二氢杨梅素对人乳牙牙髓干细胞活性及成骨分化能力影响的初步探究

发布时间：2018-08-06 21:27

【摘要】：目的本研究拟通过体外分离、培养及鉴定人乳牙牙髓干细胞后,探讨二氢杨梅素对人乳牙牙髓干细胞的活性及成骨分化能力的影响,浅探二氢杨梅素与乳牙牙髓干细胞在治疗牙周疾病中的可能性。方法1.采用组织块法及有限稀释法获取人乳牙牙髓干细胞,在倒置显微镜下观察细胞的生长状况,免疫荧光组织化学及流式细胞仪检测细胞表面标志物来鉴定细胞来源,CCK-8法绘制细胞生长曲线,并对细胞进行成脂及成骨诱导分化;2.采用CCK-8法、碱性磷酸酶试剂盒及实时荧光定量PCR检测0.02μM、0.1μM、0.5μM、2μM、10μM及50μM浓度的二氢杨梅素对人乳牙牙髓干细胞的活性及骨向分化能力的影响。结果1.镜下观察见细胞在第7 d左右从组织块中爬出,细胞生长状态良好;免疫荧光组织化学结果为:第3代SHED波形丝蛋白在胞浆内表达阳性,角蛋白表达阴性;流式细胞仪检测发现细胞CD146、CD105表达阳性,CD34、CD45表达阴性;第4代SHED生长曲线呈现“S”型;SHED成脂诱导3周后油红O染色见细胞中有红色脂滴形成;SHED成骨诱导4周后茜素红染色形成红染的矿化结节。2.二氢杨梅素对SHED细胞活性的影响CCK-8结果显示:0.02μM、0.1μM、0.5μM、2μM、10μM及50μM浓度的二氢杨梅素在第24 h、48 h及72 h时与不含二氢杨梅素对照组相比,增值能力无显著差异(P0.05),说明试验设置的各浓度的二氢杨梅素对SHED无明显的细胞毒性。3.二氢杨梅素对SHED成骨分化能力的影响⑴二氢杨梅素对SHED碱性磷酸酶活性的影响10μM及50μM浓度的二氢杨梅素组与对照组相比,可促进SHED碱性磷酸酶的活性(P0.05);而0.02μM、0.1μM、0.5μM及2μM浓度的二氢杨梅素组的ALP活性与对照组相比无统计学差异(P0.05)。⑵二氢杨梅素对SHED RUNX2及OCN mRNA表达的影响RUNX2 mRNA表达结果为:0.5μM、2μM、10μM及50μM浓度的二氢杨梅素组RUNX2 mRNA的表达与对照组相比要高(P0.05);OCN mRNA表达变化为:2μM、10μM及50μM浓度的二氢杨梅素组与对照组相比,OCN mRNA的表达成上调趋势(P0.05)。结论1.组织块法及有限稀释法可成功培养出人乳牙牙髓干细胞,人乳牙牙髓干细胞在一定诱导条件下可向成骨及成脂方向分化;2.一定浓度的二氢杨梅素对人乳牙牙髓干细胞无明显的细胞毒性;3.一定浓度的二氢杨梅素可促进人乳牙牙髓干细胞的骨向分化能力;4.本实验为二氢杨梅素与人乳牙牙髓干细胞结合应用于牙周组织的再生提供了一定的参考价值,但其中的作用机制还需进一步的研究。
[Abstract]:Objective to investigate the effects of dihydromyricetin on the activity and osteogenic differentiation of human deciduous dental pulp stem cells after isolation, culture and identification in vitro. To explore the possibility of dihydromyricetin and deciduous dental pulp stem cells in the treatment of periodontal diseases. Method 1. Human deciduous dental pulp stem cells were obtained by tissue mass method and finite dilution method. The growth of human deciduous dental pulp stem cells was observed under inverted microscope. Immunofluorescence histochemistry and flow cytometry were used to detect the cell surface markers to identify the cell origin. CCK-8 method was used to draw the cell growth curve and adipogenic and osteogenic differentiation was carried out. CCK-8 assay, alkaline phosphatase kit and real-time fluorescence quantitative PCR were used to detect the effects of 0. 02 渭 M 0. 1 渭 M, 0. 5 渭 M, 2 渭 M, 10 渭 M and 50 渭 M dihydromyricetin on the activity and osteogenic differentiation of human deciduous dental pulp stem cells. Result 1. It was observed that the cells crawled out of the tissue mass on the 7th day and the cells grew well. The results of immunofluorescence histochemistry showed that the third generation of SHED vimentin was positive in the cytoplasm, but the expression of keratin was negative. The positive expression of CD146 and CD105 was detected by flow cytometry, and the expression of CD34 and CD45 was negative. The growth curve of the fourth generation of SHED showed "S" type SHED lipid induction 3 weeks later, oil red O staining showed red lipid droplet formation in the cells after 4 weeks of osteogenesis induced by alizarin red staining to form a red-stained mineralized nodule .2. Effects of dihydromyricetin on the activity of SHED cells CCK-8 results showed that the concentrations of 0. 02 渭 M, 0. 5 渭 M, 0. 5 渭 M and 50 渭 M of dihydromyricetin were 10 渭 M and 50 渭 M at 24 h, 48 h and 72 h, respectively, compared with those of control group without dihydromyricetin. There was no significant difference in value-added ability (P0.05), which indicated that dihydromyricetin had no obvious cytotoxicity to SHED. Effects of dihydromyricetin on the osteogenic differentiation of SHED 1 the effects of dihydromyricetin on the activity of SHED alkaline phosphatase in 10 渭 M and 50 渭 M dihydromyricetin groups were compared with those in the control group. The activity of SHED alkaline phosphatase was enhanced (P0.05), while the ALP activity of the 0. 02 渭 M 0. 1 渭 M and 2 渭 M dihydromyricetin groups was not significantly different from that of the control group (P0.05). 2 the effect of dihydromyricetin on the expression of SHED RUNX2 and OCN mRNA was found to be 0. 5 渭 M 2 渭 M 10 渭 M and 50 渭 M respectively. The expression of RUNX2 mRNA in dihydromyricetin group was significantly higher than that in control group (P0.05). The expression of OCN mRNA in dihydromyricetin group (10 渭 M) and dihydromyricetin group (50 渭 M) was significantly higher than that in control group (P0.05). Conclusion 1. Human deciduous dental pulp stem cells could be successfully cultured by tissue block method and finite dilution method. Human deciduous dental pulp stem cells could differentiate into osteogenesis and adipogenesis under certain conditions. A certain concentration of dihydromyricetin has no obvious cytotoxicity to human deciduous dental pulp stem cells. A certain concentration of dihydromyricetin can promote the bone differentiation ability of human deciduous dental pulp stem cells. This experiment provides some reference value for the application of dihydromyricetin combined with human deciduous dental pulp stem cells in periodontal tissue regeneration, but the mechanism of its action needs further study.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R781.4

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