条件培养基诱导人牙髓干细胞分化为角膜上皮样细胞
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摘要
目的探讨条件培养基(CM)诱导人牙髓干细胞(DPSCs)分化为角膜上皮样细胞的可行性。方法体外分离培养DPSCs,通过流式细胞术鉴定DPSCs,细胞计数试剂盒-8(CCK-8)检测基础培养基(BM)和不同比例CM培养的DPSCs增殖活性,选用30%、60%、90%CM诱导DPSCs,BM培养的DPSCs设为阴性对照组,免疫荧光检测角膜上皮细胞标志物细胞角蛋白3(CK3)、细胞角蛋白12(CK12)的表达。结果 DPSCs增殖活性的差异无统计学意义(P> 0. 05);诱导3 d时,30%、60%、90%CM组细胞不表达CK3,60%和90%CM组细胞开始表达CK12; 7 d时,30%、60%、90%CM组细胞可见CK3、CK12的表达; 11 d和14 d时,30%、60%、90%CM组细胞继续表达CK3、CK12。BM组细胞未见CK3、CK12表达。结论在一定诱导条件下,DPSCs可分化为角膜上皮样细胞。
Objective To investigate the feasibility of inducing human dental pulp stem cells(DPSCs) to differentiate into corneal epithelial-like cells by conditioned medium(CM). Methods DPSCs were isolated and identified by flow cytometry. The effect of basal medium(BM) and different CM on the proliferation activity of DPSCs was detected by cell counting kit-8(CCK-8) assay. DPSCs were induced by 30%CM,60%CM,90%CM. The cells cultured in BM were negative control group. Corneal epithelial cells markers cytokeratin 3(CK3) and cytokeratin 12(CK12) were detected by immunofluorescence assay. Results There was no significant difference in the proliferation activity of DPSCs between BM group and different CM group(P > 0. 05). Cells in the 30%,60%,90% CM group did not express CK3 after 3 days induction,cells in the 60%and the 90% CM group began to express CK12; CK3 and CK12 were expressed in the 30%,60%,90%CM group after 7 days; At the 11 th and 14 th day,cells continued to express CK3 and CK12 in the 30%,60%,and 90% CM groups. No expression of CK3 and CK12 was observed in the BM group. Conclusion DPSCs are capable of differentiating into corneal epithelial-like cells under the induction of CM.
Objective To investigate the feasibility of inducing human dental pulp stem cells(DPSCs) to differentiate into corneal epithelial-like cells by conditioned medium(CM). Methods DPSCs were isolated and identified by flow cytometry. The effect of basal medium(BM) and different CM on the proliferation activity of DPSCs was detected by cell counting kit-8(CCK-8) assay. DPSCs were induced by 30%CM,60%CM,90%CM. The cells cultured in BM were negative control group. Corneal epithelial cells markers cytokeratin 3(CK3) and cytokeratin 12(CK12) were detected by immunofluorescence assay. Results There was no significant difference in the proliferation activity of DPSCs between BM group and different CM group(P > 0. 05). Cells in the 30%,60%,90% CM group did not express CK3 after 3 days induction,cells in the 60%and the 90% CM group began to express CK12; CK3 and CK12 were expressed in the 30%,60%,90%CM group after 7 days; At the 11 th and 14 th day,cells continued to express CK3 and CK12 in the 30%,60%,and 90% CM groups. No expression of CK3 and CK12 was observed in the BM group. Conclusion DPSCs are capable of differentiating into corneal epithelial-like cells under the induction of CM.
引文
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[25]Nowell CS,Radtke F. Corneal epithelial stem cells and their niche at a glance[J]. J Cell Sci,2017,130(6):1021-1025.
[2]Sacchetti M,Rama P,Bruscolini A,et al. Limbal stem cell transplantation:clinical results,limits,and perspectives[J]. Stem Cells Int,2018,2018:8086269.
[3]Sehic A,UtheimA,Ommundsen K,et al. Pre-clinical cellbased therapy for limbal stem cell deficiency[J]. J Funct Biomater,2015,6(3):863-888.
[4]Botelho J,Cavacas MA,Machado V,et al. Dental stem cells:recent progresses in tissue engineering and regenerative medicine[J]. Ann Med,2017,49(8):644-651.
[5]Nakashima M,Iohara K. Mobilized dental pulp stem cells for pulp regeneration:initiation of clinical trial[J]. J Endod,2014,40(4):S26-S32.
