雌激素对人牙周膜干细胞干性维持的影响
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摘要
目的研究雌激素对人牙周膜干细胞(h PDLSC)干性维持的影响。方法分离、培养原代h PDLSC并完成其干细胞鉴定;雌激素处理48 h,采用流式细胞术检测雌激素对h PDLSC细胞周期的影响;定量聚合酶链反应(q-PCR)检测人端粒酶逆转录酶(h TERT)基因、干性相关基因Oct4、Sox2、c-Myc以及衰老相关基因p16和p53的变化;雌激素长期处理后观察细胞克隆形成能力及骨向分化能力的改变。应用SPSS 17.0软件,利用配对样本t检验对组间均数进行统计学分析,P<0.05为差异具有统计学意义。结果实验获得的h PDLSC符合间充质干细胞鉴定标准。流式细胞术结果显示,雌激素处理48 h后h PDLSC增殖能力(29.730±1.845)较对照组(23.587±2.905)明显提高,差异有统计学意义(t=9.913,P=0.010)。q-PCR结果显示雌激素处理48 h后,h TERT表达量(1.958±0.338)较对照组(1.000±0.018)明显提高(t=7.00,P=0.001);Oct4表达量(2.539±0.493)较对照组(1.000±0.011)明显提高(t=6.26,P=0.001);Sox2表达量(2.234±0.255)较对照组(1.016±0.221)明显提高(t=6.26,P=0.003);c-Myc表达量(1.328±0.091)较对照组(1.003±0.088)明显提高(t=5.67,P=0.002);而衰老相关基因p16表达量(0.460±0.085)较对照组(1.009±0.163)明显降低(t=12.24,P=0.007);p53表达量(0.301±0.041)较对照组(1.004±0.115)明显降低(t=7.77,P=0.016)。雌激素长期处理后,雌激素处理组的克隆形成率(0.058±0.008)显著高于对照组(0.013±0.008),差异有统计学意义(t=5.60,P=0.028),成骨能力显著强于对照组(A_(对照组)=1.238±0.084;A_(E2)=2.460±0.182,t=16.41,P<0.001)。结论雌激素能提高h TERT和Oct4、Sox2、c-Myc的基因表达量,维持细胞增殖,抑制细胞衰老。雌激素长期处理能维持体外扩增时h PDLSC的干性。
Objective To investigate the biological effect of estrogen(E2) on the stemnessmaintenance of human periodontal ligament stem cells(h PDLSCs). Methods Primary h PDLSCs wereisolated and characterized. After treatment with estrogen for 48 h,flow cytometry was used to evaluate cellcycle. The changes in human telomerase reverse transcriptase(h TERT)genes,stemness related genesOct4,Sox2,c-Myc,aging related genes p16 and p53 were detected by q-PCR. After long-term culture withestrogen,colony formation ability and osteogenic differentiation potential were evaluated. The mean valuesbetween groups were analyzed by Paired-Samples T Test in SPSS 17.0. A significance level was set at 0.05.Results We isolated h PDLSCs and confirmed their capacity as mesenchymal stem cells. Proliferationability of h PDLSCs was increased under 48 hours estrogen treatment(t = 9.913,P = 0.010). q-PCR resultsshowed that the expression level of h TERT(1.958 ± 0.338)was higher than the control group(1.000 ±0.018),there was a statistically significant difference between the groups(t = 7.00,P = 0.001);theexpression level of Oct4(2.539 ± 0.493)was higher than the control group(1.000 ± 0.011),there was astatistically significant difference between the groups(t = 6.26,P = 0.001);the expression level of Sox2(2.234 ± 0.255)was higher than the control group(1.016 ± 0.221),there was a statistically significant difference between the groups(t = 6.26,P = 0.003);the expression level of c-Myc(1.328 ± 0.091)was higher than the control group(1.003 ± 0.088),there was a statistically significant difference between the groups(t = 5.67,P = 0.002),while senescence related genes that the expression level of p16(0.460 ±0.085)was lower than the control group(1.009 ± 0.163),there was a statistically significant difference between the groups(t = 12.24,P = 0.007)and the expression level of p53(0.301 ± 0.041)was lower than the control group(1.004 ± 0.115),there was a statistically significant difference between the groups(t =7.77,P = 0.016). After long-term culture,the ability of clone formation(0.058 ± 0.008)was stronger thanthat of the NC group(0.013 ± 0.008),there was a statistically significant difference between the groups(t = 5.60,P = 0.028). And the osteogenic ability(A_(NC)= 1.238 ± 0.084;A_(E2)= 2.460 ± 0.182,t = 16.41,P<0.001) of hPDLSCs with estrogen treatment was significantly stronger than that of the NC group.Conclusions Estrogen can increase the expression of h TERT and Oct4,Sox2,c-Myc genes,maintaincell proliferation and inhibit cell senescence. Estrogen also can maintain stemness of h PDLSCs in longterm culture in vitro.
