杨梅素对乳牙牙髓干细胞增殖、成骨分化及分泌IL-1β、IL-6的影响

发布时间：2018-06-28 10:42

本文选题：间充质干细胞 + 牙髓　；参考：《郑州大学》2017年硕士论文

【摘要】：目的使用组织块贴壁法,在体外分离、培养乳牙牙髓干细胞(stem cells from human exfoliated deciduous teeth,SHED),探讨杨梅素对乳牙牙髓干细胞增殖和成骨分化能力的影响。以及在牙髓卟啉单胞菌脂多糖刺激下杨梅素对乳牙牙髓干细胞增殖能力及分泌IL-1β、IL-6的影响。方法1、使用组织块贴壁法进行原代细胞培养,取第3代细胞应用有限稀释法进行乳牙牙髓干细胞的分离,在倒置显微镜下观察乳牙牙髓干细胞的形态学特征,流式细胞仪检测细胞表面标记物CD29、CD105、CD34、CD45的表达情况,免疫荧光检测角蛋白、波形蛋白的表达情况。2、分别用含100μmol/L、50μmol/L、20μmol/L、15μmol/L、10μmol/L、5μmol/L、1μmol/L杨梅素的培养液对乳牙牙髓干细胞进行培养,分别于24 h、48 h、72 h时用MTS法检测OD值。实验组:用含1μmol/L、5μmol/L、10μmol/L、15μmol/L、20μmol/L杨梅素的成骨诱导液对乳牙牙髓干细胞进行培养;对照组:用不含杨梅素的成骨诱导液对乳牙牙髓干细胞进行培养;分别于24 h、48 h、72h时检测乳牙牙髓干细胞碱磷酸酶活性。3、实验组:200mg/L LPS+DMEM培养基组;200mg/L LPS+20μmol/L杨梅素+DMEM培养基组;200mg/L LPS+100mg/L甲硝唑+DMEM培养基组;空白对照组:DMEM培养基;分别培养24 h、48 h、72 h后,用MTS法检测SHED的OD值。实验组:200mg/L LPS+DMEM培养液培养乳牙牙髓干细胞;20μmol/L杨梅素+200μg/ml牙髓卟啉单胞菌脂多糖培养液培养乳牙牙髓干细胞;阳性对照组:200μg/m L甲硝唑+200μg/ml牙髓卟啉单胞菌脂多糖培养液培养乳牙牙髓干细胞;空白对照组:10%FBS的DMEM培养基培养乳牙牙髓干细胞;培养48 h后收集培养上清,用ELISA kit试剂盒检测IL-1β和IL-6的蛋白含量。结果1、倒置相差显微镜下观察原代培养的乳牙牙髓干细在第9~12 d游离出组织块,胞体多为长梭形或多角形,围绕着组织块呈放射状、漩涡状排列,细胞两端伸出的胞突较细长,胞质均匀,胞核居中、圆或卵圆形,约生长20 d可达到80%汇合。免疫荧光染色结果显示:激光共聚焦显微镜下见乳牙牙髓干细胞波形丝蛋白染色荧光,少量角蛋白染色荧光。绘制第5代乳牙牙髓干细胞1~7 d的生长曲线大致呈倒“S”形。成骨诱导液培养30 d后,倒置相差显微镜下见成骨诱导组茜素红染色呈红色有大量矿化结节形成,空白对照组茜素红染色仅有少量的红染及矿化结节。流式细胞仪检测结果显示:CD29、CD105阳性表达,CD34、CD45阴性表达。2、1μmol/L、5μmol/L杨梅素与空白对照组统计学分析无明显差异(P0.05),10μmol/L、15μmol/L、20μmol/L三个浓度的杨梅素均可促进SHED增殖,并有明显的统计学差异(P0.05);50μmol/L、100μmol/L杨梅素明显抑制SHED增殖,有明显的统计学差异(P0.05);各组ALP活性在第5 d时达到最高,第7d时活性有所下降,在3 d、5 d和7 d分别与空白对照组相比,10μmol/L、15μmol/L和20μmol/L杨梅素均可明显促进ALP活性,具有明显的统计学差异(P0.05),1μmol/L、5μmol/L杨梅素与空白对照组对比对SHED的ALP活性无明显的统计学差异(P0.05)。LSD-t检验表明10μmol/L、15μmol/L和20μmol/L浓度杨梅素之间对ALP活性的影响均具有明显的统计学差异(P0.05)。3、200mg/L LPS组与空白对照组相比SHED的OD值明显下降,具有明显的统计学差异(P0.05);200 mg/L LPS+20μmol/L杨梅素组、200 mg/L LPS+100mg/L甲硝唑组与200 mg/L LPS组相比SHED的OD值明显增加,具有明显的统计学差异(P0.05);200 mg/L LPS组与空白对照组相比SHED分泌IL-1β、IL-6明显增多,具有明显的统计学差异(P0.05);200 mg/L LPS+20μmol/L杨梅素组、200 mg/L LPS+100 mg/L甲硝唑组与200 mg/L LPS组相比SHED分泌IL-1β、IL-6明显减少,具有明显的统计学差异(P0.05);结论1、10μmol/L、15μmol/L、20μmol/L杨梅素对乳牙牙髓干细胞增殖能力有促进作用,低浓度5μmol/L、1μmol/L杨梅素对SHED增殖能力没有明显促进作用,高浓度50μmol/L、100μmol/L杨梅素对乳牙牙髓干细胞增殖能力具有抑制作用;2、10μmol/L、15μmol/L和20μmol/L杨梅素可以促进乳牙牙髓干细胞成骨分化;3、20μmol/L杨梅素、100 mg/L甲硝唑能拮抗牙髓卟啉单胞菌脂多糖对乳牙牙髓干细胞增殖能力的抑制作用,且杨梅素的作用优于甲硝唑;4、牙髓卟啉单胞菌脂多糖作用下乳牙牙髓干细胞IL-1β和IL-6的分泌量明显增多,20μmol/L杨梅素、100 mg/L甲硝唑能减少牙髓卟啉单胞菌脂多糖作用下乳牙牙髓干细胞IL-1β和IL-6的分泌量,且杨梅素和甲硝唑的作用没有明显的差异。
[Abstract]:Objective to study the effect of myricetin on the proliferation and osteogenic differentiation of dental pulp stem cells (stem cells from human exfoliated deciduous teeth, SHED) in vitro, and to investigate the effect of myricetin on the proliferation of dental pulp stem cells in deciduous teeth with porphyromoninomonas sp. And the effect of IL-1 beta and IL-6. Method 1, primary cell culture was carried out by tissue block adherence method, and third