[6]Giuliani A,Manescu A,Langer M,et al. Three years after transplants in human mandibles, histological and in-line holotomography revealed that stem cells regenerated a compact rather than a spongy bone:biological and clinical implications[J].Stem Cells Transl Med,2013,2(4):316-324.
[7]Kushnerev E,Shawcross SG,Sothirachagan S,et al. Regeneration of corneal epithelium with dental pulp stem cells using a contact lens delivery system[J]. Invest Ophthalmol Vis Sci,2016,57(13):5192-5199.
[8]Kaukua N,Shahidi MK,Konstantinidou C,et al. Glial origin of mesenchymal stem cells in a tooth model system[J]. Nature,2014,513(7519):551-554.
[9]Ding G,Niu J,Liu Y. Dental pulp stem cells suppress the proliferation of lymphocytes via transforming growth factor-β1[J].Hum Cell,2015,28(2):81-90.
[10]Li S,Lin C,Zhang J,et al. Quaking promotes the odontoblastic differentiation of human dental pulp stem cells[J]. J Cell Physiol,2018,233(9):7292-7304.
[11]Ba P,Duan X,Fu G,et al. Differential effects of p38 and Erk1/2 on the chondrogenic and osteogenic differentiation of dental pulp stem cells[J]. Mol Med Rep,2017,16(1):63-68.
[12]Westin CB,Trinca RB,Zuliani C,et al. Differentiation of dental pulp stem cells into chondrocytes upon culture on porous chitosanxanthan scaffolds in the presence of kartogenin[J]. Mater Sci Eng C Mater Biol Appl,2017,80:594-602.
[13]Chun SY,Soker S,Jang YJ,et al. Differentiation of human dental pulp stem cells into dopaminergic neuron-like cells in vitro[J]. J Korean Med Sci,2016,31(2):171-177.
[14]Cho YA,Noh K,Jue SS,et al. Melatonin promotes hepatic differentiation of human dental pulp stem cells:clinical implications for the prevention of liver fibrosis[J]. J Pineal Res,2015,58(1):127-135.
[15]Kim D,Kim J,Hyun H,et al. A nanoscale ridge/groove pattern arrayed surface enhances adipogenic differentiation of human supernumerary tooth-derived dental pulp stem cells in vitro[J].Arch Oral Biol,2014,59(8):765-774.
[16]Munévar JC,Gutiérrez N,Jiménez NT,et al. Evaluation of two human dental pulp stem cell cryopreservation methods[J]. Acta Odontol Latinoam,2015,28(2):114-121.
[17]Lindemann D, Werle SB, Steffens D, et al. Effects of cryopreservation on the characteristics of dental pulp stem cells of intact deciduous teeth[J]. Arch Oral Biol,2014,59(9):970-976.
[18]Tsai CL,Chuang PC,Kuo HK,et al. Differentiation of stem cells from human exfoliated deciduous teeth toward a phenotype of corneal epithelium in vitro[J]. Cornea,2015,34(11):1471-1477.
[19]Monteiro BG,Serafim RC,Melo GB,et al. Human immature dental pulp stem cells share key characteristic features with limbal stem cells[J]. Cell Prolif,2009,42(5):587-594.
[20]Gomes JA, Geraldes Monteiro B, Melo GB, et al. Corneal reconstruction with tissue-engineered cell sheets composed of human immature dental pulp stem cells[J]. Invest Ophthalmol Vis Sci,2010,51(3):1408-1414.
[21]Huang CE,Hu FW,Yu CH,et al. Concurrent expression of Oct4and nanog maintains mesenchymal stem-like property of human dental pulp cells[J]. Int J Mol Sci,2014,15(10):18623-18639.
[22]Al-Habib M,Yu Z,Huang GT. Small molecules affect human dental pulp stem cell properties via multiple signaling pathways[J]. Stem Cells Dev,2013,22(17):2402-2413.
[23]Karner CM, Lee SY, Long F. Bmp induces osteoblast differentiation through both smad4 and m TORC1 signaling[J]. Mol Cell Biol,2017,37(4):e00253-16.
[24]Karaoz E,Okcu A,nal ZS,et al. Adipose tissue-derived mesenchymal stromal cells efficiently differentiate into insulinproducing cells in pancreatic islet microenvironment both in vitro and in vivo[J]. Cytotherapy,2013,15(5):557-570.
[25]Nowell CS,Radtke F. Corneal epithelial stem cells and their niche at a glance[J]. J Cell Sci,2017,130(6):1021-1025.