煅烧牙粉影响人牙周膜干细胞体外矿化的自噬机制研究
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摘要
目的探讨自噬在煅烧牙粉调节牙周膜干细胞体外矿化中的作用,为牙周膜干细胞的定向诱导分化和牙周病的治疗提供参考。方法选用成人完整的牙齿在300℃的条件下煅烧后,研磨成粉,与α-MEM混合制备成20μg/mL牙粉条件培养基,与人牙周膜干细胞共培养。采用流式细胞技术检测其对牙周膜干细胞凋亡的影响,通过Western blot检测、免疫荧光染色、茜素红染色和ALP染色观察检测其对牙周膜干细胞自噬活性及体外矿化的影响。结果流式细胞技术结果显示牙粉对牙周膜干细胞的凋亡无明显影响(P>0.05);Western blot结果显示煅烧牙粉显著上调人牙周膜干细胞的自噬相关蛋白Beclin1,ATG5的表达和LC3Ⅱ/LC3Ⅰ的比值,以及明显下调了P62的蛋白水平(P<0.05);免疫荧光染色显示牙粉促进人牙周膜干细胞中LC3在胞质内点状聚集;茜素红染色显示牙粉促进牙周膜干细胞的体外矿化;ALP染色显示自噬活性抑制剂氯喹降低牙粉对牙周膜干细胞体外矿化的促进作用。结论煅烧牙粉通过激活自噬调节牙周膜干细胞的体外矿化作用。
Objective To evaluate the effects of calcined tooth powder on mineralization of human periodontal ligament stem cells(hPDLSCs) in vitro and related mechanism, to provide reference for the induced directional differentiation of periodontal ligament stem cells and treatment of periodontal diseases. Methods The human intact teeth were calcined under 300 ℃ and milled into powder, then mixed with α-MEM to make 20 μg/mL tooth powder as culture medum. The hPDLSCs were isolated and cultured with or without calcined tooth powder. The apoptosis of hPDLSCs was detected by flow cytometry. Western blot, immunofluorescence staining, alizarin red staining and alkaline phosphatase staining were performed to verify the effect of calcined tooth powder on autophagy and mineralization of hPDLSCs in vitro. Results There were no significant differences apoptosis between two groups of hPDLSCs. The protein level of Beclin1, ATG5 and the ratio of LC3Ⅱ/LC3Ⅰ of hPDLSCs cultured in calcined tooth powder conditioned medium were significantly higher than the control group hPDLSCs, while the expression of P62 was significantly lower than the control group. The calcium mineralization was significantly higher after osteogenic induction in the group of hPDLSCs calcined tooth powder treated than control group. Alkaline phosphatase expression levels were inhibited by inhibition of autophagy in the process of mineralization in vitro. Conclusion The calcined tooth powder can up-regulate mineralization of hPDLSCs via inducing autophagy in vitro.
Objective To evaluate the effects of calcined tooth powder on mineralization of human periodontal ligament stem cells(hPDLSCs) in vitro and related mechanism, to provide reference for the induced directional differentiation of periodontal ligament stem cells and treatment of periodontal diseases. Methods The human intact teeth were calcined under 300 ℃ and milled into powder, then mixed with α-MEM to make 20 μg/mL tooth powder as culture medum. The hPDLSCs were isolated and cultured with or without calcined tooth powder. The apoptosis of hPDLSCs was detected by flow cytometry. Western blot, immunofluorescence staining, alizarin red staining and alkaline phosphatase staining were performed to verify the effect of calcined tooth powder on autophagy and mineralization of hPDLSCs in vitro. Results There were no significant differences apoptosis between two groups of hPDLSCs. The protein level of Beclin1, ATG5 and the ratio of LC3Ⅱ/LC3Ⅰ of hPDLSCs cultured in calcined tooth powder conditioned medium were significantly higher than the control group hPDLSCs, while the expression of P62 was significantly lower than the control group. The calcium mineralization was significantly higher after osteogenic induction in the group of hPDLSCs calcined tooth powder treated than control group. Alkaline phosphatase expression levels were inhibited by inhibition of autophagy in the process of mineralization in vitro. Conclusion The calcined tooth powder can up-regulate mineralization of hPDLSCs via inducing autophagy in vitro.
