论文部分内容阅读
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。
AIM: To investigate the promoting effect of human periodontal ligament stem cells (hPDLSCs) on osteogenic differentiation after miR-26a transfection. Methods: Human periodontal ligament stem cells were cultured in vitro from the periodontal ligament tissue in the root of the root without root caries after toothless extraction without caries and without collagenase digestion. The miRNAs were transfected by lipofectamine 2000. The transfected cells were observed by laser scanning confocal microscopy. The viability of transfected cells was detected by MTT assay. The osteogenic differentiation-related genes of miRNA-modified hPDLSCs were detected by real-time PCR after osteogenic induction BCIP / NBT, Sirius red and alizarin red S were used to observe the alkaline phosphatase, collagen and calcification respectively. Results: The hPDLSCs were transfected successfully with Lipofectamine 2000 and the transfection efficiency was high. The viability of transfected cells was decreased but still above 80%. After transfection, Osteocalcin (OCN) and osteopontin (OPN) genes were significantly upregulated, while alkaline phosphatase activity, collagen secretion and calcification ability were significantly improved. Conclusion: miR-26a can be used to modify hPDLSCs to enhance osteogenic differentiation.