静压力对牙周膜成纤维细胞p38 MAPK活性及炎性因子的影响
发布时间：2019-01-02 15:34
【摘要】：目的:通过构建体外人牙周膜成纤维细胞(Human periodontal ligament fibroblasts)静压力加载模型,探索静压力对p38MAPK磷酸蛋白和IL-17 A、IL-17R和IL-6表达的影响,并观察p38 MAPK特异性抑制剂SB203580预处理后,静压力对炎性因子表达的变化,为正畸力下牙周膜成纤维细胞生物力学的信号转导以及与炎症因子的关系提供新思路和实验基础。方法:1、收集健康前磨牙的根中1/3牙周膜,利用组织块培养法行原代培养。采用细胞免疫荧光对培养的细胞进行来源鉴定。采用CCK8法检测0-5g/cm2静压力和不同浓度的抑制剂SB203580刺激HPDLF后增殖活性的变化,从而构建体外HPDLF的静压力加载模型。2、采用Western blot(WB)法检测0-5g/cm2静压力对HPDLF内p38磷酸蛋白表达情况。3、ELISA和RT-PCR检测静压力对白介素17受体(IL-17R)、白介素17(IL-17A)、和白介素6(IL-6)蛋白和mRNA表达变化。经p38MAPK抑制剂SB203580预处理HPDLF后,检测IL-17A、IL-17R和IL-6的mRNA和蛋白表达。实验结果:1、经组织块原代培养5天后可以得到长条形的成纤维细胞。传至第四代的成纤维细胞以漩涡方式生长,此时生长状态良好。细胞免疫荧光鉴定结果显示细胞来源于中胚层,结合取材部位可鉴定为HPDLF。体外1-4 g/cm2的静压力值可以促进HPDLF生长,在2 g/cm2组细胞增殖能力最强,3-4 g/cm2组时细胞增殖变缓,并有少量下降。5g/cm2的力值作用下细胞受到明显抑制作用。体外低于8μmol/L浓度的SB2023580对细胞增殖有一定促进作用,其中在2μmol/L时增殖能力最强。而当浓度增大至16μmol/L后,细胞增殖则受到抑制。2、经WB检测发现,在0-1g/cm2组,HPDLF内p38磷酸蛋白表达无明显变化。当力值增加至2-5g/cm2时,HPDLF内p38磷酸蛋白表达增加,且随加力时间呈动态变化,其中10min时表达开始增加,刺激30min时磷酸蛋白表达量最高,至60min后磷酸蛋白表达下降。3、RT-PCR和ELISA结果发现,静压力刺激HPDLF能上调IL-6和IL-17R的mRNA及IL-6、IL-17R和IL-17A蛋白的表达。当抑制剂SB203580预处理HPDLF后,4g/cm2静压力刺激下IL-6的mRNA和蛋白表达受到抑制作用。SB203580对HPDLF表达IL-17A和IL-17R无明显抑制作用。结论:1、组织块原代细胞培养法能成功构建人牙周膜成纤维细胞系,传至第四代的细胞增殖活性增强,且生物学性状稳定。2、适宜的静压力和浓度的SB203580能促进HPDLF增殖,而过大的静压力和浓度的SB203580会抑制细胞的增殖,其中2g/cm2为HPDLF最适静压力,2μmol/L为最适浓度。3、p38 MAPK是静压力在HPDLF内转导信号分子,当静压力力值达到2g/cm2能激活HPDLF内p38 MAPK信号通路,静压力对HPDLF内p38MAPK活性呈动态变化。4、静压力能促进HPDLF对IL-17A、IL-17R和IL-6因子的表达。p38MAPK可能是静压力诱导HPDLF分泌IL-6的一条信号通路。但静压力对体外HPDLF调控IL-17可能并不是通过p38MAPK信号转导完成。
[Abstract]:Objective: to investigate the effect of static pressure on the expression of p38MAPK phosphate protein, IL-17 Agni IL-17R and IL-6 in human periodontal ligament fibroblasts (PDF) by establishing a static pressure loading model of human periodontal ligament fibroblasts (PDF) in vitro. After pretreatment with p38 MAPK specific inhibitor SB203580, the changes of inflammatory factor expression induced by static pressure were observed, which provided a new idea and experimental basis for biomechanical signal transduction of periodontal ligament fibroblasts under orthodontic force and its relationship with inflammatory factors. Methods: 1. A third of periodontal membranes in the root of healthy premolars were collected and primary culture was performed by tissue mass culture. The source of cultured cells was identified by immunofluorescence. CCK8 method was used to detect the proliferation activity of HPDLF stimulated by 0-5g/cm2 static pressure and different concentrations of SB203580 inhibitor SB203580, so as to construct a hydrostatic pressure loading model of HPDLF in vitro. 2. The expression of p38 phosphophosphate protein (p38) in HPDLF under static pressure of 0-5g/cm2 was detected by Western blot (WB) method. The expression of p38 phosphophosphate protein in HPDLF was detected by Elisa and RT-PCR respectively. And interleukin 6 (IL-6) protein and mRNA expression. HPDLF was pretreated with p38MAPK inhibitor SB203580 to detect the expression of mRNA and protein in IL-17A,IL-17R and IL-6. The results were as follows: 1. After 5 days of primary culture of tissue block, long fibroblasts could be obtained. The fourth generation of fibroblasts grew in a whirlpool fashion, and the growth state was good. The results of immunofluorescence assay showed that the cells originated from mesoderm and could be identified as HPDLF. in combination with the material taken. The static pressure of 1-4 g/cm2 in vitro could promote the growth of HPDLF. The cell proliferation was strongest in 2 g/cm2 group and slowed down in 3-4 g/cm2 group. And there was a little decrease. The cells were obviously inhibited by the force of 5g/cm2. In vitro, SB2023580 with concentration below 8 渭 mol/L could promote cell proliferation to some extent, and the ability of proliferation was strongest at 2 渭 mol/L. However, when the concentration increased to 16 渭 mol/L, cell proliferation was inhibited. 