衰老清除药物对规模化长期培养中牙髓干细胞增殖和分化功能的影响  

Senolytic effects on dental pulp stem cell′s proliferation and differentiation during long-term expansion

作　　者：王冠玉 廖立 田卫东[1] Wang Guanyu;Liao Li;Tian Weidong(Department of Trauma and Platic Surgery,West China Hospital of Stomatology,Sichuan University&State Key Laboratory of Oral Diseases&National Center for Stomatology&National Clinical Research Center for Oral Diseases&Ministry of Education&National Engineering Laboratory for Oral Regenerative Medicine,Chengdu 610041,China)

机构地区：[1]四川大学华西口腔医院创伤整形外科、口腔疾病防治全国重点实验室、国家口腔医学中心、国家口腔疾病临床医学研究中心、教育部口腔转化医学工程研究中心,成都610041

出　　处：《中华口腔医学杂志》2024年第5期444-452,共9页Chinese Journal of Stomatology

基　　金：国家自然科学基金(82071092,U21A20369)。

摘　　要：目的探讨间断清除衰老细胞对规模化长期培养中牙髓干细胞(dental pulp stem cell,DPSC)增殖和分化功能的影响,探索维持体外培养DPSC年轻状态的方法。方法从四川大学华西口腔医院口腔颌面外科提供的因正畸或阻生拔除的健康恒牙中提取人DPSC,模拟DPSC体外规模化长期培养过程,并对年轻DPSC(第5代,P5)、衰老DPSC(第25代,P25)进行细胞衰老β-半乳糖苷酶染色、集落形成实验、成骨分化诱导茜素染色实验。利用细胞衰老β-半乳糖苷酶染色比较5种衰老清除药物[那维克拉(ABT-263)、姜黄素、达沙替尼、非瑟酮、槲皮素]的清除效率,选择清除效率最高的药物进行后续实验。选取衰老细胞占比开始显著增多的代数,给予间隔3代、累计3次的衰老清除药物处理,对对照组和药物处理组细胞进行荧光细胞衰老β-半乳糖苷酶染色、实时荧光定量PCR(qPCR)、集落形成实验、细胞划痕实验、成骨诱导茜素红染色实验。体内验证使用裸鼠皮下异位成骨实验,对移植物进行HE染色、碱性磷酸酶(ALP)免疫组织化学染色。结果衰老细胞占比随培养周期延长而增加,相比年轻DPSC,衰老DPSC的增殖和成骨分化能力减退(P<0.05)。与年轻DPSC相比,衰老DPSC衰老相关蛋白p21、p16^(INK4a)、白细胞介素(IL-6)mRNA及增殖相关蛋白Ki67 mRNA表达量均发生显著改变(P<0.001)。5种衰老清除药物中,ABT-263有较高的清除效率(P<0.05)。培养过程中间断使用ABT-263后,药物处理组衰老细胞占比[(6.72±2.34)%]显著小于对照组[(31.82±0.57)%](P<0.001);qPCR证实,药物处理组p21、p16^(INK4a)、IL-6 mRNA表达量均显著小于对照组(P<0.05),Ki67 mRNA表达量显著大于对照组(P<0.01)。药物处理组的细胞愈合能力、成骨分化能力均显著大于对照组(P<0.05)。体内实验结果显示,药物处理组DPSC移植体内后相对新生骨质面积[(2.36±0.48)%]显著大于对照组[(1.00±0.46)%](P<0.05)。结论ABT-2Objective To investigate the impact of intermittent senescent cell clearance on the proliferation and differentiation of dental pulp stem cells(DPSC)in long-term,large-scale expansion,and to explore strategies for maintaining the youthful state of DPSC in vitro.Methods Human-derived dental pulp stem cells were isolated from healthy permanent teeth extracted for orthodontic or impeding eruption reasons,provided by the Department of Oral and Maxillofacial Surgery at West China Hospital of Stomatology,Sichuan University.Long-term,large-scale in vitro expansion of DPSC was conducted.The study compared young DPSC(passage 5)with aged DPSC(passage 25)using cellular senescence-associatedβ-galactosidase staining,colony formation assay,and Alizarin Red S staining for osteogenic differentiation induction.To assess the differences between the two cell populations in terms of senescence and amplification and differentiation ability.Medicine screening for the most effective senolytic was compared among 5 common senolytics[Navitoclax(ABT-263),curcumin,dasatinib,fisetin,and quercetin].The clearance efficacy was compared using cellular senescence-associatedβ-galactosidase staining to reflect the changes in senescent cell ratio.The senolytic with the highest efficacy was chosen for further experiments.The passage at which the proportion of senescent cells significantly increased was identified,and the selected senolytic was administered three times at three-generation intervals from that passage to remove senescent cells.Both the control and senolytic-treated groups were estimated by fluorescence cellular senescence-associatedβ-galactosidase staining,real-time fluorescence quantitative PCR(RT-qPCR),colony formation assay,wound healing assay,and Alizarin Red S staining for osteogenic differentiation induction.Subcutaneous heterotopic osteogenesis was performed in nude mice and the grafts were analyzed by HE staining and alkaline phosphatase(ALP)immunohistochemical staining.Results The proportion of senescent cells increased as t

关 键 词：牙髓 干细胞 细胞衰老 细胞培养技术 衰老清除药物 

分 类 号：R78[医药卫生—口腔医学]