Objective To investigate the biological effect of estrogen(E2) on the stemnessmaintenance of human periodontal ligament stem cells(h PDLSCs). Methods Primary h PDLSCs wereisolated and characterized. After treatment with estrogen for 48 h,flow cytometry was used to evaluate cellcycle. The changes in human telomerase reverse transcriptase(h TERT)genes,stemness related genesOct4,Sox2,c-Myc,aging related genes p16 and p53 were detected by q-PCR. After long-term culture withestrogen,colony formation ability and osteogenic differentiation potential were evaluated. The mean valuesbetween groups were analyzed by Paired-Samples T Test in SPSS 17.0. A significance level was set at 0.05.Results We isolated h PDLSCs and confirmed their capacity as mesenchymal stem cells. Proliferationability of h PDLSCs was increased under 48 hours estrogen treatment(t = 9.913,P = 0.010). q-PCR resultsshowed that the expression level of h TERT(1.958 ± 0.338)was higher than the control group(1.000 ±0.018),there was a statistically significant difference between the groups(t = 7.00,P = 0.001);theexpression level of Oct4(2.539 ± 0.493)was higher than the control group(1.000 ± 0.011),there was astatistically significant difference between the groups(t = 6.26,P = 0.001);the expression level of Sox2(2.234 ± 0.255)was higher than the control group(1.016 ± 0.221),there was a statistically significant difference between the groups(t = 6.26,P = 0.003);the expression level of c-Myc(1.328 ± 0.091)was higher than the control group(1.003 ± 0.088),there was a statistically significant difference between the groups(t = 5.67,P = 0.002),while senescence related genes that the expression level of p16(0.460 ±0.085)was lower than the control group(1.009 ± 0.163),there was a statistically significant difference between the groups(t = 12.24,P = 0.007)and the expression level of p53(0.301 ± 0.041)was lower than the control group(1.004 ± 0.115),there was a statistically significant difference between the groups(t =7.77,P = 0.016). After long-term culture,the ability of clone formation(0.058 ± 0.008)was stronger thanthat of the NC group(0.013 ± 0.008),there was a statistically significant difference between the groups(t = 5.60,P = 0.028). And the osteogenic ability(A_(NC)= 1.238 ± 0.084;A_(E2)= 2.460 ± 0.182,t = 16.41,P<0.001) of hPDLSCs with estrogen treatment was significantly stronger than that of the NC group.Conclusions Estrogen can increase the expression of h TERT and Oct4,Sox2,c-Myc genes,maintaincell proliferation and inhibit cell senescence. Estrogen also can maintain stemness of h PDLSCs in longterm culture in vitro.
引文
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[25]Kyo S,Takakura M,Kanaya T,et al.Estrogen activates telomerase[J].Cancer Res,1999,59(23):5917-5921.
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[27]Fafián-Labora J,Fernández-Pernas P,Fuentes I,et al.Influence of age on rat bone-marrow mesenchymal stem cells potential[J/OL].Sci Rep,2015[2015-11-19].http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4652164/pdf/srep16765.pdf.[published online ahead of print March 23,2016].
[28]Chen X,Xu H,Yuan P,et al.Integration of external signaling pathways with the core transcriptional network in embryonic stem cells[J].Cell,2008,133(6):1106-1117.
[29]Takahashi K,Tanabe K,Ohnuki M,et al.Induction of pluripotent stem cells from adult human fibroblasts by defined factors[J].Cell,2007,131(5):861-872.
[30]Jung EM,Choi KC,Yu FH,et al.Effects of 17beta-estradiol and xenoestrogens on mouse embryonic stem cells[J].Toxicol In Vitro,2010,24(6):1538-1545.
[31]Xie Y,Zhao X,Jia H,et al.Derivation and characterization of goat fetal fibroblast cells induced with human telomerase reverse transcriptase[J].In Vitro Cell Dev Biol Anim,2013,49(1):8-14.
[2]Hynes K,Menicanin D,Gronthos S,et al.Clinical utility of stem cells for periodontal regeneration[J].Periodontol 2000,2012,59(1):203-227.
[3]Zhu W,Liang M.Periodontal ligament stem cells:current status,concerns,and future prospects[J/OL].Stem Cells Int,2015[2015-03-16].http://www.hindawi.com/journals/sci/2015/972313.[published online ahead of print March 23,2016].
[4]Wada N,Menicanin D,Shi S,et al.Immunomodulatory properties of human periodontal ligament stem cells[J].J Cell Physiol,2009,219(3):667-676.
[5]Otte A,Bucan V,Reimers K,et al.Mesenchymal stem cells maintain long-term in vitro stemness during explant culture[J].Tissue Eng Part C Methods,2013,19(12):937-948.
[6]Cheng H,Qiu L,Ma J,et al.Replicative senescence of human bone marrow and umbilical cord derived mesenchymal stem cells and their differentiation to adipocytes and osteoblasts[J].Mol Biol Rep,2011,38(8):5161-5168.
[7]Hong L,Zhang G,Sultana H,et al.The effects of 17-βestradiol on enhancing proliferation of human bone marrow mesenchymal stromal cells in vitro[J].Stem Cells Dev,2011,20(5):925-931.
