Dissertation > Excellent graduate degree dissertation topics show

LEF-1 Expression of the (Like) Osteoblast under Shear Forces

Author: ChenLuoPing
Tutor: ZhangGuiRong
School: Dalian Medical University
Course: Orthodontics
Keywords: MC3T3-E1 Human periodontal ligament cells Mesenchymal cells LEF-1 shear force Gene Expression Osteogenesis Classic Wnt signaling pathway
CLC: R783.5
Type: Master's thesis
Year: 2011
Downloads: 38
Quote: 0
Read: Download Dissertation

Abstract


Background and Purpose: Mechanical stimulation of cells in the process of biological evolution is subjected to one of the most basic biological stimulation, proper mechanics stimulation can promote cell growth and differentiation in vivo experiments and clinical application discovery suitable mechanical loading in fracture repair, Orthodontic tooth movement and distraction osteogenesis (maxillary deficiency of therapy, restoration of bone defects, limb lengthening) and other treatment, can stimulate the growth of bone tissue itself and reconstruction. In vitro experiments show that while a large number, loads the appropriate stress protein may promote bone formation and its receptors, collagen, OPN, VEGF expression, etc., induce proliferation and differentiation of bone cells themselves, adjusting its physiological function, contributes to cell matrix mineralization and bone remodeling. study class osteoblasts and osteoblast precursor cells in mechanical signal transduction and biological response of bone cells for orthodontics, orthognathic surgery, dental implants, alveolar bone regeneration, seed cells Engineering is important, but the current mechanical stress stimulate osteoblast mechanical signal transduction mechanism and osteogenic cells in the biological response of the mechanical environment remains to continue in-depth. Wnt signaling pathway by regulating osteoblast proliferation, differentiation and feature to affect the individual postnatal growth and development of bone tissue. Upward or downward in this way the various factors can significantly change the expression of osteoblast function and changes the amount of change in the bone. Lymphoid enhancer factor -1 (1ymphoid enhancer factor-l, LEF-1) is a lymphoid enhancer factor / T-cell factor (LEF / TCF) member of the family, located on human chromosome 4 q23-q25 region, as an important transcription factor is a core component of the Wnt signaling pathway, is a high mobility component (high mobility group, HMG). Studies have shown that LEF-1 in the Wnt signaling pathway, IL-7 receptor signaling pathway, TGF-β signaling pathway and several other signaling pathway, is complex and multi-promoter features, multi-domain, multi-isomers Features . LEF-1 protein abnormalities in a number of tooth development, the development of bone, oral malignant tumors such as malignant melanoma cells were found and attention on the case in a variety of bone (class) cells after stimulation mechanics accepted expression, at home and abroad has not been reported by domestic and foreign core journals literature search, reflecting the Wnt pathway upstream research include the identification of target gene transfer, the membrane receptor binding, the medium in the cytoplasm of the transfer and the transfer of research involved many aspects activate the Wnt signaling pathway to promote intracellular expression of genes sensitive [2]. ago Osx osteoblast gene under the control of differentiation typical osteoblast differentiation and maturation, synthesis and secretion of extracellular matrix, including early maturity bone sialoprotein, osteocalcin mid and late osteopontin [3-4]. Based on the above experimental basis, the present study, the mechanical loading apparatus from different species of osteoblast precursor cells: rat bone marrow derived Mesenchymal stem cells (Marrow Mesenchymal Stem Cells, MSCs), human periodontal ligament cells (human periodontal ligament cells, hPDLC), mouse-derived MC3T3-E1 osteoblast-like cell line cross-sectional study, the level of genes from the cells and protein horizontal force through the cell before and after LEF-1 expression changed, and explore three types of osteoblasts in mechanical stress LEF-1 expression of downstream target effector mechanisms and tension force on the characteristics of cell differentiation and proliferation, from the Wnt signaling pathway perspective to explore the biological function of LEF-1 and mechanical stress to induce bone formation into molecular mechanisms. MATERIALS AND METHODS: a bone marrow mesenchymal stem cells (BMSCs) of primary cultured bone marrow adherent France cultured rat BMSCs. After rats were sacrificed rat femur and tibial epiphyseal side cut, PBS marrow repeated washing, rinsing fluid collected in the centrifuge tube, centrifuged