| Objective:1.To study the influence of TGF-β1 on biologic activity of HPDLCs in primary culture.2.To determine the effects of TGF-β1on the expressions ofIL-6,BGP in periodontal tissue of rat periodontitis model.Methods:1.Human periodontal ligament cells were obtained from healthy periodontium using the explant technique.The effect of TGF-β1 on the proliferative ability of HPDLCs was evaluated with MTT assay;The effects of TGF-β1on the total amount of protein and the cell cycle were detected by coomassie brilliant blue(CBB)method and flow cytometry;The effects of TGF-β1 on the alkaline phosphatase(ALP)activity and expression of ALP mRNA were measured with the p-nitrophenylphosphate(PNPP)and reverse -transcription polymerase chain reaction(RT-PCR).2.Based on the successful rat periodontitis model,the experimental rats were randomized into different group followed by oral treatment with TGF-β1 for 1,2,3,4 weeks and then the rats were sacrificed and analyzed.Pathological assay and HIE staining were used to detect the general conditions and pathological changes of rats periodontal tissues.And immunohistochemical staining was conducted to determine the expressions ofIL-6,BGP in rats periodontitis model.Result:1.The growth condition of the human periodontal ligament cellsThe time was 3～14 days for the primarily cultured cells growing out from tissue. Elongated fusiform cells or astrocytes could be seen through inverted microscope.The cell which was sudden slender,radial array,and its body fullness,uniform cytoplasm,and nuclear round or oval was consided as the fibroblast.2.The effect of TGF-β1 on the proliferation of HPDLCsTreatment with TGF-β1 in 2.0μg/L,5.0μg/L and 10.0μg/L increased cell proliferation significantly compared with the control group(P＜0.05).At 3 days 10.0μg/L TGF-β1 showed a higher cell proliferation compared with the other groups.0.5μg/L TGF-β1did not positively influnce the cell proliferation in this study.3.The effect of TGF-β1 on the total amount of protein of HPDLCsCompared with the control group,the total amount of protein of HPDLCs treated with TGF-β1 in 2.0μg/L,5.0μg/L and 10.0μg/L was increased dramatically((P＜0.05).The cells with 10.0μg/L TGF-β1 treated produced statistically more protein than with other treatments. 4.The effect of TGF-β1 on the alkaline phosphatase(ALP)activity of HPDLCs The ALP activity of HPDLCs was positively affected by TGF-β1 in 2.0μg/L,5.0μg/L and 10.0μg/L at 48 hours compared with the control group,and 10.0μg/L TGF-β1 treated was enhanced more significantly than with other treatments.5.The effect of TGF-β1on the expression of ALP mRNA of HPDLCsThe expression of ALPmRNA was significantly up-regulated by TGF-β1 in 2.0μg/L,5.0μg/L and 10.0μg/L at 48 hours((P＜0.05).Compared with the control group,optical density respectively increased 1.34,1.66,1.75 times through image analysis.6.The effect of TGF-β1 on cell cycle of HPDLCsEmploying the FCM technique,we observed the decreased G1%,the increased S%and(S +G2/M)%when the cells were stimulated by TGF-β1(10.0μg/L)after 48 hours.7.The levels of IL-6 in the periodontitis tissues were significantly higher than that in control group and the level of BGP was lower.Treatment with TGF-β1 decreased the levels of IL-6 and increased the level of BGP in periodontitis tissue((P＜0.05).The general conditions and pathological changes in the control group and groups treated with TGF-β1 were much better than that in periodontitis group.Conclusion:These findings suggested that TGF-β1 was capable of producing an accelerative effect on the proliferation and differentiation functions on HPDLFs,It may be related to TGF-β1 enhancing cell DNA and protein synthesis,ALP mRNA expression and ALP activity.Moreover,it inhibited the expressions of IL-6 and increased the level of BGP in periodontal tissues of rats periodontitis model and promoted the regeneration of the periodontal tissues.In a word,our studies suggested that TGF-β1 may have potential clinical application to periodontal disease.
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