摘要
目的探讨miR-149-3p靶向调节蛋白激酶B(protein kinase B,Akt1)介导牙周膜干细胞(periodontal ligament stem cells,PDLSCs)成骨分化的作用及机制。方法选取2022-01月在作者医院口腔科就诊的18~25岁患者因正畸而拔除的前磨牙或第三磨牙,分离牙周膜组织,提取人PDLSCs。免疫组织化学染色法检测角蛋白(pan-cytokeratin)和波形蛋白(vimentin)鉴定人PDLSCs。将人PDLSCs分为对照组、空载组、miR-149-3p inhibitor组和miR-149-3p mimic组。对照组人PDLSCs不进行处理,空载组、miR-149-3p inhibitor组和miR-149-3p mimic组按Lipofectamine 3000说明书方法分别将空载质粒和miR-149-3p inhibitor、miR-149-3p mimic转染到人PDLSCs。实时荧光逆转录聚合酶链反应(real-time reverse transcription polymerase chain reaction,RT-PCR)检测细胞内miR-149-3p、碱性磷酸酶(alkaline phosphatase,ALP)、Runt相关转录因子2(runt-related transcription factor 2,Runx2)、Akt1 mRNA表达;Western blot检测细胞内ALP、Runx2、Akt1蛋白表达。采用Fluo-3 AM探针检测细胞内钙离子。结果免疫组织化学结果显示,人PDLSCs二氨基联苯胺(diaminobenzidine,DAB)染色vimentin呈阳性,pan-cytokeratin呈阴性,提示PDLSCs提取成功。与对照组和空载组相比,miR-149-3p inhibitor组miR-149-3p表达显著降低,ALP、Runx2、Akt1 mRNA和蛋白表达、细胞内钙离子荧光强度均显著升高(P均<0.05);miR-149-3p mimic组miR-149-3p表达显著升高,ALP、Runx2、Akt1 mRNA和蛋白表达、细胞内钙离子荧光强度均显著降低(P均<0.05)。与miR-149-3p inhibitor组相比,miR-149-3p mimic组miR-149-3p表达显著升高,ALP、Runx2、Akt1 mRNA和蛋白表达、细胞内钙离子荧光强度均显著降低(P均<0.05)。对照组与空载组各指标比较差异均无统计学意义(P>0.05)。结论过表达miR-149-3p可靶向抑制Akt1蛋白的表达,从而抑制成骨标志物ALP和Runx2表达,降低人PDLSCs内钙离子浓度,不利于人PDLSCs成骨分化。
Objective To explore the role and mechanism of miR-149-3p targeting regulating protein kinase B(Akt1)in the osteogenic differentiation of periodontal ligament stem cells(PDLSCs).Methods The premolars or third molars of 18-25-year-old patients who were extracted for orthodontic treatment in the department of stomatology of au-thor's hospital in January 2022 were selected,the periodontal ligament tissues were isolated and human PDLSCs were ex-tracted.Human PDLSCs were identified by immunohistochemical staining of pan-cytokeratin and vimentin.Human PDLSCs were divided into control group,empty plasmid group,miR-149-3p inhibitor group and miR-149-3p mimic group.The human PDLSCs in control group were not treated,the empty plasmid,miR-149-3p inhibitor and miR-149-3p mimic groups were transfected into human PDLSCs according to the instructions of Lipofectamine 3000 in the empty plas-mid group,miR-149-3p inhibitor group and miR-149-3p mimic group.The mRNA expressions of miR-149-3p,alkaline phosphatase(ALP),runt-related transcription factor 2(Runx2)and Akt1 were detected by real-time reverse transcrip-tion polymerase chain reaction(RT-PCR).Protein expressions of ALP,Runx2 and Akt1 were detected by Western blot.Intracellular calcium ion was detected by Fluo-3 AM probe.Results The results of immunohistochemistry showed that the diaminobenzidine(DAB)staining of hu-man PDLSCs was positive for vimentin and negative for pan-cytokinetin which indicated the successful exrtaction of PDLSCs.Compared with the control group and the empty plasmid group,the expression of miR-149-3p in the miR-149-3p inhibitor group significantly decreased,the mRNA and protein expressions of ALP,Runx2,Akt1,and the intra-cellular calcium ion fluorescence intensity significantly increased(all P<0.05);the expression of miR-149-3p in the miR-149-3p mimic group significantly increased,and the mRNA,protein expressions and the intracellular calcium ion fluores-cence intensity of ALP,Runx2 and Akt1 significantly decreased(all P<0.05).Compared with miR-149-3p inhibitor group,miR-149-3p expression significantly increased in the miR-149-3p mimic group,mRNA,protein expressions and in-tracellular calcium ion fluorescence intensity of ALP,Runx2 and Akt1 significantly decreased(all P<0.05).There was no significant difference between the control group and the empty plasmid group(P>0.05).Conclusion Over-expressed miR-149-3p can target the expression of Akt1 protein,thus inhibiting the expression levels of osteogenic markers ALP and Runx2,decreasing calcium ion concentration in human PDLSCs,which is not conducive to the osteogenic differentia-tion of human PDLSCs.
作者
袁琴
张玲玲
戴丽
章茜
卫峥
YUAN Qin;ZHANG Lingling;DAI Li;ZHANG Qian;WEI Zheng(Department of Stomatology,Nanjing Stomatological Hospital,Medical School of Nanjing University,Nanjing Jiangsu 210000,China)
出处
《联勤军事医学》
CAS
2024年第1期1-5,共5页
Military Medicine of Joint Logistics
基金
国家自然科学基金项目(82103516)。