2. The expression of p38 phosphophosphate protein in HPDLF was not significantly changed in 0-1g/cm2 group by WB assay. When the force value increased to 2-5g/cm2, the expression of p38 phosphophosphate protein in HPDLF increased, and the expression of p38 protein increased dynamically with the application time. The expression of p38 protein began to increase at the time of 10min, and reached the highest level when 30min was stimulated, and decreased after 60min. RT-PCR and ELISA showed that HPDLF could up-regulate the expression of mRNA, IL-6,IL-17R and IL-17A in IL-6 and IL-17R. When HPDLF was pretreated with SB203580, the expression of mRNA and protein of IL-6 was inhibited under static pressure of 4g/cm2, but SB203580 had no obvious inhibitory effect on HPDLF expression of IL-17A and IL-17R. Conclusion: 1. Human periodontal ligament fibroblasts can be successfully constructed by tissue block primary cell culture method. The proliferation activity of the cells transferred to the fourth passage is enhanced, and the biological properties are stable. 2. The optimal static pressure and concentration of SB203580 can promote the proliferation of HPDLF. However, excessive static pressure and concentration of SB203580 could inhibit cell proliferation. 2g/cm2 was the optimal static pressure for HPDLF and 2 渭 mol/L for optimal concentration. 3p38 MAPK was the signal molecule transduced by static pressure in HPDLF. When the static pressure reached 2g/cm2, the p38 MAPK signaling pathway in HPDLF could be activated, and the activity of p38MAPK in HPDLF changed dynamically under static pressure. 4. Static pressure could promote HPDLF to IL-17A,. Expression of IL-17R and IL-6 factors. P38MAPK may be a signal pathway for HPDLF to secrete IL-6 under static pressure. However, the regulation of IL-17 by static pressure on HPDLF in vitro may not be accomplished by p38MAPK signal transduction.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R783.5
[Abstract]:Objective: to investigate the effect of static pressure on the expression of p38MAPK phosphate protein, IL-17 Agni IL-17R and IL-6 in human periodontal ligament fibroblasts (PDF) by establishing a static pressure loading model of human periodontal ligament fibroblasts (PDF) in vitro. After pretreatment with p38 MAPK specific inhibitor SB203580, the changes of inflammatory factor expression induced by static pressure were observed, which provided a new idea and experimental basis for biomechanical signal transduction of periodontal ligament fibroblasts under orthodontic force and its relationship with inflammatory factors. Methods: 1. A third of periodontal membranes in the root of healthy premolars were collected and primary culture was performed by tissue mass culture. The source of cultured cells was identified by immunofluorescence. CCK8 method was used to detect the proliferation activity of HPDLF stimulated by 0-5g/cm2 static pressure and different concentrations of SB203580 inhibitor SB203580, so as to construct a hydrostatic pressure loading model of HPDLF in vitro. 