摘要
目的探讨含DEP结构域mTOR结合蛋白(DEPTOR)与人表皮生长因子受体2(ErbB2)的相互作用对人牙周膜干细胞(hPDLSCs)增殖和成骨分化能力的影响。方法收集联勤保障部队第九〇〇医院口腔科就诊的3例青少年患者正常人牙周膜组织,采用组织块培养方法分离hPDLSCs,并利用流式细胞术进行细胞鉴定。通过慢病毒将FLAG-DEP、FLAG-PDZ、FLAG-DEPTOR转染至P3代hPDLSCs,通过免疫共沉淀实验检测DEPTOR与ErbB2之间的结合关系。使用脂质体转染法将si-DEPTOR、si-ErbB2、si-NC转染至hPDLSCs,采用CCK8检测各组细胞的增殖能力;对细胞进行成骨诱导培养,采用茜素红染色观察细胞第21天成骨矿化形成情况,并用分光光度计法对矿化结节进行定量分析;利用RT-qPCR检测各组细胞Runt相关转录因子2(Runx2)、骨钙素(OCN)、1型胶原蛋白(COL1)、碱性磷酸酶(ALP)mRNA表达水平;采用Western blot检测各组细胞DEPTOR、Erb-b2受体酪氨酸激酶2(ErbB2)、磷酸化磷脂酰肌醇3激酶(p-PI3K)蛋白表达水平。多组间比较采用单因素方差分析,组间两两比较采用LSD-t检验。结果培养的P3代PDLSCs CD90和CD105表达呈阳性,CD34表达呈阴性。在过表达FLAG-DEPTOR和FLAG-DEP的免疫沉淀产物中可检测到ErbB2b蛋白。与si-NC比较,转染si-DEPTOR后DEPTOR蛋白表达水平(1.00±0.18比0.23±0.11)降低,差异有统计学意义(P<0.05);与si-NC比较,转染si-ErbB2和si-DEPTOR后ErbB2蛋白表达水平(1.00±0.07比0.50±0.10、0.23±0.05)均降低,差异有统计学意义(P<0.05)。与si-NC比较,转染si-DEPTOR、si-ERB2的细胞在第7天增殖能力(246.45±8.66比208.33±2.89、216.67±5.77)减弱,矿化结节形成OD值(1.23±0.09比0.78±0.06、0.64±0.09)减少,Runx2、OCN、COL1、ALP mRNA表达水平、p-PI3K蛋白表达水平(1.00±0.16比0.36±0.05、0.16±0.06;1.00±0.27比0.38±0.08、0.21±0.12;1.00±0.15比0.51±0.07、0.51±0.09;1.00±0.17比0.70±0.03、0.64±0.08;1.00±0.15比0.44±0.05、0.23±0.05)降低。结论在hPDLSCs中,DEPTOR的PDZ端与ErbB2结合。沉默DEPTOR能够降低其与ErbB2的相互作用,抑制PI3K信号通路,抑制细胞增殖和成骨分化。
Objectiv To investigate the effects of the interaction between DEP domain-containing mTOR interactingprotein(DEPTOR)and epidermal growth factor receptor 2(ErbB2)on the proliferation and osteogenic differentiation of human periodontal ligament stem cells(hPDLSCs).Methods Normal human periodontal tissues were collected from Department of Stomatology,900th Hospital of Joint Logistic Support Force.hPDLSCs were isolated by tissue block culture method and cell identification was performed using flow cytometry.FLAG-DEP,FLAG-PDZ,and FLAG-DEPTOR were transfected into P3 generation hPDLSCs by lentivirus,and the binding relationship between DEPTOR and ErbB2 was detected by immunoprecipitation assay.Using liposome transfection method to transfect si-DEPTOR,si-ErbB2,and si-NC into hPDLSCs.The osteogenic induction culture was performed for each group of cells,and the proliferation ability of both groups was detected by CCK8 assay;the formation of osteogenic mineralization at d 21 was observed by alizarin red staining,and the mineralized nodules were quantified by spectrophotometric method.The mRNA expression levels of runt-related transcription factor 2(Runx2),osteocalcin(OCN),collagen-1(COL1)and alkaline phosphatase(ALP)were measured by RT-qPCR,and the protein expression levels of DEPTOR,erb-b2 receptor tyrosine kinase 2(ErbB2),phospho-phosphatidylinositol 3-kinase(p-PI3K)were detected by Western blot.One-way ANOVA was used for comparison between multiple groups,and LSD-t test was used for pairwise comparison between groups.Results The expression of CD90 and CD105 was positive,while CD34 was negative in cultured P3 PDLSCs.ErbB2 protein could be detected in immunoprecipitation products by overexpressing Flag-DEPTOR and Flag-DEP.Compared with si-NC group,the protein expression level of DEPTOR was decreased after transfection of si-DEPTOR(1.00±0.18 vs 0.23±0.11),with statistical significance(P<0.05).Compared with si-NC group,the protein expression level of ErbB2 was decreased after transfection of si-ErbB2 and si-DEPTOR(1.00±0.07 vs 0.50±0.10 and 0.23±0.05),with statistical significance(P<0.05).In addition,compared with the si-NC group,the si-DEPTOR and si-ErbB2 transfected cells showed diminished cell proliferation(7 d:246.45±8.66 vs 208.33±2.89 and 216.67±5.77),reduced mineralized nodule formation OD value(1.23±0.09 vs 0.78±0.06 and 0.64±0.09),reduced the mRNA expression levels of Runx2,OCN,COL1,ALP(1.00±0.16 vs 0.36±0.05 and 0.16±0.06,1.00±0.27 vs 0.38±0.08 and 0.21±0.12,1.00±0.15 vs 0.51±0.07 and 0.51±0.09,1.00±0.17 vs 0.70±0.03 and 0.64±0.08)and p-PI3K(1.00±0.15 vs 0.44±0.05 and 0.23±0.05).Conclusion In human periodontal stem cells,the PDZ domain of DEPTOR binds to ErbB2.Silencing DEPTOR was able to reduce its interaction with ErbB2,inhibit PI3K signaling pathway,and suppress cell proliferation and osteogenic differentiation.
作者
周冰
王琤
Zhou Bing;Wang Zheng(Department of Stomatology,900th Hospital of Joint Logistic Support Force,Fuzhou 350025,China)
出处
《中华细胞与干细胞杂志（电子版）》
2021年第6期358-364,共7页
Chinese Journal of Cell and Stem Cell（Electronic Edition）
关键词
人牙周膜干细胞
含DEP结构域蛋白
人表皮生长因子受体2
成骨分化
Human periodontal ligament stem cells
DEP domain-containing mTOR interacting protein
Epidermal growth factor receptor 2
Osteogenic differentiation