generation cells were used to separate the dental pulp stem cells from dental pulp by finite dilution method. The morphological characteristics of the pulp stem cells were observed under the inverted microscope. The expression of cell surface markers, CD29, CD105, CD34, CD45, was detected by the inverted microscope. Conditions, immunofluorescence detection of keratin, vimentin expression.2, respectively using 100 mu mol/L, 50 mu mol/L, 20 u mol/L, 15 mu mol/L, 10 mu mol/L, 5 mu mol/L, and 1 u myricetin to culture the pulp stem cells of the tooth teeth, respectively, when 24 h, 48 h, 72 h were detected by MTS method. Ol/L, 20 mu mol/L myricetin induction liquid was cultured on the pulp stem cells of the deciduous teeth. The control group was cultured with the osteogenic induction solution without myricetin. The alkaline phosphatase activity of the pulp stem cells.3 was detected at 24 h, 48 h and 72h respectively. The experimental group: 200mg/L LPS+DMEM medium group; 200mg/L LPS+20 mu mol/L poplar. The myricin +DMEM medium group; 200mg/L LPS+100mg/L metronidazole +DMEM medium group; blank control group: DMEM culture medium; 24 h, 48 h, 72 h, respectively, the MTS method was used to detect the OD value of SHED. Experimental group: 200mg/L LPS+DMEM culture medium was used to cultivate pulp stem cells in deciduous teeth; 20 Mu myricetin Dental pulp stem cells; positive control group: 200 g/m L metronidazole +200 mu g/ml lipopolysaccharide culture medium to cultivate dental pulp stem cells in deciduous teeth; blank control group: 10%FBS DMEM medium was used to cultivate pulp stem cells in deciduous teeth; culture supernatant was collected after 48 h and ELISA kit kit was used to detect IL-1 beta and IL-6 protein content. Results 1, inversion Under the phase contrast microscope, the primary dental pulp dry in the primary culture was observed to free the tissue block in 9~12 D. The cell body was mostly long shuttle or polygon, and the tissue mass was arranged radially and swirled around. The cells at both ends were elongated and homogeneous, the nucleus was in the middle, round or oval, and about 20 D could reach 80% confluence. Immunofluorescence staining. The results showed that the coloring fluorescence of the pulp stem cells in the deciduous tooth pulp stem cells was observed under the confocal laser confocal microscope, and a small amount of keratin was stained. The growth curve of 1~7 D of the fifth generation deciduous dental pulp stem cells was roughly inverted "S" shape. After 30 d culture of the osteogenic induction solution, the coloring of alizarin red in the osteogenic induction group was greatly red under the inverted phase contrast microscope. The mineralized nodules were formed in the blank control group with alizarin red staining only a small amount of red dye and mineralized nodules. Flow cytometry showed that CD29, CD105 positive expression, CD34, CD45 negative expression.2,1 micron mol/L, 5 mu mol/L myricetin and blank control group had no significant difference (P0.05), 10 u mol/L, 15 mu mol/L, 20 mu mol/L three concentrations of Yang Meetin could promote the proliferation of SHED, and there was a significant statistical difference (P0.05). 