supernatant solution was added to the culture to form a single cell suspension by pipetting, inoculated in the culture bottle of conventional liquid passage .2 for human periodontal ligament cells in primary culture collection of the clinical need for orthodontic aged 11-14 periodontal health caries removal fresh premolars, immediately after removal of dual anti-PBS solution and DMEM medium repeated cleaning teeth scraped root 1/3 periodontal ligament tissue, without cutting, directly placed in a centrifuge tube, add type Ⅰ collagenase digestion method combined with enzyme mixed tissue culture method hPDLC. Take the second generation of cells were detected by immunocytochemistry SABC confirmed periodontal ligament fibroblasts .3 MC3T3-E1 osteoblast cell cultures of mouse embryonic cell line MC3T3-E1 cells after resuscitation with DMEM culture medium (containing 10% FBS ), medium was changed every 3 days 1, 80% confluent cells, digested with 0.25% trypsin, according to 5 × 105 cells were seeded to a 25 cm culture flask subculture. 4 reverse transcription polymerase chain reaction detection afterburner around three kinds (classes) of osteoblasts LEF-1 expression in a) primer design and synthesis; 2) total cellular DNA extraction and reverse transcription; 3) the total DNA reverse transcriptase the cDNA; 4) The cDNA for PCR amplification product after electrophoresis of the PCR amplification product: PCR reaction was completed, a 1.2% agarose gel electrophoresis, using the DL-2000 DNA Marker, pictures and scan results. 5 double labeling immunofluorescence after afterburner with 4% paraformaldehyde, 0. 2% TritonX-100 permeabilization to crosslinking, during which 2% bovine serum albumin closed non-specific sites. Each climbing film Add 30μ12% bovine serum albumin (BSA) diluted non-phosphorylated β-catenin polyclonal antibody (1:100), into the wet box 1h, light 30μl2% BSA was added diluted labeled goat anti RBITC rabbit secondary antibody (1:150), placed in a humidified chamber 1h. DAPI role in 5 min, the slides Flip the new slide, middle MOVO (fluorescence quencher) 10 ml, 95% glycerol were mounted. Inverted fluorescence microscope, respectively 495 nm, 527 nm excitation light was observed in the expression of the same field of view and camera. The experiment was repeated three times. Each level of each photo selected three different horizons, 50 cells per field, with Image2pro2plus image analysis software to analyze the level of expression of each integrated optical density (IOD) .6 ALP staining the cells with 1 × 10 ~ 8 / ml was inoculated slides → PBS wash coverslips 3 times → 10% formaldehyde solution, rinsed with distilled water fixed 30min → 5 min → into incubation medium, 37 ℃ incubated for 2h → distilled water rinse 5min → 2% nitric drilling fluid effect 2min → distilled water rinse 5min → 2% amine curing effect 2min → distilled water rinse excess amine disulfide dye, to be dry → 2% methylene blue stained → gum sealed and placed in an optical microscope. 7 Statistical analysis of the results Bandscan5.0 gray value analysis LEF-1 in the sample with the same sample, the gray value of the gray value of the ratio of GAPDH as an evaluation indicator of LEF-1. (T test) Results RT -PCR, showed that cells from different species of MSCs, hPDLC cells, MC3T3 cell memory in LEF-1 mRNA expression; role in FSS LEF-1 mRNA expression increased with regularity, but there are some differences. in human periodontal ligament cells and control groups LEF-1 mRNA expression was weak. Afterburner 2h, 4h later, LEF-1 mRNA expression levels tended to increase afterburner 8h LEF-1 mRNA expression levels increased to a peak, 12h LEF-1 mRNA expression levels begin to decline, but still higher; in MSCs cells control groups and 2h LEF-1 mRNA expression was weak, 4h group, no expression, 8h group LEF-1 mRNA expression levels increased to a peak; MC3T3-E1 cells in the control group and 2h group of LEF-1 mRNA have a weak expression, half an hour and 4h group LEF-1 mRNA expression was decreased, continuing 12dyn/cm 8h group LEF-1 mRNA expression levels increased to a peak. Conclusions This study found that LEF-1 mRNA in normal human periodontal ligament cells expression (not reported). role in FSS within 12 h, MSCs cells, hPDLC cells, MC3T3 cells LEF-1 mRNA expression was enhanced volatility trend, and in different sizes FSS force value, the premise of different species origin arrive at 8h under both LEF-1 mRNA expression peak, the results showed that the shear stress is enhanced LEF-1 expression, we believe that LEF-1 mRNA expression in osteoblasts enhancement is the class of canonical Wnt signaling pathway responses to shear stress . LEF-1 protein low expression of osteoblast maturation is a necessary condition. LEF-1 expression was no species difference.