2. The expression of p38 phosphophosphate protein (p38) in HPDLF under static pressure of 0-5g/cm2 was detected by Western blot (WB) method. The expression of p38 phosphophosphate protein in HPDLF was detected by Elisa and RT-PCR respectively. And interleukin 6 (IL-6) protein and mRNA expression. HPDLF was pretreated with p38MAPK inhibitor SB203580 to detect the expression of mRNA and protein in IL-17A,IL-17R and IL-6. The results were as follows: 1. After 5 days of primary culture of tissue block, long fibroblasts could be obtained. The fourth generation of fibroblasts grew in a whirlpool fashion, and the growth state was good. The results of immunofluorescence assay showed that the cells originated from mesoderm and could be identified as HPDLF. in combination with the material taken. The static pressure of 1-4 g/cm2 in vitro could promote the growth of HPDLF. The cell proliferation was strongest in 2 g/cm2 group and slowed down in 3-4 g/cm2 group. And there was a little decrease. The cells were obviously inhibited by the force of 5g/cm2. In vitro, SB2023580 with concentration below 8 渭 mol/L could promote cell proliferation to some extent, and the ability of proliferation was strongest at 2 渭 mol/L. However, when the concentration increased to 16 渭 mol/L, cell proliferation was inhibited. 2. The expression of p38 phosphophosphate protein in HPDLF was not significantly changed in 0-1g/cm2 group by WB assay. When the force value increased to 2-5g/cm2, the expression of p38 phosphophosphate protein in HPDLF increased, and the expression of p38 protein increased dynamically with the application time. The expression of p38 protein began to increase at the time of 10min, and reached the highest level when 30min was stimulated, and decreased after 60min. RT-PCR and ELISA showed that HPDLF could up-regulate the expression of mRNA, IL-6,IL-17R and IL-17A in IL-6 and IL-17R. When HPDLF was pretreated with SB203580, the expression of mRNA and protein of IL-6 was inhibited under static pressure of 4g/cm2, but SB203580 had no obvious inhibitory effect on HPDLF expression of IL-17A and IL-17R. Conclusion: 1. Human periodontal ligament fibroblasts can be successfully constructed by tissue block primary cell culture method. The proliferation activity of the cells transferred to the fourth passage is enhanced, and the biological properties are stable. 2. The optimal static pressure and concentration of SB203580 can promote the proliferation of HPDLF. However, excessive static pressure and concentration of SB203580 could inhibit cell proliferation. 2g/cm2 was the optimal static pressure for HPDLF and 2 渭 mol/L for optimal concentration. 3p38 MAPK was the signal molecule transduced by static pressure in HPDLF. When the static pressure reached 2g/cm2, the p38 MAPK signaling pathway in HPDLF could be activated, and the activity of p38MAPK in HPDLF changed dynamically under static pressure. 4. Static pressure could promote HPDLF to IL-17A,. Expression of IL-17R and IL-6 factors. P38MAPK may be a signal pathway for HPDLF to secrete IL-6 under static pressure. However, the regulation of IL-17 by static pressure on HPDLF in vitro may not be accomplished by p38MAPK signal transduction.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R783.5
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