摘要
目的:探讨miR-30d拮抗剂(antagomir-30d)正性调控大鼠骨髓基质干细胞(BMSCs)的成骨分化作用并确认转染的最佳浓度。方法:BMSCs培养并分组,分为不同浓度的antagomir-30d(antagomir-30d组)、阴性对照(NC组)和未做转染的BMSCs(空白组);将不同浓度的antagomir-30d及其NC转染至BMSCs后进行成骨诱导。通过RT-PCR技术进行比较,测定成骨基因碱性磷酸酶(ALP)、骨钙素(OC)、Runt相关转录因子2(RUNX2)的mRNA的表达情况,并确定最佳转染浓度及其转染效率。通过ALP染色验证antagomir-30d对成骨分化的调控作用。结果:RT-PCR结果表明,antagomir-30d体外可以促进BMSCs的成骨分化。不同浓度antagomir-30d组的成骨基因表达量随浓度变化而改变,当antagomir-30d转染浓度为150 nmol/L时,ALP、OC、RUNX2的mRNA水平均显著高于空白组和NC组(P<0.05)。Antagomir-30d的转染最佳浓度为150 nmol/L,此时ALP染色结果显示其活性最高,成骨分化作用好。结论:Antagomir-30d体外正性调控BMSCs的成骨分化,其转染至BMSCs的最佳浓度为150 nmol/L。
Objective:To verify the upregulating effect of antagomir-30d(miR-30d)on osteogenic differentiation of bone mesenchymal stem cells(BMSCs)and to confirm the optimum transfection concentration.Methods:Antagomir-30d of different concentrations was divided into antagomir-30d(antagomir-30d group),negative control(NC group)and untransfected BMSCs(blank group).Transfecting different concentrations of antagomir-30d/NC/blank to BMSCs,gene expression of osteoblast marker genes(ALP,OC and RUNX2)was analyzed using RT-PCR and then optimum transfection concentration and transfection efficiency were confirmed.The role of antagomir-30d in regulating osteogenesis was verified by ALP staining.Results:The results of RT-PCR showed that antagomir-30d enhanced osteogenic differentiation of BMSCs in vitro.The transfection effect changed according to transfection concentration.When transfected with 150 nmol/L,the expression of ALP,OC and RUNX2 in the antagomir-30d group was significantly higher than the other groups.Meanwhile,there was no significant change in the proliferation and apoptosis of BMSCs when transfected with 150 nmol/L.Therefore,150 nmol/L proved to be optimum ALP staining showed ALP activity of antagomir-30d group was significantly upregulated.Conclusion:The antagonist of miR-30d(antagomir-30d)promotes osteogenic differentiation of BMSCs in vitro and 150 nmol/L is the optimum transfected concentration.
作者
汪艳
金琼
陈威
黄慧
WANG Yan;JIN Qiong;CHEN Wei;HUANG Hui(Department of Implant Prosthodontics,School&Hospital of Stomatology,Wenzhou Medical University,Wenzhou 325027,China;Department of Pediatric Dentistry,School&Hospital of Stomatology,Wenzhou Medical University,Wenzhou 325027,China;Department of Prosthodontics,Shanghai Ninth People’s Hospital,Shanghai Jiao Tong University School of Medicine,Shanghai 200011,China)
出处
《温州医科大学学报》
2021年第6期478-482,共5页
Journal of Wenzhou Medical University
关键词
miR-30d拮抗剂
骨髓基质干细胞
成骨分化
最佳转染浓度
antagomir-30d
bone mesenchymal stem cells
osteogenic differentiation
optimum transfection concentration
