發(fā)布時(shí)間：2018-03-21 23:18

  本文選題：牙周膜干細(xì)胞　切入點(diǎn)：腫瘤壞死因子-α　出處：《天津醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:本研究擬采用喪失性功能實(shí)驗(yàn)探討胰島素樣生長(zhǎng)因子結(jié)合蛋白5(IGFBP5)和組蛋白去甲基化酶6A(KDM6A)在炎癥微環(huán)境下對(duì)牙周膜干細(xì)胞(PDLSCs)成骨分化的影響及分子調(diào)控機(jī)制,為炎癥微環(huán)境下牙周組織再生尋找治療靶點(diǎn)。方法:1.PDLSCs的收集與分離培養(yǎng),并通過流式細(xì)胞術(shù)檢測(cè)其表面抗原標(biāo)志物。2.分別建立IGFBP5穩(wěn)定敲低的細(xì)胞系,KDM6A穩(wěn)定敲低的細(xì)胞系,并進(jìn)行成骨誘導(dǎo)分化。在成骨誘導(dǎo)條件培養(yǎng)下過通過CCK-8檢測(cè)細(xì)胞增殖能力,Annexin V檢測(cè)凋亡變化,堿性磷酸酶活性、堿性磷酸酶染色檢測(cè)、茜素紅染色檢測(cè)TNF-α、IL-17對(duì)牙周膜干細(xì)胞分化及生物學(xué)特性的影響。3.通過Real-Time PCR檢測(cè)IGFBP5喪失性功能實(shí)驗(yàn)中的PDLSCs在體外成骨誘導(dǎo)條件培養(yǎng)下,炎癥因子對(duì)PDLSCs的成骨分化相關(guān)標(biāo)志性基因及相關(guān)的組蛋白去甲基化酶表達(dá)的影響。檢測(cè)KDM6A低表達(dá)后PDLSCs的成骨分化基因表達(dá)變化,并通過流式細(xì)胞儀微球捕獲芯片技術(shù)分析其相關(guān)炎癥因子的變化。4.通過Western-blot檢測(cè)炎癥因子作用下成骨誘導(dǎo)條件培養(yǎng)下KDM6A低表達(dá)的PDLSCs,細(xì)胞質(zhì)內(nèi)p-p65、β-catenin蛋白及細(xì)胞核內(nèi)p-p65蛋白的表達(dá)變化。結(jié)果:1.通過成骨誘導(dǎo)分化培養(yǎng)基定向誘導(dǎo)IGFBP5低表達(dá)的PDLSCs,在炎癥因子作用下,堿性磷酸酶染色檢測(cè)顯示藍(lán)染程度減弱。堿性磷酸酶活性檢測(cè)發(fā)現(xiàn)TNF-α、IL-17刺激后的IGFBP5低表達(dá)的PDLSCs堿性磷酸酶活性降低。實(shí)時(shí)定量Real-time RT-PCR結(jié)果顯示成骨誘導(dǎo)分化組中,TNF-α、IL-17處理后成骨分化的相關(guān)基因OCN及BSP表達(dá)顯著降低,組蛋白去甲基化酶KDM2A、KDM3B、KDM5B、JMJD6也有顯著變化。(P0.05,n=3)2.磷酸酶染色結(jié)果顯示KDM6A低表達(dá)的PDLSCs在TNF-α、IL-17刺激后染色效果減弱;茜素紅染色結(jié)果顯示炎癥因子刺激下KDM6A低表達(dá)后染色效果并無顯著變化;通過細(xì)胞增殖檢測(cè)結(jié)果可以確定KDM6A喪失性功能實(shí)驗(yàn)中的PDLSCs在成骨誘導(dǎo)分化過程中加入TNF-α、IL-17刺激后,與對(duì)照組相比并無明顯差異。通過細(xì)胞凋亡檢測(cè)結(jié)果可以確定KDM6A低表達(dá)后的PDLSCs,加入TNF-α、IL-17刺激后細(xì)胞凋亡略有增加。Real-Time PCR結(jié)果顯示在TNF-α、IL-17刺激后,成骨分化重要標(biāo)志基因RUNX2、BSP、OCN m RNA的表達(dá)顯著下降。流式細(xì)胞儀微球捕獲芯片技術(shù)檢測(cè)結(jié)果顯示KDM6A低表達(dá)后的PDLSCs,炎癥因子IL-6的表達(dá)顯著升高。(P0.05,n=3)3.Western-blot檢測(cè)結(jié)果顯示,在KDM6A敲低的PDLSCs成骨誘導(dǎo)分化條件培養(yǎng)下,加入TNF-α、IL-17刺激后,細(xì)胞質(zhì)中β-catenin蛋白水平顯著降低。結(jié)論:1.IGFBP5可以促進(jìn)炎癥因子TNF-α、IL-17刺激下的PDLSCs成骨分化。2.KDM6A可以促進(jìn)炎癥因子TNF-α、IL-17刺激下的PDLSCs成骨分化。3.KDM6A對(duì)炎癥因子TNF-α、IL-17刺激下PDLSCs成骨分化的促進(jìn)作用,可能與其促進(jìn)Wnt/β-catenin信號(hào)通路有關(guān)。
[Abstract]:Objective: to investigate the effects of insulin-like growth factor-binding protein (IGFBP5) and histone demethylase (6A- KDM6A) on the osteogenic differentiation of periodontal ligament stem cells (PDLSCs) in inflammatory microenvironment. Methods: 1. The collection, isolation and culture of PDLSCs were used to detect the surface antigen markers of PDLSCs by flow cytometry. The KDM6A stable knockout cell lines were established by flow cytometry. Under the condition of osteogenic induction, apoptosis, alkaline phosphatase activity and alkaline phosphatase staining were detected by CCK-8 and Annexin V, respectively. Effects of TNF- 偽 IL-17 on differentiation and biological characteristics of periodontal ligament stem cells by alizarin red staining. 