The Study Of Biological Function And LncRNA Expression Of PDLSCs Derived From Healthy And Periodontitis Microenvironment Under Static Mechanical Strain

As the orthodontics could improve the profile and smile of patients by moving teeth, more and more adult patients are searching for comprehensive orthodontic treatment. However, the high morbidity of periodontitis is an intractable problem in adult orthodontics. Periodontitis leads to loss of periodontal tissue and even the teeth because of localchronic inflammation. So far, the treatment of periodontitis includes inflammation control and periodontal tissue regeneration. Periodontal ligament stem cells(PDLSCs) are considered to be one of the most promising seed cells because of its high capacity of proliferation, differentiation and tissue homology. During the period of orthodontic tooth movement, PDLSCs play an important role in transduction of mechanical stimulation and tissue remodeling. Studies have shown that the regeneration ability of PDLSCs could be damaged by periodontitis microenvironment. However, the response of PDLSCs to mechanical stimulation in the inflammatory microenvironment is still unknow. Lnc RNA has been demonstrated to play a crucial role in inflammatory diseases, and researches reported that there were many differently expressed Lnc RNAs between healthy andinflammatory periodontal tissues. Nevertheless, it is unclear how Lnc RNAs regulate the response to static mechanical strain(SMS) of PDLSCs obtained from healthy periodontal tissues(HPDLSCS) and PDLSCs obtained from periodontal tissues of periodontitis patients(PPDLSCs). Accordingly,, we designed two parts of studies :Part I The study of the response of HPDLSCs and PPDLSCs to different magnitudes of SMS and the filtration of optimal SMSObjective 1. To isolated and culture the HPDLSCs and PPDLSCs, and identify their biological characteristics. 2. To make sure the effect of different magnitudes of SMS on the proliferative and osteogenic capacities of HPDLSCs and PPDLSCs. 3. To make sure the effect of different magnitudes of SMS on the osteoclastogenic capacities and inflammatory reactions of HPDLSCs and PPDLSCs. 4. To find out the optimal SMS values for promoting the regeneration ability of HPDLSCs and PPDLSCs.Methods 1. HPDLSCs and PPDLSCs were isolated and cultured by low density inoculation. The expressions of mesenchymal stem cell markers were detected by flow cytometry, proliferative activity was determined by colony-forming assay, and osteogenic capacity was tested by alizarin red staining. 2. 6%、8%、10%、12%、14% SMS was exerted to the the HPDLSCs and PPDLSCs by Flexcell Tension Unit. 3. After loading of different SMS, cell cycles of HPDLSCs and PPDLSCs were tested by flow cytometry, cell viabilities were examined by MTT assay, and osteogenic capacities were determined by ALP staining, ALP activity, alizarin red staining and real time RT-PCR. 4. After loading of different SMS, expressions of osteoclastogenic genes Rankl and C-fos were detected by real time RT-PCR, and secretions of inflammatory cytokines IL-6, IL-8, IL-1β, and TNF-α were examined by elisa.Results 1. Both HPDLSCs and PPDLSCs strongly expressed the mesenchymal stem cell markers Stro-1, CD146, CD90, CD29, and were negative for hematopoietic markers CD31 and CD14. The expressions of Stro-1, CD146, CD90, CD29 were higher in HPDLSCs than PPDLSCs. Colony-forming assay showed that the colony-forming rate of HPDLSCs was 37%, and that of PPDLSCs was 64%. Alizarin red staining indicated that both HPDLSCs and PPDLSCs generated mineralized nodules, and the nodules in HPDLSCs were more and larger. 2. Cell cycle and MTT assay showed that, the proliferative ability of PPDLSCs was higher than HPDLSCs without SMS. When SMS was loaded, all the SMS from 6% to 14% improved the proliferative abilities of HPDLSCs, while 12% SMS presented the best effect. For PPDLSCs, only 6% and 8% SMS promoted the proliferative capacity, and 8% showed a better effect. When SMS was larger than 8%, the proliferative ability of PPDLSCs decreased in a magnitude-dependent manner. The results of ALP staining, ALP activity, alizarin red staining and real time RT-PCR consistently demonstrated that, PPDLSCs had a worse osteogenic capacity than HPDLSCs when there was no SMS loading, while every index of HPDLSCs increased significantly when the SMS was loaded, in which 12% SMS was optimal. For PPDLSCs, 8% SMS improved the osteogenic capacity to the greatest extent, and when the force was larger than 8%, the the osteogenic capacity of PPDLSCs was inhibited obviously. 3. Real time RT-PCR was used for detecting the expressions of Rankl and C-fos in HPDLSCs and PPDLSCs. It showed that when there was no SMS loading, expressions of Rankl and C-fos in PPDLSCs were significantly higher than HPDLSCs. When the SMS was loaded, the expressions of Rankl and C-fos didn’t change obviously under force values lower than 12%. When SMS was larger than 12%, the expressions of Rankl and C-fos up-regulated significantly. In the groups of PPDLSCs, the expressions of Rankl and C-fos were not activated when SMS was lower than 8%, but these genes were obviously activitaed to a high level when SMS was higher than 8%. The Elisa results showed that, the secretions of IL-6, IL-8, IL-1β and TNF-α were higher in PPDLSCs than in HPDLSCs.When SMS was loaded, the expressions of IL-1β and TNF-α increased a little, but there was no difference in each SMS group. However, the expressions of IL-6 and IL-8 up regulated to a considerable high level. Specifically speaking, for HPDLSCS, when SMS was lower than 12%, the expressions of IL-6 and IL-8 showed no differences in each group, and when SMS was higher than 12%, the expressions increased. For PPDLSCs, SMS of 6% and 8% had no difference in the expressions of IL-6 and IL-8, but when SMS was larger than 8%, the secretions of the two nflammatory cytokines showed a magnitude-dependent up-regulation.Conclusions 1. Both HPDLSCs and PPDLSCs expressed the mesenchymal stem cell markers, demonstrating they had the self-renewal and pluripotential capacities. However, the periodontitis microenvironment stimulated the proliferative capacity, decreased the expression rate of mesenchymal stem cell markers and damaged the osteogenic capacity of PPDLSCs. 