摘要
目的利用小鼠骨髓间充质干细胞(BMSCs)的成骨分化模型,探讨miR-26a在成骨分化中的表达趋势及调控作用。方法提取雌性C57/BL6J小鼠双侧下肢股骨骨髓分离培养BMSCs,诱导分化为成骨细胞,通过实时定量PCR检测miR-26a在BMSCs及其诱导分化细胞中的表达。使用miR-26a的促进物miR-RiboTMmicroRNA-26a mimics通过siPORTTMNeoFXTMagent转染试剂瞬时转染BMSCs并加入成骨诱导剂培养,同时设立转染microRNA-26a mimics NC的对照组。培养7 d、14 d后,进行细胞形态、碱性磷酸酶(ALP)染色、钙盐结节(茜素红染色)等项目的检测。另外制造绝经后骨质疏松小鼠模型(OVX组)与假手术动物模型(sham组),RT-PCR检测OVX组中miR-26a的表达。结果 miR-26a的表达在BMSCs向成骨分化过程中上升约25倍,成骨基因的表达也随着诱导时间的延长逐渐升高。转染miR-26a mimics后的BMSCs在培养7d后逐渐由梭形变为多角形,与阴性对照组类似;ALP染色显示阴性对照组培养7 d后为阳性,而实验组在培养7 d时呈强阳性;茜素红染色显示实验组培养14d后出现数量较多的钙盐沉积结节,而阴性对照组则较少。miR-26a mimics组成骨基因的表达均高于对照组。RT-PCR检测结果显示OVX组中miR-26a的表达低于sham组4倍。结论 miR-26a mimics的转染可以促进BMSCs的成骨分化潜能,在骨质疏松的环境中miR-26a表达下降,间接证实了miR-26a对小鼠BMSCs成骨潜能的促进作用。
Objective To establish an osteogenic differentiation model and investigate the expression pattern and the regulative effect of miR-26a during osteogenesis. Methods BMSCs were isolated and cultured by extracting femoral bone marrow in bilateral lower limbs of female C57BL/6J mice, and were then induced into osteoblasts. The expression level of miR-26a was detected by real-time quantitative PCR in both BMSCs and osteoblasts. To investigate the function of miR-26a in BMSCs, BMSCs were transfected with miR-RiboTM microRNA-26a mimics （the augmenter of miR-26a）, using siPORTrMNeoFXTM agent as the transfection reagent, and were cultured with osteogenesis inducers. Meanwhile, the control group was established. After 7 days and 14 days of culture, the cells were collected and examined by cell morpholo- gy, alkaline phosphatase staining, calcium nodules （Alizarin red staining） and so on. In vivo expression of miR-26a was detected by real-time PCR in the BMSCs of both the animal postmenopausal osteoporosis models （OVX group） and the sham group. Results There was an ap- proximately 25-fold increase of miR-26a expression in BMSCs during osteogenesis. Meanwhile, the expression of osteogenetic genes including OCN, Runx-2 and Co1-1 also increased following a time-dependent pattern during the osteogenetic induction. BMSCs transfected with miR- 26a mimics changed from the fusiform shape into the polygonal shape after being cultured for 7 days, which was the same as the control group. ALP staining revealed that after 7 days, the ALP activity of BMSCs transfected with miR-26a mimics was higher than that of BMSC transfected with mimics control. Alizarin red staining showed that calcium deposits were more and bigger in experiment group compared with control group. The PCR showed that the expression of osteogenetic genes （ OCN, Runx-2 and Col-1 ） were higher in miR-26a mimics-tmnsfectinggroup. 31ae expression of miR-26a in vivo was 4 times lower in OVX group than in sham group. Conclusion miR-26a mimics can enhance os-
出处
《中国医科大学学报》
CAS
CSCD
北大核心
2012年第7期591-595,606,共6页
Journal of China Medical University
基金
国家基础研究项目(973项目)(2011CB964700)
