發(fā)布時間：2018-06-16 19:32

  本文選題：脂多糖 + 牙髓干細胞�。� 參考：《南通大學》2014年碩士論文

【摘要】：目的研究脂多糖(LPS)對牙髓干細胞(DPSCs)進行反復刺激后其衰老程度。方法分離、培養(yǎng)DPSCs后進行形態(tài)觀察。甲苯胺藍染色檢測DPSCs成軟骨分化,油紅O染色檢測DPSCs成脂肪分化,DPSCs成骨分化液,茜素紅染色檢測鈣結(jié)節(jié)形成。LPS刺激0、1、3和6次,觀察細胞形態(tài),細胞計數(shù)、流式細胞儀檢測LPS對DPSCs增殖的影響,檢測β-gal的表達情況,檢測LPS對DPSCs免疫熒光檢測LPS對DPSCs細胞骨架的影響、ROS、γ-H2A.X、p16INK4A表達。運用Western blot測定TLR4、p16INK4A、cyclinD1、Rb、p-Rb、γ-H2A.X蛋白表達,RT-PCR測定p16INK4A、γ-H2A.X基因水平表達。結(jié)果DPSCs呈成纖維細胞樣梭形外觀,具有向成軟骨分化、成脂肪、成骨分化的特征。LPS反復刺激后,細胞體積增大、β-gal染色加深、細胞增殖能力降低、G1/G0期細胞比例增加,隨著LPS刺激數(shù)目增多,LPS衰老增強。在此過程中,ROS、TLR4表達增高,DNA損傷標志γ-H2A.X表達上調(diào),p16INK4A信號通路被激活,抑制p16INK4A信號通路可以逆轉(zhuǎn)LPS誘導的DPSCs衰老。結(jié)論我們的研究表明LPS反復刺激DPSCs,誘導DPSCs衰老,為DPSCs的自體移植提供了理論基礎(chǔ)。目的探討腫瘤壞死因子α(TNF-α)對牙髓干細胞(DPSCs)成骨分化、增殖的影響及其機制的研究。方法分離、培養(yǎng)DPSCs后進行形態(tài)觀察。甲苯胺藍染色檢測DPSCs成軟骨分化,油紅O染色檢測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,茜素紅染色檢測鈣結(jié)節(jié)形成,ALP染色檢測ALP活性,運用Western blot分析BMP2蛋白表達情況,RT-PCR測定BMP2、ALP、RUNX2、COL I等成骨標志物m RNA的表達情況。通過細胞計數(shù)和MTT法分析TNF-α對DPSCs增殖的影響。運用Western blot和免疫熒光檢測NF-κB信號通路中p65、IκBα、p-p65和p-IκBα表達情況。結(jié)果DPSCs呈成纖維細胞樣梭形外觀,具有向成軟骨分化、成脂肪分化的特征。TNF-α促進DPSCs鈣結(jié)節(jié)形成、ALP活性高表達及BMP2、ALP、RUNX2、COL I成骨標志物的表達。在此過程中NF-κB信號通路p-p65、p-IκBα表達增高,p65入核,IκBα降解,加入NF-κB信號通路抑制劑PDTC后,TNF-α促進DPSCs成骨分化作用可以被有效逆轉(zhuǎn)。細胞計數(shù)及MTT方法檢測結(jié)果顯示TNF-α不影響DPSCs的增殖。結(jié)論我們的研究表明TNF-α促進DPSCs成骨分化,并誘導DPSCs礦化相關(guān)標志物的表達,為DPSCs的自體移植提供了理論基礎(chǔ)。
[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, 緯 -H2A.X was detected by Western blot. The expression of p16INK4A, 緯 -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 緯 -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- 偽 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- 偽 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- 偽 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 偽 -p65 and p-I 魏 B 偽 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- 偽 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 偽 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- 偽 did not affect the proliferation of DPSCs. Conclusion our results suggest that TNF- 偽 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.
【學位授予單位】：南通大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R781.31
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本文編號：2027860