2. HPDLSCs and PPDLSCs responded differently to SMS. PPDLSCs were less tolerant and more sensitive to SMS attributed to the inflammatory microenvironment. Any excessive force could lead to a decrease of proliferation, an unbalance between osteogenesis and osteoclastogenesis, and an activation of inflammatory reactions. 3. For improving periodontal regeneration and tissue remodeling, the optimal SMS for HPDLSCs was 12%, whilt it was 8% for PPDLSCs.Part II The study of Lnc RNA expression of HPDLSCs and PPDLSCs under 12% SMSObjective 1. To select the differently expressed Lnc RNAs under 12% SMS 2. To analyze the expressions and functions of Lnc RNAs in HPDLSCs and PPDLSCsMethods 1. After 12% SMS loading, the total RNA in HPDLSCs and PPDLSCs was extracted by TRIzol, and RNA was purified by RNasey Mini Kit. The quality control and concentrationdetermination of RNA were performed using spectrophotometer. Agarose gel electrophoresis was used for detecting the integrity of the subjects and DNA contamination. After c DNA synthesis, Nano Drop ND-1000 was performed for the quality control. Then Nimble Gen Hybridization and Nimble Gen wash buffer were used for chip hybridization and washing, Axon Gene Pix 4000 B was used for chip scanning, Nimble Scan software and Agilent Gene Spring GX software for image and data analysis. 2. KEGG pathway analysis, Go analysis and CNC analysis were used for bioinformatics analysis. 3. The reliability of Lnc RNA arrays was testified by real time RT-PCR.Results 1. The results examined by spectrophotometer showed that, the absorbances of the extracted RNA from HPDLSCs and PPDLSCs were both between 1.8-2.1, and that were close to 2.0. Meanwhile, the extracted RNA was not contaminated by g DNA and not obviously degraded confirmed by Agarose gel electrophoresis, which could be used for the further array study. The box plot showed that all the six subjects had consistent fluorescence signals, so that the results could not be disturbed by discrepant signals. The scatter plot, volcano plot and clustering plot exhibited visually that there did exist abundant differently expressed Lnc RNAs between HPDLSCs and PPDLSCs when 12% SMS was loading. 2. When 12% SMS was loaded, there were 8847 differently expressed Lnc RNAs in HPDLSCs which were statistically significant and changedover 2 folds. The number was 9772 for PPDLSCs. The number of co-expressed Lnc RNAs in HPDLSCs and PPDLSCs was 7223. the number of Lnc RNAs which changed only in HPDLSCs was 1624(the number of up-regulated change that over 20 folds was 27) and changed only in PPDLSCs was 2549(the number of changeover 20 folds was 16 in which 9 were up-regulated and 7 were down-regulated). 3. KEGG pathway analysis indicated that the differently expressed m RNAs relatedbiological pathways in HPDLSCs included fatty acid degradation pathway, MAPK signaling pathway, vascular smooth muscle contraction pathway, fatty acid metabolism pathway, starch and sucrose metabolism pathway, glycine pathway, serine and threonine metabolism pathway, mineral absorption pathway, calcium signaling pathway and protein processing in endoplasmic reticulum; while in PPDLSCs the related biological pathways included sphingolipid metabolism pathway, cocaine addiction pathway, Erb B signaling pathway, Huntington’s disease pathway, lysine biosynthesis pathway, B cell receptor signaling pathway, bladder cancer pathway, homologous recombination pathway, non-small cell lung cancer and non-alcoholic fatty liver disease pathway. Go analysis showed that several differently expressed BP items in HPDLSCs played parts in mechanical stimulus and extracellular signal transduction, for example, “response to stress”、“regulation of response to stimulus”, etc.., however these items didn’t change in PPDLSCs. CNC analysis showed that there were 1250 m RNAs coexpressed with the most differently expressed 10 Lnc RNAs. KEGG analysis indicated that these m RNAs were mainly related to pathways such as arginine and proline mtabolism, cell adhesion molecules and leukocyte transendothelial migration. 4. The real time RT-PCR showed that, the four randomly selected Lnc RNAs(TCONS_ 00008604, ENST00000428781, Uc004 arq.1, ENST00000445814) expressed consistently with the Lnc RNA microarray results.Conclusions 1. The extracted RNA samples passed the quality control and could be used for the Lnc RNA microarray detection. 2. Under 12% SMS there exited massive differently expressed Lnc RNAs. 3. The pathways related to differently expressed m RNAs in HPDLSCs were mostly normal biological pathways, while in PPDLSCs they were related to diseases. 4. The pathways related to mechanical stimulus and extracellular signal transduction changed obviously in HPDLSCs but not in PPDLSCs, which indicated that periodontitismicroenvironment lead to abnormal response of PPDLSCs to mechanical stimulus. 5. The results of real time RT-PCR were consistent with the Lnc RNA microarray data, and the expression fold change in ENST00000445814 was up to 250 times, which could be our further research target.