In vitro transformation of periodontal ligament fibroblasts ( PF cells) and characterization
Date Issued
2004
Date
2004
Author(s)
Mi, Hsin-Wu
DOI
zh-TW
Abstract
The core of periodontal therapy is to ensure the regeneration of the tissue that has been destroyed by periodontitis. So the periodontal regeneration requires restitution of the periodontal attachment apparatus, i.e., new bone formation, new cementum deposition upon the denuded root surface, and insertion of functionally-oriented new collegen fibers targeting the periodontal ligament（PL）into the new bone and new cementum. The origins of progenitor cells responsible for the formation of these tissues have long been suggested to be mainly derived from PL tissue. Therefore, cells derived from there must play a pivotal role in the process of periodontal regeneration. Gene or cell therapy is a recognized methodology in delivering the therapeutic-level proteins to the targeted tissue for extended period of time. To date, ex-vivo gene therapy in the area of periodontal bone repair has remained a challenge, primarily due to the difficulty of achieving high-efficiency gene transfer into primary autologous cells derived from periodontal ligament tissue with non-viral techniques. Although viral vectors have been shown an efficacy in transforming cultured cells with high efficiency and have also been regarded as very useful gene therapy tools, the use of non-viral methods for gene delivery in non life-threatening situations remains a desirable goal. For instance, it is potentially considered as an effective method for repairing various periodontal bone defects. However, adenovirus vectors may induce host immune- response in some cases, while retroviral vectors demand dividing cells for integration of transfected gene. Moreover, there exists a chance that viral vectors may randomly be integrated into the host genome, posing a risk of neoplastic transformation. If such is the case, that the quest for a high-efficient non-viral gene transfer methods aiming at the primary autologous cells would preclude the possibility of an immune-response. The introduction of exogenous DNA into these periodontal ligament fibroblasts is controversially discussed, mainly due to the lack of systematic analysis. Therefore, in the present study we examined comparatively four non-viral gene transfer methods: calcium phorsphate reagent, electroporotion, LipofectamineTM reagent, and LipofectamineTM 2000 reagent, as potential tools for a efficient delivery of DNA to the culture of the PL fibroblasts (PF cells). The conditions of cell transfection were determined by using enhanced green fluorescent protein (GFP) as a reporter gene that expressed under the transcriptional control of the human cytomegalovirus promoter/enhancer. Quantitative evaluation of representative transfection experiments by flow cytometry revealed that out of four non-viral gene transfer methods tested, LipofectamineTM 2000 reagent allowed for a significantly higher transfection efficiency (up to 66﹪). Transfection efficiency with calcium phorsphate reagent or electroporotion was＜10％, and approximately 25％ of the PF cells were transfected by LipofectamineTM reagent. Transformation of the PF cells was made with plasmid encoded SV40 large T and GFP and the transformed PF cells were characterized by RT-PCR (mRNA level) and western blotting (protein level) techniques. By way of this effort, long-term persistent expression of the exogenous DNA in PF cells were monitored. In conclusion: firstly, transfection with GFP reporter gene by liposome technique (LipofectamineTM 2000 reagent) resulted in a higher efficiency (up to 66﹪) than other nonviral gene transfer methods. Secondly, by liposome method, we have successfully obtained two stable transfectants with GFP and SV40 large T, respectively. Both cell lines were able to express the exogenous DNA at least 3 months at the protein level. Thirdly, the results of RT-PCR showed that some osteoblast-specific markers, such as Type I collagen (Hcol), alkaline phosphatase (ALPase), osteocalcin (OCN) were able to be expressed both in cultured PF cells and the stable SV40 large T transfectants. The PF cells transfected with SV40 large T, which have propagated over 20 passages, seemed to have little change of its phenotypes. Thus, the immortalization-competent PF cells via lipofactamine 2000 reagent would provide a useful cell source for research of periodontal regeneration in vitro.
Subjects
牙周韌帶纖維母細胞轉型
PF cells； transformation
Type
other
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