Expression of M-CSF from cryopreserved teeth-derived human periodontal ligament cells with different freezing protocols
ABSTRACT: This study investigated which genes regarding root resorption are upregulated by cryopreservation and whether cryopreservation affects the expression of Macrophage –colony stimulating factor. We manufactured the customized template which was made of genes selected regarding root resorption including osteoprotegerin (OPG), receptor activator of nuclear factor-kappa B ligand (RANKL), RANKL’s cognate receptor (RANK), macrophage colony-stimulating factor (M-CSF), interleukin-1β (IL-1β), and tumor necrosis factor-alpha (TNF-α), and bone morphogenetic proteins (BMP) and analyzed gene expression. cultured human periodontal ligament cells (control) VS cryopreserved and cultured periodontal ligament cells(cryopreserved group): 3 control replicates, 3 cryopreserved replicates
Project description:Dental pulp cells of cryopreserved teeth (slow and rapid speed) were examined with microarray for screening which gene is involve in the inflammation process during the cryopreservation process. Intact caries-free, freshly extracted premolars (n=6) were collected from 3 patients for microarray assay analysis. They were classified as control and cryopreserved groups. Cryopreserved groups were divided into rapid freezing and slow freezing group.
Project description:The aim of this study was to evaluate and compare the gene expression profiles of dental follicle and periodontal ligament in humans, which can possibly explain their functions of dental follicle and PDL such as eruption coordination and stress resorption. That may apply this information to clinical problem like eruption disturbance and to periodontal tissue engineering. PDL samples were obtained from permanent premolars (n=11) and dental follicle samples were obtained during extraction of supernumerary teeth (n=4). Comparative cDNA microarray analysis revealed several differences in gene expression between permanent PDL and dental follicles.
Project description:Excessive MS is known to result in disappearance of the alveolar hard line, enlargement of thePDL space, and destruction of alveolar bone, leading to occlusal traumatism. The regulatory role of MS is believed to play a critical role in the process of alveolar bone remodeling. However, little is known about the effect of excessive MS on expression of osteoclastogenesis-related genes in human PDL cells. Human PDL cells were cultured in silicon chambers, which was attached to a stretching apparatus (STB-140; Strex Co. Ltd., Osaka, Japan) The cells were allowed to attach to the chamber base for 48 hours, after which uniaxial sinusoidal stretching (conditions: 60 sec/returns, resting time; 29 sec, stretch length; 1.6 mm, stretch ratio; 105%) was applied at 37°C, 5% CO2. We compared to the genes expression between 0 and 48 hours after MS stimulation.
Project description:Periodontitis can impair the osteogenic differentiation of human periodontal mesenchymal stem cells, but the underlying molecular mechanisms are still poorly understood. Long noncoding RNAs (lncRNAs) have been demonstrated to play significant roles under both physiologic and pathological conditions. We performed comprehensive lncRNAs profiling by lncRNA microarray to identify differentially expressed long noncoding RNA expression between Periodontal ligament stem cells from healthy Periodontal tissue and periodontal ligament stem cells from inflammatory periodontal tissue. Our analysis identified 233 lncRNAs and 423 mRNAs that were differently expressed (fold change >2.0, p-value < 0.05) between the two groups of cells. The GO analysis revealed that the significantly down-regulated biological processes included multicellular organismal process, developmental process and multicellular organismal development and the significantly up-regulated biological processes included cellular process, biological regulation and response to stimulus in periodontal ligament stem cells from inflammatory periodontal tissue. The Pathway analysis revealed that the differentially expressed mRNAs may involved in Focal adhesion, ECM-receptor interaction, Bacterial invasion of epithelial cells, Long-term depression, Circadian entrainment and HIF-1 signaling pathway. Two-condition experiment, periodontal ligament stem cells from healthy periodontal tissue (hPDLSCs) vs. periodontal ligament stem cells from inflammatory periodontal tissue (pPDLSCs), Biological replicates: 3 control replicates (hPDLSCs), 3 testing replicates (pPDLSCs).