引文
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[2] Chong LY,Chien LY,Chung MC,et al.Controlling the proliferation and differentiation stages to initiate periodontal regeneration[J].Connect Tissue Res,2013,54(2):101-107.
[3] 徐婕,刘宏伟,徐斌.人牙周膜干细胞的分离培养及体外诱导分化研究[J].中国组织工程研究,2011,27(8):702-705.
[4] 许生敏,朱凌兰.牙周膜干细胞分化、增殖特性与牙周组织再生[J].中国组织工程研究,2015,19(45):7369-7373.
[5] Cao F,Zhan J,Chen X,et al.miR-214 promotes periodontal ligament stem cell osteoblastic differentiation by modulating Wnt/βcatenin signaling[J].Mol Med Rep,2017,6(16):9301-9308.
[6] Wang YQ,Zhou YX,Jin L,et al.Mineral trioxide aggregate enhances the osteogenic capacity of periodontal ligament stem cells via NF-κB and MAPK signaling pathways[J].J Cell Physiol.,2018,233(3):2386-2397.
[7] 刘虹,邵荣光.自噬在肿瘤发生与发展过程中的调节作用[J].药学学报,2016,51(1):23-28.
[8] Eisenberg-Lerner A,Bialik S,Simon HU,et al.Life and death partners:apoptosis,autophagy and the cross-talk between them[J].Cell Death Differ,2009,16(7):966-975.
[9] Bronietzki AW,Schuster M,Schmitz I.Autophagy in T-cell development,activation and differentiation[J].Immunol Cell Biol,2015,93(1):25-34.
[10] Zhao Y,Huang Q,Yang J,et al.Autophagy impairment inhibits differentiation of glioma stem/progenitor cells[J].Brain Res,2010,1313(2010):250-258.
[11] 王凌,王菲菲,邱学敏.自噬对骨代谢的调节作用[J].老年医学与保健,2017,23(6):596-599.
[12] Nuschke A,Rodrigues M,Stolz DB,et al.Human mesenchymal stem cells/multipotent stromal cells consume accumulated autophagosomes early in differentiation[J].Stem Cell Res Ther,2014,5(6):140.
[13] Chen J,Long F.mTORC1 signaling promotes osteoblast differentiation from preosteoblasts[J].PloS One,2015,10(6):e130627.
[14] 楼剑书,应美丹,何俏军,等.低氧激活自噬促进人间充质干细胞成骨分化的研究[J].中国现代医学杂志,2016,33(3):284-289.
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[16] Siqueira L,Passador FR,Costa MM,et al.Influence of the addition of β-TCP on the morphology,thermal properties and cell viability of poly (lactic acid) fibers obtained by electrospinning[J].Mater Sci Eng C Mater Biol Appl,2015(52):135-143.
[17] Kim YK,Kim SG,Byeon JH,et al.Development of a novel bone grafting material using autogenous teeth[J].Oral Surg Oral Med Oral Pathol Oral Radiol Endod,2010,4(109):496-503.
[18] 杨海龙,张明宇,常再平.葛根素促进成骨细胞复合β-磷酸三钙的成骨[J].中国组织工程研究,2018,28(22):4447-4451.
[19] 梁志刚,陈浩东,姚金凤.磷酸钙的固有骨诱导性及其应用[J].中国组织工程研究,2016,20(25):3785-3792.
[20] Yuan H,Fernandes H,Habibovic P,et al.Osteoinductive ceramics as a synthetic alternative to autologous bone grafting[J].Proc Natl Acad Sci USA,2010,107(31):13614-13619.
[21] Gerber T,Traykova T,Henkel KO.Development andin vivo test of sol-gel derived bone grafting materials[J].J Sol-Gel Sci Techn,2003,3(26):1173-1178.
[22] 孟许亚,布文奂,刘杰,等.高尔基体通过自噬调控成骨分化的研究进展[J].吉林大学学报(医学版),2018,44(4):864-868.
[23] 张创杰,张连峰,周琳.自噬相关蛋白Beclin1、LC3和P62在进展期胰腺癌中的表达及临床意义[J].世界华人消化杂志,2015,23(2):318-323.
[24] 高艳,程晨,李静,等.骨形态发生蛋白2诱导C2C12和MC3T3-E1的成骨分化与自噬[J].中国组织工程研究,2014,20(18):3236-3241.
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