miR-210促进人牙周膜干细胞血管向分化作用的初步研究  

The preliminary research of miR-210 promoting human periodontal ligament stem cells the differentiation of angiogenesis

作　　者：古月[1] 王银龙[1] 

机构地区：[1]安徽医科大学附属口腔医院口腔颌面外科,合肥230032

出　　处：《安徽医科大学学报》2015年第10期1381-1385,共5页Acta Universitatis Medicinalis Anhui

基　　金：安徽省科技厅重点课题(编号:10021303003)

摘　　要：目的构建miR-210的慢病毒载体(Lenti-miR-210-Luciferase),转染人牙周膜干细胞(h PDLSCs)后,检测成血管因子低氧诱导因子-1α(HIF-1α)及血管内皮生长因子(VEGF)在h PDLSCs的表达。方法分离培养h PDLSCs,并根据人miR-210基因(NC_000011.9)序列行引物设计及序列片段的PCR扩增,将目的基因PCR产物连接到载体p LVX-EGFP-3FLAG-EF1-Luc上。将目的基因PCR产物和目的载体用EcoRⅠ和XbaⅠ分别进行酶切,对质粒进行鉴定。采用LR重组系统构建慢病毒载体Lenti-miR-210-Luciferase(Lenti-Lac Z-Luciferase为对照组)。转染h PDLSCs后,通过q PCR和免疫组化法检测HIF-1α及VEGF的表达。结果通过对构建质粒克隆进行测序及酶切,证实Lenti-miR-210-Luciferase构建成功。Lenti-miR-210-Luciferase转染h PDLSCs,0、1、4、7、14 d后行q PCR检测,结果表明第4天HIF-1α和VEGF明显过表达,且持续到第14天。免疫组化结果显示,miR-210转染h PDLSCs后,抗HIF-1α和抗VEGF染色呈阳性,对照组呈阴性。结论成功构建了慢病毒载体Lenti-miR-210-Luciferase,并且miR-210具有促进h PDLSCs血管向分化的作用。Objective To construct the lentiviral vector Lenti-miR-210-Luciferase, and to detect angiogenic factors＆amp;nbsp;HIF-1α and VEGF expression in hPDLSCs transduced by Lenti-miR-210-Luciferase. Methods hPDLSCs were iso-lated and cultured, and according to human miR-210 gene sequence（NC_000011. 9）, its primer was designed and amplified through PCR. The PCR products of the target gene were connected to the vector pLVX-EGFP-3FLAG-EF1-Luc. To identify the plasmid, target gene PCR product and the purpose vector were digested by EcoRⅠand XbaⅠ. Lenti-miR-210-Luciferase （ the control group was Lenti-LacZ-Luciferase） was constructed using the LR re-combination system. After hPDLSCs was transduced by Lenti-miR-210-Luciferase, the analysis of HIF-1α and VEGF expression was done with qPCR and the immunohistochemistry examination. Results The results of plasmid sequencing and digestion confirmed that the vector Lenti-miR-210-Luciferase was successfully constructed. After Lenti-miR-210-Luciferase was transduced to hPDLSCs on 0, 1, 4, 7 and 14 d, the results of qPCR showed that the over-expression of HIF-1αand VEGF was detected on 4 d, and continued until 21 d. Immunohistochemical results showed that after hPDLSCs were transduced by Lenti-miR-210-Luciferase, hPDLSCs were positive for HIF-1α and VEGF antibody, and the control group was negative. Conclusion The Lenti-miR-210-Luciferase is successfully constructed, and miR-210 can promote hPDLSCs the differentiation of angiogenesis.

关 键 词：牙周膜干细胞 MIR-210 低氧诱导因子-1Α 血管内皮生长因子 慢病毒载体 

分 类 号：Q782[生物学—分子生物学]