3. PDLSCs in IGFBP5 loss function experiment was detected by Real-Time PCR under osteogenic induction condition in vitro. Effects of inflammatory factors on the expression of osteogenic differentiation related signature genes and histone demethylase in PDLSCs. The expression of osteogenic differentiation genes in PDLSCs was detected after low expression of KDM6A. Flow cytometry microsphere capture chip technique was used to analyze the changes of inflammatory cytokines. 4. Western-blot was used to detect PDLSCs, p-p65, 尾 -catenin protein in cytoplasm and p-p65 eggs in nucleus of osteoblasts induced by inflammatory factors. Results 1. The low expression of IGFBP5 was induced by osteogenic differentiation medium. Alkaline phosphatase staining showed that the degree of blue staining was weakened. Alkaline phosphatase activity test showed that the low expression PDLSCs alkaline phosphatase activity of IGFBP5 stimulated by TNF- 偽 IL-17 was decreased. Real-time quantitative Real-time RT-PCR results showed that TNF- 偽 -IL-17 in osteogenic differentiation group was located in TNF- 偽 IL-17. The expression of OCN and BSP in osteogenic differentiation was significantly decreased. The histone demethylase KDM2An KDM3Bm5BHJMJD6 was also significantly changed. The results of phosphatase staining showed that the PDLSCs with low expression of KDM6A decreased after stimulation of TNF- 偽 -IL-17, while the staining effect of alizarin red showed no significant change after the low expression of KDM6A stimulated by inflammatory factors. The results of cell proliferation test showed that PDLSCs in KDM6A loss function test was stimulated by TNF- 偽 -IL-17 during osteogenic induction. Compared with the control group, there was no significant difference between the two groups. The results of apoptosis detection could determine the PDLSCsafter low expression of KDM6A, and increase the apoptosis slightly after adding TNF- 偽 -IL-17. The results of Real-Time PCR showed that the cells were stimulated by TNF- 偽 -IL-17. The expression of OCN m RNA, an important marker of osteogenic differentiation, was significantly decreased. The results of flow cytometry microsphere capture chip showed that the expression of inflammatory factor IL-6 was significantly increased after low expression of KDM6A. Under the condition of KDM6A knockout PDLSCs osteogenesis induced differentiation, TNF- 偽 -IL-17 was added to stimulate the differentiation. Conclusion: 1. IGFBP5 can promote the osteogenic differentiation of PDLSCs stimulated by TNF- 偽 and IL-17. 2. KDM6A can promote the osteogenic differentiation of PDLSCs stimulated by TNF- 偽 -IL-17. 3. KDM6A can promote the osteogenic differentiation of PDLSCs stimulated by TNF- 偽 IL-17. It may be related to the promotion of Wnt/ 尾-catenin signaling pathway.
【學(xué)位授予單位】：天津醫(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)】：R781.4

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相關(guān)期刊論文 前1條

1 Sarah K Johnson;Randy S Haun;;Insulin-like growth factor binding protein-5 influences pancreatic cancer cell growth[J];World Journal of Gastroenterology;2009年27期

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本文編號(hào)：1646013