本文選題：間充質(zhì)干細(xì)胞 + 牙髓　； 參考：《鄭州大學(xué)》2017年碩士論文

【摘要】：目的使用組織塊貼壁法,在體外分離、培養(yǎng)乳牙牙髓干細(xì)胞(stem cells from human exfoliated deciduous teeth,SHED),探討楊梅素對(duì)乳牙牙髓干細(xì)胞增殖和成骨分化能力的影響。以及在牙髓卟啉單胞菌脂多糖刺激下楊梅素對(duì)乳牙牙髓干細(xì)胞增殖能力及分泌IL-1β、IL-6的影響。方法1、使用組織塊貼壁法進(jìn)行原代細(xì)胞培養(yǎng),取第3代細(xì)胞應(yīng)用有限稀釋法進(jìn)行乳牙牙髓干細(xì)胞的分離,在倒置顯微鏡下觀察乳牙牙髓干細(xì)胞的形態(tài)學(xué)特征,流式細(xì)胞儀檢測(cè)細(xì)胞表面標(biāo)記物CD29、CD105、CD34、CD45的表達(dá)情況,免疫熒光檢測(cè)角蛋白、波形蛋白的表達(dá)情況。2、分別用含100μmol/L、50μmol/L、20μmol/L、15μmol/L、10μmol/L、5μmol/L、1μmol/L楊梅素的培養(yǎng)液對(duì)乳牙牙髓干細(xì)胞進(jìn)行培養(yǎng),分別于24 h、48 h、72 h時(shí)用MTS法檢測(cè)OD值。實(shí)驗(yàn)組:用含1μmol/L、5μmol/L、10μmol/L、15μmol/L、20μmol/L楊梅素的成骨誘導(dǎo)液對(duì)乳牙牙髓干細(xì)胞進(jìn)行培養(yǎng);對(duì)照組:用不含楊梅素的成骨誘導(dǎo)液對(duì)乳牙牙髓干細(xì)胞進(jìn)行培養(yǎng);分別于24 h、48 h、72h時(shí)檢測(cè)乳牙牙髓干細(xì)胞堿磷酸酶活性。3、實(shí)驗(yàn)組:200mg/L LPS+DMEM培養(yǎng)基組;200mg/L LPS+20μmol/L楊梅素+DMEM培養(yǎng)基組;200mg/L LPS+100mg/L甲硝唑+DMEM培養(yǎng)基組;空白對(duì)照組:DMEM培養(yǎng)基;分別培養(yǎng)24 h、48 h、72 h后,用MTS法檢測(cè)SHED的OD值。實(shí)驗(yàn)組:200mg/L LPS+DMEM培養(yǎng)液培養(yǎng)乳牙牙髓干細(xì)胞;20μmol/L楊梅素+200μg/ml牙髓卟啉單胞菌脂多糖培養(yǎng)液培養(yǎng)乳牙牙髓干細(xì)胞;陽(yáng)性對(duì)照組:200μg/m L甲硝唑+200μg/ml牙髓卟啉單胞菌脂多糖培養(yǎng)液培養(yǎng)乳牙牙髓干細(xì)胞;空白對(duì)照組:10%FBS的DMEM培養(yǎng)基培養(yǎng)乳牙牙髓干細(xì)胞;培養(yǎng)48 h后收集培養(yǎng)上清,用ELISA kit試劑盒檢測(cè)IL-1β和IL-6的蛋白含量。結(jié)果1、倒置相差顯微鏡下觀察原代培養(yǎng)的乳牙牙髓干細(xì)在第9~12 d游離出組織塊,胞體多為長(zhǎng)梭形或多角形,圍繞著組織塊呈放射狀、漩渦狀排列,細(xì)胞兩端伸出的胞突較細(xì)長(zhǎng),胞質(zhì)均勻,胞核居中、圓或卵圓形,約生長(zhǎng)20 d可達(dá)到80%匯合。免疫熒光染色結(jié)果顯示:激光共聚焦顯微鏡下見(jiàn)乳牙牙髓干細(xì)胞波形絲蛋白染色熒光,少量角蛋白染色熒光。繪制第5代乳牙牙髓干細(xì)胞1~7 d的生長(zhǎng)曲線大致呈倒“S”形。成骨誘導(dǎo)液培養(yǎng)30 d后,倒置相差顯微鏡下見(jiàn)成骨誘導(dǎo)組茜素紅染色呈紅色有大量礦化結(jié)節(jié)形成,空白對(duì)照組茜素紅染色僅有少量的紅染及礦化結(jié)節(jié)。