Project description:The periodontium are the tissues supporting and investing the tooth and consists of the periodontal ligament, the gingiva, the root cementum, and the alveolar bone. The functions of the cell populations in health and disease regarding the host-mediated tissue destruction are not well understood. To get a first idea, of which genes might play a distinct role in chronic periodontal disease in vivo, we compared the genom-wide gene expressions of chronic inflamed and healthy periodontal ligament cells by microarray analysis and validated the data by real-time RT-PCR. The expression rates of 14.239 genes were investigated and 3.018 of them were found differentially expressed by at least two-fold, the expression rates of 1.451 genes were significantly up-regulated and the expression rates of 1.567 genes were significantly down-regulated in inflamed PDL cells. We focused on mainly structural components, for example, laminins and integrins, as well as degrading enzymes, for example, MMPs and cathepsins. The molecular composition of the laminin network varies in chronic inflamed compared to healthy PDL cells in vivo. Furthermore, integrin alpha6beta4, together with laminin-332, might be involved in chronic periodontal inflammation. Findings that diverse keratins were upregulated in chronic disease indicate that the epithelial cell rests of Malassez might also be involved in chronic periodontal inflammation. Also cathepsin B and cathepsin C might participate in the connective tissue destruction. The microarray analysis has identified a profile of genes potentially involved in chronic periodontal inflammation in vivo. Further studies are needed to entirely understand cellular activities during chronic periodontal inflammation in vivo. Experiment Overall Design: Periodontal tissue was collected from 32 patients at the Dental Medical School of the University of Goettingen (12 men, 20 women, aged between 18 and 72 years) from March 2005 to Dezember 2005. The tissue probes were taken from teeth, either extracted for orthodontic reasons (healthy periodontium) or because of chronic periodontal lesions. The differentiation between both collectives was performed using clinical and radiological parameters (clinical attachment loss, increase in probing depth, and radiographic bone loss). A detailed anamnesis of each patient was explored. All patients were without a medical history and were not on medication. In addition, the tissue samples used in our study were only taken from non-smokers. The extracted teeth were immediately frozen and stored at -80°C. All patients who participated in the study were informed about the nature and aim of this project and gave their written informed consent. The study was approved according to the regulations of the Ethics Committee of the Medical Faculty of the University of Goettingen.
Project description:Our recent studies suggested that the freezability of carp semen is related to seminal plasma protein profiles. Here, we aimed to compare the spermatozoa proteomes of good (GF) and poor (PF) freezability semen of carp. To achieve this, we used two-dimensional difference in gel electrophoresis followed by MALDI-TOF/TOF mass spectrometry. The semen was classified as GF or PF based on sperm motility after freeze/thawing. We identified proteins enriched in spermatozoa of GF (22 proteins) and PF (18 proteins) semen. We also identified 12 proteins enriched in the supernatant after cryopreservation of PF semen. Good freezability is related to high concentrations of proteins involved in the maintenance of flagella structure, membrane fluidity, efficient control of Ca2+ and sperm motility, energy production, and antioxidative protection, which likely reflects the full maturation status of spermatozoa of GF semen. On the other hand poor freezability seems to be related to the presence of proteins identified as released in high quantities from cryopreserved sperm of PF. Thus, the identified proteins might be useful bioindicators of freezing resilience and could be used to screen carp males before cryopreservation, thus improve long-term sperm preservation in carp.
Project description:Analysis of effect of long-term cryopreservation on peripheral blood mononuclear cells at gene expression level. The hypothesis tested in the present study was that long-term cryopreservation has an influence on the transcriptome profile of peripheral blood mononuclear cells. Results indicated remarkable changes in expression patterns upon cryopreservation of PBMCs, with decreasing signal intensities over time. Overall design: Total RNA obtained from isolated peripheral blood mononuclear cells of melanoma patients cryopreserved in liqiud nitrogen from 20 to 60 months.
Project description:Cryopreservation is a routinely used methodology for prolonged storage of viable cells. The use of cryo-protective agents (CPAs) such as dimethylsulfoxide (DMSO), glycerol or trehalose is paramount to reduce cellular cryo-injury but their effectiveness is still limited. The current study focuses on establishing and modulating the proteomic and the corresponding biological profiles associated with the cryo-injury of human leukaemia (HL-60) cells cryopreserved in DMSO alone or DMSO +/- novel CPAs [e.g. nigerose (Nig) or salidroside (Sal)].
Project description:Analysis of cryopreservation effects on peripheral blood mononuclear cells at gene expression level. The hypothesis tested in the present study was that cryopreservation has an influence on the transcriptome profile of peripheral blood mononuclear cells. Results indicated remarkable changes in expression patterns upon cryopreservation of PBMCs, with a strong loss of signal intensities to background levels for several transcripts. Overall design: Total RNA obtained from isolated peripheral blood mononuclear cells of healthy human subjects eihter cryopreserved in liqiud nitrogen (frozen) or direclty lysed in Trizol after isolation (fresh).
Project description:Analysis of effect of long-term cryopreservation on peripheral blood mononuclear cells at gene expression level. The hypothesis tested in the present study was that long-term cryopreservation has an influence on the transcriptome profile of peripheral blood mononuclear cells. Results indicated remarkable changes in expression patterns upon cryopreservation of PBMCs, with decreasing signal intensities over time. Total RNA obtained from isolated peripheral blood mononuclear cells of melanoma patients cryopreserved in liqiud nitrogen from 20 to 60 months.