| Periodontitis is a complex etiology, which is subject to the regulation of body defense mechanisms and a variety of systemic and local factors,caused by the interaction of many factors of destructive periodontal disease. As one of the local factors, trauma from occlusion plays a very important role. Human periodontal ligament fibroblasts( hPDLF), as the major susceptibility cells and effector cells of periodontal ligament,and its proliferation is also a direct reflection of changes in the healthy state of periodontal tissue.Currently, studies on the cell proliferation and its complex regulaion mechanism of mechanical stimulation is still superficial. NF-кB(unclear factorκB,NF-κB)is an important class of transcription factors of eukaryotic cells. There will be guiding significance to the mechanism of whether the mechanical signal transduction can go through the NF-кB signaling pathways and make the extracellular signal transduction to the nucleus caused hPDLF proliferation.Objective: First, the experiment applys the multi-channel cell stretch stress loading system in vitro hPDLF impose cyclic stretch at different times and observes the the cyclic stretch on the hPDLF proliferation.Secondly, it clarifys effects and mechanism of NF-кB signaling pathways in hPDLF proliferation of cyclic stretch and explicits the relationship between cyclic stretch, hPDLF proliferation and NF-кB signaling pathways.To fully elucidate the pathogenic mechanism of occlusal trauma and periodontitis caused by traumatic occlusion explore the intervention of the key targets to provide a theoretical basis and new ideas.Methods:1. This experiment established the in vitro culture-tensile stimulate models of hPDLF by using a multi-passage load adding system.2. The control group were given stimulation of cyclic stretch 1, 2, 4, 6, 12, 24h the load was set for 10% surface elongation (each cycle including 3s-stretch/3s-relaxation) with frequency 0.1 Hz(6cycles / Points) while the no strength is considered as control group. First of all, the hPDLF proliferate is detected by cell counting kit-8 (CCK-8) method,as the hPDLF cells cycle is analyzed by flow cytometry. In the next place, the expression of mRNA and nuclear factor-κB (NF-κB) are detected by RT-PCR technology. At last, the hPDLF proliferate is detected by the pretreated cells (control group) of specific inhibitor of NF-кB-pyrrolidine dithiocarbamate (PDTC) .3. Detect protein expression of I-кBαby Western-blot technique in hPDLF of each group. The utility of pretreated cells by PDTC is to detect protein expression of I-кBαas the control group.Results:1. In vitro can get a typical and stable proliferation characteristic hPDLF, which can be used for the mechanical loading on the cell experiments.2. Compared with static group, the proliferation activity of hPDLF was decreased in 1, 2 h making no statistic meaning in their differences(P>0.05); hPDLF began to proliferate in 6 h, and reached the peak in 12 h,the proliferation of hPDLF were inhibited obviously. PDTC inhibited the cells proliferation and the expression of PCNA and mRNA which made the differences had statistic significance(P<0.05).3. The expression of IκBαin the static group is strong and after 12h loaded,the protein expression of IκBαwas significantly reduced. Comparing with PDTC group, no PDTC group has a increasing protein expression of IκBαwhich reflected the significance between two group(P<0.05).Conclusion:1. The model of the in vitro culture-tensile stimulate models of hPDLF is constructed successfully.2. The cyclic stretch can promote the in vitro hPDLF proliferation in a certain time with loading range of 10% surface elongation and frequency 0.1 Hz.The cell proliferation is inhibited obviously when extended the certain time. NF-κB signaling pathway played an important role in regulating the cells'proliferation condition.3. The cyclic stretch can reduce the level of protein expression of I-κBα. Meanwhile, I-κBαof hPDLF degradation is one of the mechanisms of NF-κB signaling pathways' activity. |
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