流式細(xì)胞儀檢測(cè)結(jié)果顯示:CD29、CD105陽(yáng)性表達(dá),CD34、CD45陰性表達(dá)。2、1μmol/L、5μmol/L楊梅素與空白對(duì)照組統(tǒng)計(jì)學(xué)分析無(wú)明顯差異(P0.05),10μmol/L、15μmol/L、20μmol/L三個(gè)濃度的楊梅素均可促進(jìn)SHED增殖,并有明顯的統(tǒng)計(jì)學(xué)差異(P0.05);50μmol/L、100μmol/L楊梅素明顯抑制SHED增殖,有明顯的統(tǒng)計(jì)學(xué)差異(P0.05);各組ALP活性在第5 d時(shí)達(dá)到最高,第7d時(shí)活性有所下降,在3 d、5 d和7 d分別與空白對(duì)照組相比,10μmol/L、15μmol/L和20μmol/L楊梅素均可明顯促進(jìn)ALP活性,具有明顯的統(tǒng)計(jì)學(xué)差異(P0.05),1μmol/L、5μmol/L楊梅素與空白對(duì)照組對(duì)比對(duì)SHED的ALP活性無(wú)明顯的統(tǒng)計(jì)學(xué)差異(P0.05)。LSD-t檢驗(yàn)表明10μmol/L、15μmol/L和20μmol/L濃度楊梅素之間對(duì)ALP活性的影響均具有明顯的統(tǒng)計(jì)學(xué)差異(P0.05)。3、200mg/L LPS組與空白對(duì)照組相比SHED的OD值明顯下降,具有明顯的統(tǒng)計(jì)學(xué)差異(P0.05);200 mg/L LPS+20μmol/L楊梅素組、200 mg/L LPS+100mg/L甲硝唑組與200 mg/L LPS組相比SHED的OD值明顯增加,具有明顯的統(tǒng)計(jì)學(xué)差異(P0.05);200 mg/L LPS組與空白對(duì)照組相比SHED分泌IL-1β、IL-6明顯增多,具有明顯的統(tǒng)計(jì)學(xué)差異(P0.05);200 mg/L LPS+20μmol/L楊梅素組、200 mg/L LPS+100 mg/L甲硝唑組與200 mg/L LPS組相比SHED分泌IL-1β、IL-6明顯減少,具有明顯的統(tǒng)計(jì)學(xué)差異(P0.05);結(jié)論1、10μmol/L、15μmol/L、20μmol/L楊梅素對(duì)乳牙牙髓干細(xì)胞增殖能力有促進(jìn)作用,低濃度5μmol/L、1μmol/L楊梅素對(duì)SHED增殖能力沒(méi)有明顯促進(jìn)作用,高濃度50μmol/L、100μmol/L楊梅素對(duì)乳牙牙髓干細(xì)胞增殖能力具有抑制作用;2、10μmol/L、15μmol/L和20μmol/L楊梅素可以促進(jìn)乳牙牙髓干細(xì)胞成骨分化;3、20μmol/L楊梅素、100 mg/L甲硝唑能拮抗牙髓卟啉單胞菌脂多糖對(duì)乳牙牙髓干細(xì)胞增殖能力的抑制作用,且楊梅素的作用優(yōu)于甲硝唑;4、牙髓卟啉單胞菌脂多糖作用下乳牙牙髓干細(xì)胞IL-1β和IL-6的分泌量明顯增多,20μmol/L楊梅素、100 mg/L甲硝唑能減少牙髓卟啉單胞菌脂多糖作用下乳牙牙髓干細(xì)胞IL-1β和IL-6的分泌量,且楊梅素和甲硝唑的作用沒(méi)有明顯的差異。
[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.
【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R781

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