| Periodontal disease is what happens in the gum, periodontalmembrane, alveolar bone, cementum and periodontal tissue, chronicnonspecific, infectious diseases. According to different degrees, theclinical manifestations and treatment each different. Main clinicalfeature are gum inflammation, hemorrhage, periodontal bag forming,alveolar bone absorption and even loose teeth fall out. For severeperiodontal disease,we reconstruct the alveolar bone,cementum andperiodontal membrane fiber usually through bone graft or Guided tissueregeneration, finally forming new periodontal tissue and new attached.Currently a lot of periodontal tissue engineering researchs abouttransplanting marrow stem cell, periodontal ligament cells, dentalfollicle cells in periodontal defect bone to promoting alveolar boneregenration.The dental follicle,a loose connective tissue that surroundsthe dental papilla and enamel organ,is part of the tooth germ, derivingfrom the embryos outside mesenchymal, playing an important role in theteeth formation, will eventually form the periodontal tissue (alveolar bone, periodontal membrane, cementum). The dental follicle cellscomes from the gum pocket.It is one of the seed cells in periodontaltissue engineering,has a variety of biological function and thedifferentiation potential.In addition,it can differentiate into osteoblast,cementoblast,periodontal ligaments cells and undifferentiatedmesenchymal cells forming the alveolar bone and cementum,periodontal membrane. It has the extremely vital role in periodontaldisease treatment,and is the cytology base of periodontal tissueregeneration. One of the key factors in periodontal tissue regeneration isreconstrunting alveolar bone.It has important research value forperiodontal therapy through the induction of DFCS differentiate intoosteoblast and using its ossification in periodontal defect to increase theamount of bone.However, the dental follicle cells easily lost update ability, aging,and purification difficult in vitro, difficult to achieve a large expansion,restricting its research and application in the periodontal tissueengineering.Cloud immortalized the somatic cells,remained itscharacteristics and no carcinogenicity for the first time. These daysresearches immortaliz cells to provide enough cells for research.Immortalizaton is the process of cells culture in vitro after thespontaneous or interference to avoid aging, have the ability of infiniteproliferation. The experimental study the method of immortalize dental follicle cells and its ossify characteristics after immortalizing. Studieshave confirmed that the osteogenesis ability of dental follicle cells willincrease after immortalizing, may be helpful for periodontal tissueengineering research. And providing enough number of dental folliclecells for later related experimentsPurpose:1． This study will be proposed to infect SV40T-Ag virus to the ratdental follicle cells culturation in vitro, and set up the immortal ratdental follicle cell lines.2．This study will observe the biochemical characteristics and itsdifferentiation potential of immortal rat dental follicle cells.3．This study can provided enough amount dental follicle cells forthe research of periodontal tissue engineering.Methods:1．Cultivate the original generations rat dental follicle cells byusing the method of the enzyme digestion and establishment the limitedrat denta follicle cell lines, separation and purification rat dental folliclecells through the way of differential stick wall.Morphologicalobservation (light and electron microscopy), the cells histogenesisidentification, drawing cell growth curve, provide cells for the next step.2．Using293cells building virus retroviruses carrier containingSV40T-Ag,infect the carrier to3or4generation dental follcle cells, and set up the immortal rat dental follicle cell lines.3．Detect the integration of SV40T-Ag with the dental follicle cellsby Peal-Time PCR.4．Analysis the length of telomeres in dental follicle cells byWesten Blot.5．The ossify characteristic （ALP,OC,BMP2，RUNX2） ofimmortal dental follicle cells6．Draw the growth curve and observe the proliferation features ofthe immortal rat dental follicle cells.7．Detect the cloning ability of immortal rat dental follicle cells.Results:1．The formation and growth curve of rat dental follicle cells aresimilar with the report, it confirmed by immunohistochemical that thedental follicle cells derived from the ectoderm, this comfirm dentalfollicle cells can be used to further research.2. Success infect SV40T-Ag retroviruses carrierinto rat dentalfollicle cells, and enhanced telomerase activity of the dental folliclecells.3. The ossify characteristic of ALP, OC, BMP2, runx2between thedental follicle cells and immortal dental follicle cells have no differenceConclusion1. successful building immortal dental follicle cells using SV40T-Ag..2.The dental follicle cells have no changing about ossifycharacteristic after immortalling... |
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