安徽医科大学学报
安徽醫科大學學報
안휘의과대학학보
Acta Universitatis Medicinalis Anhui
2015年
10期
1381-1385
,共5页
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
牙週膜榦細胞%miR-210%低氧誘導因子-1α%血管內皮生長因子%慢病毒載體
아주막간세포%miR-210%저양유도인자-1α%혈관내피생장인자%만병독재체
periodontal ligament stem cells%miR-210%hypoxia inducible factor-1α%vascular endothelial growth factor%lentiviral vector
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 ( hPDLSCs)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 hPDLSCs 的表达. 方法 分离培养hPDLSCs,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pLVX-EGFP-3FLAG-EF1-Luc上. 将目的基因 PCR 产物和目的载体用 EcoR Ⅰ和 Xba Ⅰ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-Lucif-erase ( Lenti-LacZ-Luciferase为对照组). 转染 hPDLSCs后,通过qPCR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 hP-DLSCs,0、1、4、7、14 d 后行 qPCR 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 hPDLSCs 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 hPDLSCs血管向分化的作用.
目的 構建miR-210 的慢病毒載體 ( Lenti-miR-210-Luciferase) ,轉染人牙週膜榦細胞 ( hPDLSCs)後,檢測成血管因子低氧誘導因子-1α ( HIF-1α)及血管內皮生長因子( VEGF)在 hPDLSCs 的錶達. 方法 分離培養hPDLSCs,併根據人miR-210 基因(NC_000011. 9)序列行引物設計及序列片段的 PCR 擴增,將目的基因 PCR 產物連接到載體pLVX-EGFP-3FLAG-EF1-Luc上. 將目的基因 PCR 產物和目的載體用 EcoR Ⅰ和 Xba Ⅰ分彆進行酶切,對質粒進行鑒定. 採用LR重組繫統構建慢病毒載體Lenti-miR-210-Lucif-erase ( Lenti-LacZ-Luciferase為對照組). 轉染 hPDLSCs後,通過qPCR和免疫組化法檢測HIF-1α及VEGF 的錶達. 結果 通過對構建質粒剋隆進行測序及酶切,證實Lenti-miR-210-Luciferase構建成功. Lenti-miR-210-Luciferase 轉染 hP-DLSCs,0、1、4、7、14 d 後行 qPCR 檢測,結果錶明第 4 天HIF-1α 和 VEGF 明顯過錶達,且持續到第14天. 免疫組化結果顯示,miR-210 轉染 hPDLSCs 後,抗 HIF-1α和抗 VEGF染色呈暘性,對照組呈陰性. 結論 成功構建瞭慢病毒載體Lenti-miR-210-Luciferase,併且 miR-210 具有促進 hPDLSCs血管嚮分化的作用.
목적 구건miR-210 적만병독재체 ( Lenti-miR-210-Luciferase) ,전염인아주막간세포 ( hPDLSCs)후,검측성혈관인자저양유도인자-1α ( HIF-1α)급혈관내피생장인자( VEGF)재 hPDLSCs 적표체. 방법 분리배양hPDLSCs,병근거인miR-210 기인(NC_000011. 9)서렬행인물설계급서렬편단적 PCR 확증,장목적기인 PCR 산물련접도재체pLVX-EGFP-3FLAG-EF1-Luc상. 장목적기인 PCR 산물화목적재체용 EcoR Ⅰ화 Xba Ⅰ분별진행매절,대질립진행감정. 채용LR중조계통구건만병독재체Lenti-miR-210-Lucif-erase ( Lenti-LacZ-Luciferase위대조조). 전염 hPDLSCs후,통과qPCR화면역조화법검측HIF-1α급VEGF 적표체. 결과 통과대구건질립극륭진행측서급매절,증실Lenti-miR-210-Luciferase구건성공. Lenti-miR-210-Luciferase 전염 hP-DLSCs,0、1、4、7、14 d 후행 qPCR 검측,결과표명제 4 천HIF-1α 화 VEGF 명현과표체,차지속도제14천. 면역조화결과현시,miR-210 전염 hPDLSCs 후,항 HIF-1α화항 VEGF염색정양성,대조조정음성. 결론 성공구건료만병독재체Lenti-miR-210-Luciferase,병차 miR-210 구유촉진 hPDLSCs혈관향분화적작용.
Objective To construct the lentiviral vector Lenti-miR-210-Luciferase, and to detect angiogenic factors 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.
