WW域转录调节因子1对人牙髓干细胞的衰老调控作用研究  

Effect of WW-domain transcription regulator 1 on aging regulation of human dental pulp stem cells

作　　者：李丹丹 刘慧娟 王燕[1] 陈增国 张雪 李文静[1] Li Dandan;Liu Huijuan;Wang Yan;Chen Zengguo;Zhang Xue;Li Wenjing(Department of Stomatology,The Second Hospital of Hebei Medical University,Shijiazhuang 050000,China;Department of Key Laboratory,School and Hospital of Stomatology,Hebei Medical University&Hebei Key Laboratory of Stomatology&Hebei Clinical Research Center for Oral Diseases,Shijiazhuang 050017,China)

机构地区：[1]河北医科大学第二医院口腔内科,石家庄050000 [2]河北医科大学口腔医学院·口腔医院重点实验室、河北省口腔医学重点实验室、河北省口腔疾病临床医学研究中心,石家庄050017

出　　处：《中华口腔医学杂志》2024年第12期1240-1247,共8页Chinese Journal of Stomatology

基　　金：2022年河北省政府资助临床医学优秀人才项目(303-2022-27-41)。

摘　　要：目的探讨人牙髓干细胞(hDPSC)随传代次数增加衰老相关表型和分子的改变,探究含WW域转录调节因子1(WWTR1)在hDPSC衰老中的作用。方法组织块法原代培养hDPSC,根据培养出的hDPSC来源受试者的年龄、细胞代数及细胞敲低和过表达WWTR1后将细胞分为4组。Ⅰ组:人牙来源的hDPSC根据受试者不同年龄分为青年组(15~25岁)和中年组(40~50岁)。Ⅱ组:根据hDPSC不同传代分为年轻细胞组(传至第3代)和年老细胞组(传至第10代)。Ⅲ组:hDPSC敲低WWTR1,分为敲低组和敲低空载体组。Ⅳ组:hDPSC过表达WWTR1,分为过表达组和过表达空载体组。实时荧光定量PCR(RT-qPCR)检测Ⅰ、Ⅱ组WWTR1表达量的变化,对Ⅱ、Ⅲ、Ⅳ组通过细胞计数试剂盒(CCK-8)检测细胞增殖能力、茜素红染色检测细胞成骨分化能力、衰老相关β-半乳糖苷酶染色检测细胞衰老阳性率、RT-qPCR检测衰老相关基因p53、p21表达量的变化。结果衰老细胞占比随连续传代培养逐渐增加,与年轻细胞组相比,年老细胞组hDPSC的增殖及成骨分化能力均显著降低(P<0.001),年老细胞组hDPSC衰老相关基因p53(2.09±0.24)、p21(4.91±0.54)的表达量均显著高于年轻细胞组[p53:(1.08±0.09),p21:(1.09±0.08)](P<0.01,P<0.001)。与青年组和年轻细胞组hDPSC相比,中年组和年老细胞组hDPSC的WWTR1表达量均显著降低(P<0.01)。敲低组(44.50±2.42)较敲低空载体组(22.27±0.56)衰老细胞占比显著增加(P<0.001),hDPSC敲低WWTR1后衰老相关基因p53、p21的表达水平显著上调(P<0.001),敲低组hDPSC的增殖及成骨分化能力较敲低空载体组显著降低(P<0.001)。过表达空载体组(20.40±0.79)较过表达组(10.07±0.61)衰老细胞占比显著增加(P<0.001),hDPSC过表达WWTR1后衰老相关基因p53、p21的表达水平显著下调,过表达组hDPSC的增殖及成骨分化能力较过表达空载体组显著升高(P<0.001)。结论WWTR1可以抑制衰老相关基因p53、p21的表达进而延缓hDPObjective Investigating the changes of phenotype and moleculars associated with aging with the increase of passage times of human dental pulp stem cells(hDPSC),to explore the role of WW-containing transcriptional regulator 1(WWTR1)in the aging mechanism.Methods hDPSCs were cultured by tissue block method,and were divided into 4 groups according to the age,algebra,cell knockdown and overexpression of WWTR1 in hDPSCs.GroupⅠ:hDPSCs from human teeth were further divided into youth group(15-25 years old)and group middle-aged group(40-50 years old)according to different ages.GroupⅡ:according to different passage,hDPSCs were divided into young cells group(hDPSCs were transmitted to P3 generation),and old cells group(hDPSCs were transmitted to P10 generation).GroupⅢ:hDPSCs were knocked down of WWTR1,which were further divided into knockdown group and knockdown carrier group.GroupⅣ:hDPSCs were overexpressed of WWTR1,which were further divided into overexpression group and overexpression carrier group.Real-time fluorescence quantitative PCR(RT-qPCR)was used to detect the changes of WWTR1 expression in groupsⅠandⅡ,and cell counting kit-8(CCK-8)was used for groupsⅡ,Ⅲ,andⅣ.Cell proliferation capacity was detected by CCK-8 assay.The ability of osteogenic differentiation was detected by alizarin red staining.Cell senescence positive rate was detected by age-relatedβ-galactosidase staining.The expression levels of age-related genes p53 and p21 were detected by RT-qPCR.Results The proportion of senescent cells increased gradually with continuous culture.The proliferation and osteogenic differentiation of hDPSCs in the old group were significantly lower than those in the young group(P<0.001).The expression levels of senescence related genes p53(2.09±0.24)and p21(4.91±0.54)in old cell group were higher than those in young cell group respectively[p53:(1.08±0.09)and p21:(1.09±0.08)](P<0.01,P<0.001).The WWTR1 expression levels of hDPSCs in middle-aged group and old cells group were both decreased compared with t

关 键 词：细胞衰老 人牙髓干细胞 含WW域转录调节因子1 增殖 成骨分化 连续传代 间充质干细胞 

分 类 号：R781.3[医药卫生—口腔医学]