低氧增强人牙周膜干细胞干性促进骨再生的作用研究  

The study on the effect of hypoxia on enhancing the stemness of hPDLSCs to promote bone regeneration

作　　者：马佳慧 肖轶婧 李梓睿 陈旭[1,2,3] 徐艳 MA Jiahui;XIAO Yijing;LI Zirui;CHEN Xu;XU Yan(Department of Periodontology,The Affiliated Stomatological Hospital of Nanjing Medical University,Nanjing 210029,China;State Key Laboratory Cultivation Base of Research,Prevention and Treatment for Oral Diseases,Nanjing 210029,China;Jiangsu Province Engineering Research Center of Stomatological Translational Medicine,Nanjing 210029,China)

机构地区：[1]南京医科大学附属口腔医院牙周病科,江苏南京210029 [2]口腔疾病研究与防治国家级重点实验室培育建设点,江苏南京210029 [3]江苏省口腔转化医学工程研究中心,江苏南京210029

出　　处：《口腔生物医学》2025年第2期72-80,共9页Oral Biomedicine

基　　金：国家自然科学基金(82370961,82170962);江苏省研究生实践创新计划(SJCX23_0713);江苏省卫健委重点项目(ZD2021025,BJ19033);江苏省科教能力提升工程——江苏省研究型医院(YJXYYJSDW4);江苏省医学创新中心(CXZX202227)。

摘　　要：目的:研究低氧培养环境下人牙周膜干细胞(hPDLSCs)干性特征的改变及其对体内外成骨分化的影响。方法:常氧和低氧环境下培养hPDLSCs,通过CCK-8和集落形成实验研究其增殖能力,β-半乳糖苷酶染色检测细胞衰老情况,实时荧光定量PCR和Western blot实验检测干性相关基因性别决定区Y框蛋白2(SOX2)、八聚体结合转录因子4(OCT4)和NANOG表达水平。收集两种培养环境下的hPDLSCs上清液作为条件培养基配制成骨诱导液,在常氧环境下对hPDLSCs进行成骨诱导,利用碱性磷酸酶(ALP)染色、茜素红染色及实时荧光定量PCR实验检测成骨相关基因Ⅰ型胶原(COL-1)、骨桥蛋白(OPN)、骨钙蛋白(OCN)、Runt相关转录因子2(RUNX2)表达研究hPDLSCs体外促成骨分化能力。将两种培养环境预处理的hPDLSCs细胞膜片植入大鼠颅骨临界骨缺损,8周后取材进行显微计算机断层扫描(micro-CT)、HE染色、Masson染色、免疫组化染色和荧光染料标记新骨实验,检测hPDLSCs体内促成骨分化能力。结果:低氧环境下,hPDLSCs增殖能力增强(P<0.05),衰老水平下降(P<0.05),SOX2、OCT4、NANOG基因和蛋白表达均增强(P<0.05)。低氧培养hPDLSCs条件培养基体外促进hPDLSCs成骨分化,ALP活性增高、钙结节形成增多(P<0.05),COL-1、OPN、OCN、RUNX2基因表达升高(P<0.05)。低氧培养hPDLSCs细胞膜片植入骨缺损区后,新骨形成增多,镜下新生骨发出荧光面积更大、强度更强,大鼠颅骨骨体积分数(BV/TV)明显升高(P<0.05),HE和Masson染色观察骨缺损边缘可见大量新生骨组织,周围可见骨细胞及大量的胶原纤维。免疫组化结果显示OPN、OCN蛋白表达增高(P<0.05)。结论:在低氧环境下,hPDLSCs的干性增强,低氧预处理的hPDLSCs体内外促进细胞成骨分化。Objective:To investigate the changes in stemness characteristics of human periodontal ligament stem cells(hPDLSCs)under hypoxia environment and their effects on osteogenic differentiation in vitro and in vivo.Methods:hPDLSCs were cultured under normoxia and hypoxia environment,and their proliferative ability was studied by CCK-8 and colony forming unit experiments.The se-nescence was detected byβ-galactosidase staining.The expression of stemness-related genes sex determining region of Y-box protein 2(SOX2),octamer-binding transcription factor 4(OCT4)and NANOG was studied by qRT-PCR and Western blot experiments.The su-pernatant of hPDLSCs under two culture environments was collected and used as the conditioned medium to configure the osteogenic in-duction medium,then osteogenic induction of hPDLSCs was performed under normoxia.The alkaline phosphatase(ALP)staining,alizarin red staining and the expression of osteogenic-related genes collagen typeⅠ(COL-1),osteopontin(OPN),osteocalcin(OCN)and runt-related transcription factor-2(RUNX2)detected by qRT-PCR assay were used to study the ability of hPDLSCs to promote os-teogenic differentiation in vitro.Cell membrane sheets of hPDLSCs pretreated in two culture environments were implanted into the criti-cal bone defects of rats skulls,tissue samples were collected after 8 weeks,and microcomputed tomography(micro-CT),HE staining,Masson staining,immunohistochemical staining and fluorescent dyes labeling new bone experiment were used to study the ability of hP-DLSCs to promote osteogenic differentiation in vivo.Results:Under hypoxia conditions,the proliferation ability of hPDLSCs was in-creased(P<0.05),the level of senescence was decreased(P<0.05),and the expressions of SOX2,OCT4,and NANOG genes and proteins were all enhanced(P<0.05).Using hPDLSCs conditioned medium cultured in hypoxia to promote hPDLSCs osteogenic differ-entiation in vitro,the ALP activity increased,calcium nodule formation increased(P<0.05),and the expressions of COL-1,OPN,OCN and RUNX2 were increased(P<0

关 键 词：人牙周膜干细胞 低氧 干性 骨再生 

分 类 号：R780.2[医药卫生—口腔医学]