炎症微环境对牙髓干细胞生物学特性影响

发布时间：2018-06-16 19:32

  本文选题：脂多糖 + 牙髓干细胞　； 参考：《南通大学》2014年硕士论文

【摘要】：目的研究脂多糖(LPS)对牙髓干细胞(DPSCs)进行反复刺激后其衰老程度。方法分离、培养DPSCs后进行形态观察。甲苯胺蓝染色检测DPSCs成软骨分化,油红O染色检测DPSCs成脂肪分化,DPSCs成骨分化液,茜素红染色检测钙结节形成。LPS刺激0、1、3和6次,观察细胞形态,细胞计数、流式细胞仪检测LPS对DPSCs增殖的影响,检测β-gal的表达情况,检测LPS对DPSCs免疫荧光检测LPS对DPSCs细胞骨架的影响、ROS、γ-H2A.X、p16INK4A表达。运用Western blot测定TLR4、p16INK4A、cyclinD1、Rb、p-Rb、γ-H2A.X蛋白表达,RT-PCR测定p16INK4A、γ-H2A.X基因水平表达。结果DPSCs呈成纤维细胞样梭形外观,具有向成软骨分化、成脂肪、成骨分化的特征。LPS反复刺激后,细胞体积增大、β-gal染色加深、细胞增殖能力降低、G1/G0期细胞比例增加,随着LPS刺激数目增多,LPS衰老增强。在此过程中,ROS、TLR4表达增高,DNA损伤标志γ-H2A.X表达上调,p16INK4A信号通路被激活,抑制p16INK4A信号通路可以逆转LPS诱导的DPSCs衰老。结论我们的研究表明LPS反复刺激DPSCs,诱导DPSCs衰老,为DPSCs的自体移植提供了理论基础。目的探讨肿瘤坏死因子α(TNF-α)对牙髓干细胞(DPSCs)成骨分化、增殖的影响及其机制的研究。方法分离、培养DPSCs后进行形态观察。甲苯胺蓝染色检测DPSCs成软骨分化,油红O染色检测DPSCs成脂肪分化。DPSCs成骨分化液中加入10 ng/ml TNF-α,分化3、5、7、14天,茜素红染色检测钙结节形成,ALP染色检测ALP活性,运用Western blot分析BMP2蛋白表达情况,RT-PCR测定BMP2、ALP、RUNX2、COL I等成骨标志物m RNA的表达情况。通过细胞计数和MTT法分析TNF-α对DPSCs增殖的影响。运用Western blot和免疫荧光检测NF-κB信号通路中p65、IκBα、p-p65和p-IκBα表达情况。结果DPSCs呈成纤维细胞样梭形外观,具有向成软骨分化、成脂肪分化的特征。TNF-α促进DPSCs钙结节形成、ALP活性高表达及BMP2、ALP、RUNX2、COL I成骨标志物的表达。在此过程中NF-κB信号通路p-p65、p-IκBα表达增高,p65入核,IκBα降解,加入NF-κB信号通路抑制剂PDTC后,TNF-α促进DPSCs成骨分化作用可以被有效逆转。细胞计数及MTT方法检测结果显示TNF-α不影响DPSCs的增殖。结论我们的研究表明TNF-α促进DPSCs成骨分化,并诱导DPSCs矿化相关标志物的表达,为DPSCs的自体移植提供了理论基础。
[Abstract]:Objective to study the senescence of dental pulp stem cells (DPSCs) after repeated stimulation with lipopolysaccharide (LPS). Methods DPSCs were isolated and cultured for morphological observation. The cartilage differentiation of DPSCs was detected by toluidine blue staining, the osteogenic fluid of DPSCs by oil red O staining, calcium nodule formation by alizarin red staining, and lipopolysaccharide (LPS) stimulation for 3 and 6 times, and cell morphology and cell count were observed. The effects of LPS on the proliferation of DPSCs and the expression of 尾 -gal were detected by flow cytometry. The effects of LPS on the cytoskeleton of DPSCs were detected by immunofluorescence. The expression of p16INK4A, 纬 -H2A.X was detected by Western blot. The expression of p16INK4A, 纬 -H2A.X was detected by RT-PCR. Results DPSCs had the appearance of fusiform fibroblast, characterized by differentiation into cartilage, fat and osteogenesis. After repeated stimulation of LPS, cell volume increased, 尾 -gal staining deepened, cell proliferation decreased and the proportion of cells in G1 / G0 phase increased. With the increase of LPS stimulation, LPS senescence increased. During this process, the expression of TLR4 increased and the expression of 纬 -H2A.X up-regulated the activation of p16INK4A signaling pathway. Inhibition of p16INK4A signaling pathway could reverse the senescence of DPSCs induced by LPS. Conclusion our study shows that LPS repeatedly stimulates DPSCs and induces the senescence of DPSCs, which provides a theoretical basis for autotransplantation of DPSCs. Objective to investigate the effect of TNF- 伪 on osteogenic differentiation and proliferation of dental pulp stem cells (DPSCs) and its mechanism. Methods DPSCs were isolated and cultured for morphological observation. Toluidine blue staining was used to detect the cartilage differentiation of DPSCs, oil red O staining was used to detect the adipogenic differentiation of DPSCs, 10 ng/ml TNF- 伪 was added to the osteogenic differentiation liquid of DPSCs, and 3 days after differentiation, the activity of ALP was detected by alizarin red staining for calcium nodule formation and ALP staining. The expression of BMP2 protein was analyzed by Western blot and the mRNA expression of osteoblastic markers such as RUNX2COL I was detected by RT-PCR. The effects of TNF- 伪 on the proliferation of DPSCs were analyzed by cell count and MTT assay. Western blot and immunofluorescence were used to detect the expression of p65 I 魏 B 伪 -p65 and p-I 魏 B 伪 in NF- 魏 B signaling pathway. Results DPSCs had the appearance of fusiform fibroblasts and had the characteristics of cartilage differentiation and adipogenic differentiation. TNF- 伪 promoted the high expression of ALP activity and the expression of osteoblastic marker RUNX2COL I in calcium nodules of DPSCs. During this process, the expression of p-p65 and p-I 魏 B 伪 increased in the NF- 魏 B signaling pathway. The osteogenic differentiation of DPSCs could be effectively reversed by the addition of NF- 魏 B signaling pathway inhibitor PDTC. Cell count and MTT assay showed that TNF- 伪 did not affect the proliferation of DPSCs. Conclusion our results suggest that TNF- 伪 promotes osteogenic differentiation of DPSCs and induces the expression of biomarkers related to mineralization of DPSCs, which provides a theoretical basis for autotransplantation of DPSCs.
【学位授予单位】：南通大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R781.31
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本文编号：2027860