Related Dissertations

  1. Research on Combinatorial Regulation of Multiple Transcription Factors,Q78
  2. On Explanatory Power of Conceptual Integration Theory for the Ttanslation of Chinese Ancient Classic Through the Analysis of the English Versions of Tao Te Ching,H315.9
  3. The Effect of Overexpression of sFrp2 and sFrp3 in the Development of Mouse Molar,Q954.48
  4. The Expression Survey of BMP Signalling Pathway in the Human Embryonic Tooth Germ,R78
  5. Study on Synthesis and Physiological Function of N-carbamoyl-glutamate,R914
  6. Functional Analysis of Two Rice Nitrate Transporter Genes OsNRT1.1a and OsNRT1.1b,S511
  7. Coparative Analyses of Carbon and Nitrogen Metabolism of Flue-cured Tobacco Growing in Henan and Yunnan,S572
  8. Study of Chicken Δ~6 Fatty Acid Desaturase Gene’s Promotor Region Polymorphism and Gene Expression,S831
  9. The Expression of CPV-2 VP2 Gene in SF9 and Development of Indirect Elisa for Serum Antibodys,S852.65
  10. Comparison of Gene Expression Data Cluster Methods and Gene Network Construction for Phytophthora Sojae Genes,S435.651
  11. Functional Identification of Two Tomoto Phosphater Transporter Genes LePT1 and LePT2 in Rice,S511
  12. Analysis for Intron 1 and 2 of Rice Transformation to Promoter Activity,S511
  13. Research on Characteristics of Growth, Development and Material Metabolism of Flue Cured Tobacco Leaves in Huili, Sichuan,S572
  14. Studies on Extracting Mitochondrial DNA and Gene Expressions in Brassica Napus L,S565.4
  15. Functional Characterization of Wheat Na~+ /H~+ Antiporter TaNHX2 and Study of TaNHX2 C- Terminus Domain,S512.1
  16. Identification and Analysis of miRNA and Flower Development Specific Genes in Wheat,S512.1
  17. Molecular Characterization, Expression, and Promoter Methylation Status of the Bovine Synaptonemal Complex Protein 3 Gene,S823
  18. The Deposition Rule and Gene Expression Involved in Yolk Cholesterol in Different Layer Breeds,S831
  19. Study on the mRNA Expression Level and the Polymorphism of Follicle-Stimulating Hormone Receptor Gene in Taihu Pig’s Ovary,S828
  20. The Effects of Don on Proliferation, Differentiation, and Apoptosis of Chondrocytes in Chicken,S858.31
  21. Expression of WW Domain Containing Oxidoreductase Gene in Cholangiocarcinoma and Its Effect on the Biological Behavior of Cancer Cell Line QBC939,R735.8

CLC: > Medicine, health > Oral Sciences > Oral orthotics > Journal of Orthodontics
© 2012 www.DissertationTopic.Net  Mobile