【摘要】：目的:通過構(gòu)建體外人牙周膜成纖維細(xì)胞(Human periodontal ligament fibroblasts)靜壓力加載模型,探索靜壓力對(duì)p38MAPK磷酸蛋白和IL-17 A、IL-17R和IL-6表達(dá)的影響,并觀察p38 MAPK特異性抑制劑SB203580預(yù)處理后,靜壓力對(duì)炎性因子表達(dá)的變化,為正畸力下牙周膜成纖維細(xì)胞生物力學(xué)的信號(hào)轉(zhuǎn)導(dǎo)以及與炎癥因子的關(guān)系提供新思路和實(shí)驗(yàn)基礎(chǔ)。方法:1、收集健康前磨牙的根中1/3牙周膜,利用組織塊培養(yǎng)法行原代培養(yǎng)。采用細(xì)胞免疫熒光對(duì)培養(yǎng)的細(xì)胞進(jìn)行來源鑒定。采用CCK8法檢測(cè)0-5g/cm2靜壓力和不同濃度的抑制劑SB203580刺激HPDLF后增殖活性的變化,從而構(gòu)建體外HPDLF的靜壓力加載模型。2、采用Western blot(WB)法檢測(cè)0-5g/cm2靜壓力對(duì)HPDLF內(nèi)p38磷酸蛋白表達(dá)情況。3、ELISA和RT-PCR檢測(cè)靜壓力對(duì)白介素17受體(IL-17R)、白介素17(IL-17A)、和白介素6(IL-6)蛋白和mRNA表達(dá)變化。經(jīng)p38MAPK抑制劑SB203580預(yù)處理HPDLF后,檢測(cè)IL-17A、IL-17R和IL-6的mRNA和蛋白表達(dá)。實(shí)驗(yàn)結(jié)果:1、經(jīng)組織塊原代培養(yǎng)5天后可以得到長條形的成纖維細(xì)胞。傳至第四代的成纖維細(xì)胞以漩渦方式生長,此時(shí)生長狀態(tài)良好。細(xì)胞免疫熒光鑒定結(jié)果顯示細(xì)胞來源于中胚層,結(jié)合取材部位可鑒定為HPDLF。體外1-4 g/cm2的靜壓力值可以促進(jìn)HPDLF生長,在2 g/cm2組細(xì)胞增殖能力最強(qiáng),3-4 g/cm2組時(shí)細(xì)胞增殖變緩,并有少量下降。5g/cm2的力值作用下細(xì)胞受到明顯抑制作用。體外低于8μmol/L濃度的SB2023580對(duì)細(xì)胞增殖有一定促進(jìn)作用,其中在2μmol/L時(shí)增殖能力最強(qiáng)。而當(dāng)濃度增大至16μmol/L后,細(xì)胞增殖則受到抑制。2、經(jīng)WB檢測(cè)發(fā)現(xiàn),在0-1g/cm2組,HPDLF內(nèi)p38磷酸蛋白表達(dá)無明顯變化。當(dāng)力值增加至2-5g/cm2時(shí),HPDLF內(nèi)p38磷酸蛋白表達(dá)增加,且隨加力時(shí)間呈動(dòng)態(tài)變化,其中10min時(shí)表達(dá)開始增加,刺激30min時(shí)磷酸蛋白表達(dá)量最高,至60min后磷酸蛋白表達(dá)下降。3、RT-PCR和ELISA結(jié)果發(fā)現(xiàn),靜壓力刺激HPDLF能上調(diào)IL-6和IL-17R的mRNA及IL-6、IL-17R和IL-17A蛋白的表達(dá)。當(dāng)抑制劑SB203580預(yù)處理HPDLF后,4g/cm2靜壓力刺激下IL-6的mRNA和蛋白表達(dá)受到抑制作用。SB203580對(duì)HPDLF表達(dá)IL-17A和IL-17R無明顯抑制作用。結(jié)論:1、組織塊原代細(xì)胞培養(yǎng)法能成功構(gòu)建人牙周膜成纖維細(xì)胞系,傳至第四代的細(xì)胞增殖活性增強(qiáng),且生物學(xué)性狀穩(wěn)定。2、適宜的靜壓力和濃度的SB203580能促進(jìn)HPDLF增殖,而過大的靜壓力和濃度的SB203580會(huì)抑制細(xì)胞的增殖,其中2g/cm2為HPDLF最適靜壓力,2μmol/L為最適濃度。3、p38 MAPK是靜壓力在HPDLF內(nèi)轉(zhuǎn)導(dǎo)信號(hào)分子,當(dāng)靜壓力力值達(dá)到2g/cm2能激活HPDLF內(nèi)p38 MAPK信號(hào)通路,靜壓力對(duì)HPDLF內(nèi)p38MAPK活性呈動(dòng)態(tài)變化。4、靜壓力能促進(jìn)HPDLF對(duì)IL-17A、IL-17R和IL-6因子的表達(dá)。p38MAPK可能是靜壓力誘導(dǎo)HPDLF分泌IL-6的一條信號(hào)通路。但靜壓力對(duì)體外HPDLF調(diào)控IL-17可能并不是通過p38MAPK信號(hào)轉(zhuǎn)導(dǎo)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.
【學(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R783.5

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