摘要
目的:研究人牙周膜干细胞(hPDLSCs)在转染miR-26a后成骨分化的促进效果。方法:从因正畸拔除的无龋坏、无牙周疾病离体牙的牙根中部牙周膜组织中分离、胶原酶消化,进行人牙周膜干细胞的体外培养。使用脂质体2000进行miRNA转染,采用激光共聚焦观察转染效果;MTT方法检测转染后的细胞活力;成骨诱导后使用实时定量PCR技术检测miRNA修饰的hPDLSCs成骨分化相关基因的表达;采用BCIP/NBT、天狼星红、茜素红S分别对碱性磷酸酶、胶原以及钙化进行染色观察。结果:采用脂质体2000能够成功转染hPDLSCs,且转染效率较高;转染后的细胞活力有所下降,但仍在80%以上;转染后的细胞在经成骨诱导分化过程中骨钙素(OCN)和骨桥蛋白(OPN)基因表达显著上调,同时碱性磷酸酶活性、胶原分泌以及钙化能力均得到显著提升。结论:miR-26a可以用于修饰hPDLSCs以提高其成骨分化能力。
Objective： To investigate the osteogenic differentiation capacity of human periodontal ligament stem cells（hPDLSCs）after modification with miR-26 a. Methods： The periodontal ligament tissues from no-caries-no-periodontal-lesions teeth obtained from orthodontic treatment were isolated and digested with collagenase to perform the in vitro culture. The miRNA transfection was performed by lipofectamine 2000 according to the manufacturer＇s instructions. Confocal laser scanning microscope was used to observe the transfection efficiency and the cell viability was monitored by MTT assay. The real time quantitative PCR was used to check the osteogenic differentiation alteration after miRNA modification. Meanwhile, the alkaline phosphatase activity, collagen secretion and calcium deposition were visualized by BCIP/NBT, Sirius Red, Alizarin Red S staining,respectively. Results： The lipofectamine 2000 was able to transfect hPDLSCs and the transfection efficiency was remarkable. Cell viability was suppressed after transfection, but it was still more than 80 percent. During the osteogenic induction, cells modified with miR-26 a could enormously improve the osteocalcin（OCN） and osteopontin（OPN） m RNA level. In the meanwhile, the alkaline phosphatase activity, collagen secretion and calcium deposition were all augmented significantly. Conclusion： The miR-26 a can be used to modify hPDLSCs in order to improve their osteogenic differentiation ability.
出处
《现代生物医学进展》
CAS
2016年第3期445-448,456,共5页
Progress in Modern Biomedicine
关键词
人牙周膜干细胞
成骨分化
miR-26a
细胞培养
Human periodontal ligament stem cells
Osteogenic differentiation
miR-26a
Cell culture
作者简介
米修奎（1965-），副主任医师，电话：13677106331，E-mail：mixiukui@126．com