[8]Cha Y,Kwon SJ,Seol W,et al.Estrogen receptor-alpha mediates the effects of estradiol on telomerase activity in human mesenchymal stem cells[J].Mol Cells,2008,26(5):454-458.
[9]Kim BC,Bae H,Kwon IK,et al.Osteoblastic/cementoblastic and neural differentiation of dental stem cells and their applications to tissue engineering and regenerative medicine[J].Tissue Eng Part B Rev,2012,18(3):235-244.
[10]Yeasmin S,Ceccarelli J,Vigen M,et al.Stem cells derived from tooth periodontal ligament enhance functional angiogenesis by endothelial cells[J].Tissue Eng Part A,2014,20(7-8):1188-1196.
[11]Wu Y,Yang Y,Yang P,et al.The osteogenic differentiation of PDLSCs is mediated through MEK/ERK and p38 MAPK signalling under hypoxia[J].Arch Oral Biol,2013,58(10):1357-1368.
[12]Ding G,Liu Y,Wang W,et al.Allogeneic periodontal ligament stem cell therapy for periodontitis in swine[J].Stem Cells,2010,28(10):1829-1838.
[13]Mrozik KM,Wada N,Marino V,et al.Regeneration of periodontal tissues using allogeneic periodontal ligament stem cells in an ovine model[J].Regen Med,2013,8(6):711-723.
[14]Dominici M,Le Blanc K,Mueller I,et al.Minimal criteria for defining multipotent mesenchymal stromal cells.The International Society for Cellular Therapy position statement[J].Cytotherapy,2006,8(4):315-317.
[15]Asselin-Labat ML,Vaillant F,Sheridan JM,et al.Control of mammary stem cell function by steroid hormone signalling[J].Nature,2010,465(7299):798-802.
[16]Sun H,Wang H,Hu S.Effects of estrogen on diverse stem cells and relevant intracellular mechanisms[J].Sci China Life Sci,2010,53(5):542-547.
[17]Liang L,Yu J,Wang Y,et al.Effect of estrogen receptor beta on the osteoblastic differentiation function of human periodontal ligament cells[J].Arch Oral Biol,2008,53(6):553-557.
[18]Pan F,Zhang R,Wang G,et al.Oestrogen receptors are involved in the osteogenic differentiation of periodontal ligament stem cells[J].Biosci Rep,2011,31(2):117-124.
[19]Cha Y,Kwon SJ,Seol W,et al.Estrogen receptor-alpha mediates the effects of estradiol on telomerase activity in human mesenchymal stem cells[J].Mol Cells,2008,26(5):454-458.
[20]Zhang B,Li Y,Zhou Q,et al.Estrogen deficiency leads to impaired osteogenic differentiation of periodontal ligament stem cells in rats[J].Tohoku J Exp Med,2011,223(3):177-186.
[21]Cai C,Yuan GJ,Huang Y,et al.Estrogen-related receptorαis involved in the osteogenic differentiation of mesenchymal stem cells isolated from human periodontal ligaments[J].Int J Mol Med,2013,31(5):1195-1201.
[22]Bayne S,Jones ME,Li H,et al.Estrogen deficiency leads to telomerase inhibition,telomere shortening and reduced cell proliferation in the adrenal gland of mice[J].Cell Res,2008,18(11):1141-1150.
[23]Estrada JC,Torres Y,Benguría A,et al.Human mesenchymal stem cell-replicative senescence and oxidative stress are closely linked to aneuploidy[J].Cell Death Dis,2013(4):e691.
[24]Piper SL,Wang M,Yamamoto A,et al.Inducible immortality in h TERT-human mesenchymal stem cells[J].J Orthop Res,2012,30(12):1879-1885.
[25]Kyo S,Takakura M,Kanaya T,et al.Estrogen activates telomerase[J].Cancer Res,1999,59(23):5917-5921.
[26]Zhou C,Steplowski TA,Dickens HK,et al.Estrogen induction of telomerase activity through regulation of the mitogen-activated protein kinase(MAPK)dependent pathway in human endometrial cancer cells[J].PLo S One,2013,8(2):e55730.
[27]Fafián-Labora J,Fernández-Pernas P,Fuentes I,et al.Influence of age on rat bone-marrow mesenchymal stem cells potential[J/OL].Sci Rep,2015[2015-11-19].http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4652164/pdf/srep16765.pdf.[published online ahead of print March 23,2016].
[28]Chen X,Xu H,Yuan P,et al.Integration of external signaling pathways with the core transcriptional network in embryonic stem cells[J].Cell,2008,133(6):1106-1117.
[29]Takahashi K,Tanabe K,Ohnuki M,et al.Induction of pluripotent stem cells from adult human fibroblasts by defined factors[J].Cell,2007,131(5):861-872.
[30]Jung EM,Choi KC,Yu FH,et al.Effects of 17beta-estradiol and xenoestrogens on mouse embryonic stem cells[J].Toxicol In Vitro,2010,24(6):1538-1545.
[31]Xie Y,Zhao X,Jia H,et al.Derivation and characterization of goat fetal fibroblast cells induced with human telomerase reverse transcriptase[J].In Vitro Cell Dev Biol Anim,2013,49(1):8-14.