| Periodontal regeneration is expected to reconstitute the injured periodontal tissues physiologically and functionally, including cementum, periodontal ligament and alveolar bone. It is difficult for the traditional treatment to attain this goal. Although the guided tissue regeneration(GTR) achieved the periodontal regeneration, it can not fulfill this goal entirely. To treat the periodontitis with tissue engineering established the new field for the therapy of periodontal regeneration. Nowadays the form styles of tissue engineering have included cell/scaffolds, growth factors/ scaffolds and cells/ growth factors/ scaffolds. But the proteins have to expose in high temperature and high concentrations of surfactants when the protein incorporated into scaffolds. These conditions can promote the protein degradation, and in turn, decrease potency and increase risk of immune toxicity. Moreover, protein successfully incorporated in scaffolds may undergo irreversible aggregation, deamidation and oxidation because of elevated levels of moisture and evolution of harsh microclimate. So to maintain full bioactivity and to sustain the release of growth factors, we need a large amounts of recombinant cytokine. The proteins are expensive to manufacture. High-dose proteins bring us an additional safety risk: the cytokine may diffuse from the wound bed into the bloodstream. If the cells in the scaffolds can highly express the recombinant cytokine, the disadvantages of using the protein will be overcome. So wecombine gene therapy with tissue engineering. Dog's Insulin-like growth factor I (IGF-1) gene was transfected into bone marrow stem cells (MSCs). The transfected MSCs with ostrich true bone ceramics(OTBC) were used to repair the periodontal defects. This study is to estimate the potential effects of IGF-1 gene transfected MSCs and to provide a new way for restoring the periodontal defects. This study has included five experiments:1. Total RNA was extracted from dog's left ventricular myocardium. The desired DNA product was obtained from the total RNA by RT-PCR. The segment (about 297bp) was inserted into pcDNA3.1(+) vector and the inserted plasmid was transformed into E.coli DH5 a . The positive clone was analyzed by restriction endonuclease mapping and DNA sequencing. The restriction endonuclease map and sequence of dog IGF-I functional fragment were consistent with those of the published. The IGF-1 gene in the pcDNA3.1(+)-IGF-l were subcloned into pIRES2-EGFP expressing vector. The plasmid was transformed into E.coli DH5 a . Restrictive enzyme(Nhe I, EcoR I) digestion analysis showed that recombinant expression vector pIRES2-EGFP-IGF-l has been constructed successfully.2. pIRES2-EGFP-IGF-l was transfected into MSCs with lipofectamine. After 48h transfection, the Insulin-like growth factor I (IGF-1) expression in the transfected MSCs was detected under fluorescence microscope and by immunohistochemistry. The supernatant of the transfected MSCs cultured with L929 fibroblasts. The effect of the recombinant IGF-1 on the L929's cell cycle was observed by flow cytometer analysis. Result: The transfected MSCs displaying green fluorescence were observed under fluorescence microscope. The immunohistochemical staining showed that positive reactant of brown-yellow can be seen in the cytoplasm of MSCs transfected by pIRES2-EGFP-IGF-l. The supernatant of the transfected MSCs can increase the L929's proliferation.3. pIRES2-EGFP-IGF-l was transfected into MSCs with lipofectamine. Positive clones were selected with G418. The expression of IGF-1 protein in the MSCs was determined by immunohistochemistry and Western blot analysis. The IGF-1 in the supernatant of the transfected MSCs was detectedby sandwich-in ELISA. The transfected MSCs' ALP activity were investigated. Add culture medium of stably transfected MSCs cells to periodontal ligament cells (PDLCs). The change of PDLCs' proliferation was observed by MTT.The results of immunohistochemistry and Western blot analysis suggested that IGF-1-trnsfected MSCs could e...
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