50 mu mol/L, 100 mol/L myricetin obviously inhibited SHED proliferation, and there was a significant statistical difference (P0.05). The activity of ALP in each group was highest at fifth D, and the activity decreased at 7d, at 3 D, 5 D and 7, respectively, 10 mu, 15 mu and 20 mu respectively. Myricetin could obviously promote the activity of ALP, with significant statistical difference (P0.05), 1 mu mol/L, 5 mol/L myricetin and blank control group compared with the blank control group, there was no significant difference in ALP activity of SHED (P0.05).LSD-t test showed that 10 mu mol/L, 15 mu mol/L and 20 mu mol/L concentration of myricetin had significant difference in the activity of ALP. The O (P0.05).3200mg/L LPS group was significantly lower in SHED than that in the blank control group, with significant statistical difference (P0.05); 200 mg/L LPS+20 mu mol/L myricetin group and 200 mg/L LPS+100mg/L metronidazole group were significantly higher than 200 mg/L LPS groups. Compared with the SHED secretion of IL-1 beta, the group IL-6 increased significantly and had significant statistical differences (P0.05); 200 mg/L LPS+20 mu mol/L myricetin group and 200 mg/L LPS+100 mg/L metronidazole group secreted the beta group compared with the 200 mg/L LPS group. The proliferation ability of dental pulp stem cells was promoted. The low concentration of 5 mol/L, 1 mol/L myricetin did not promote the proliferation of SHED. The high concentration of 50 u mol/L, 100 mu mol/L myricetin could inhibit the proliferation of dental pulp stem cells, and 2,10 mu mol/L, 15 mu mol/L and 20 UU myricetin could promote the osteogenesis of the pulp stem cells. Differentiation; 3,20 mu mol/L myricetin and 100 mg/L metronidazole can antagonize the inhibition of the proliferation of dental pulp stem cells by porphyromoninomonas pulpium lipopolysaccharide, and the effect of myricetin is better than metronidazole; 4, the secretion of IL-1 beta and IL-6 in the pulp stem cells of the deciduous teeth is significantly increased under the action of porphyromoninomonas pulpium lipopolysaccharide, 20 mu mol/L myricetin and 100 mg/ L metronidazole can reduce the secretion of IL-1 beta and IL-6 in the pulp stem cells of the deciduous teeth, and there is no significant difference in the effect of myricetin and metronidazole.
【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

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