lentivirus,the expression of LINC00638 decreased by 2.67 times compared with NC group.2.After PPDLSCs was loaded with 12% SMS for 12 h,the results of PCR showed that after overexpressing LINC00638,the gene expression of Runx2,ALP and Col1 in the 12% SMS+LINC00638 group was significantly higher than that in the control group?P<0.05?,among which Runx2 The most significant increase was observed;after silencing LINC00638,the gene expression of the 12% SMS+LINC00638 RNAi group was lower than that of the control group?P<0.05?.ALP staining and alizarin red staining results also support this trend.3.After silencing LINC00638,the expression of Runx2,ALP and Col1 in the 8% SMS group also decreased,and the results of ALP and alizarin red staining were the same.4.Bioinformatics prediction results showed that LINC00638 can compete with FGFR1 for adsorption of miR-424-5p,forming a ceRNA effect.5.anti-miR-424-5p can effectively reduce the expression of miR-424-5p in PPDLSCs.At the same time,the expression level of LINC00638 increased?1.65 times?;after transfection of LINC00638 RNAi,the expression level of miR-424-5p also increased.6.Overexpression of LINC00638 can promote the expression of FGFR1 in gene and protein level;silencing LINC00638 will inhibit the expression of FGFR1 in gene and protein level.Conclusions 1.LINC00638 fully participates in the regulation of osteogenic differentiation of PPDLSCs under 12% SMS loading.Overexpression of LINC00638 can promote osteogenic differentiation of PPDLSCs under 12% SMS stimulation in vitro.2.Silencing LINC00638 can inhibit the osteogenic differentiation of PPDLSCs under 8% SMS;3.LINC00638 may form a negative loop with miR-424-5p,which is involved in the regulation of osteogenic differentiation by FGFR1. |
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