Mechanism of miR-26a-5p/cAMP response element binding protein 1 molecular axis regulating osteogenic differentiation of adipose-derived mesenchymal stem cells / 中国修复重建外科杂志

OBJECTIVE@#To investigate the regulatory effects of miR-26a-5p on the osteogenic differentiation of adipose-derived mesenchymal stem cells (ADSCs) by regulating cAMP response element binding protein 1 (CREB1).@*METHODS@#The adipose tissues of four 3-4 weeks old female C57BL/6 mice were collected and the cells were isolated and cultured by digestion separation method. After morphological observation and identification by flow cytometry, the 3rd-generation cells were subjected to osteogenic differentiation induction. At 0, 3, 7, and 14 days after osteogenic differentiation induction, the calcium deposition was observed by alizarin red staining, ALP activity was detected, miR- 26a-5p and CREB1 mRNA expressions were examined by real-time fluorescence quantitative PCR, and CREB1 protein and its phosphorylation (phospho-CREB1, p-CREB1) level were measured by Western blot. After the binding sites between miR-26a-5p and CREB1 was predicted by the starBase database, HEK-293T cells were used to conduct a dual-luciferase reporter gene experiment to verify the targeting relationship (represented as luciferase activity after 48 hours of culture). Finally, miR-26a-p inhibitor (experimental group) and the corresponding negative control (control group) were transfected into ADSCs. Alizarin red staining, ALP activity, real-time fluorescent quantitative PCR (miR-26a-5p) and Western blot [CREB1, p-CREB1, Runt-related transcription factor 2 (RUNX2), and osteocalcin (OCN)] were performed at 7 and 14 days after osteogenic induction culture.@*RESULTS@#The cultured cells were identified as ADSCs. With the prolongation of osteogenic induction culture, the number of calcified nodules and ALP activity significantly increased ( P<0.05). The relative expression of miR-26a-5p in the cells gradually decreased, while the relative expressions of CREB1 mRNA and protein, as well as the relative expression of p-CREB1 protein were increased. The differences were significant between 7, 14 days and 0 day ( P<0.05). There was no significant difference in p-CREB1/CREB1 between different time points ( P>0.05). The starBase database predicted that miR-26a-5p and CREB1 had targeted binding sequences, and the dual-luciferase reporter gene experiment revealed that overexpression of miR-26a-5p significantly suppressed CREB1 wild-type luciferase activity ( P<0.05). After 7 and 14 days of osteogenic induction, compared with the control group, the number of calcified nodules, ALP activity, and relative expressions of CREB1, p-CREB1, OCN, and RUNX2 proteins in the experimental group significantly increased ( P<0.05). There was no significant difference in p-CREB1/CREB1 between the two groups ( P>0.05).@*CONCLUSION@#Knocking down miR-26a-5p promoted the osteogenic differentiation of ADSCs by up-regulating CREB1 and its phosphorylation.

Anatomical locking compression plate augmented with anchor nail versus calvicular hook plate for Neer Ⅱb distal calvicular fractures / 中华创伤骨科杂志

Objective To compare the clinical efficacy of anatomic locking compression plate augmented with anchor nail versus calvicular hook plate in the treatmeut of Neer type Ⅱb distal calvicular fractures.Methods The data of 65 patients were retrospectively analyzed who had undergone surgery in our hospital from January 2012 to January 2015 for Neer type Ⅱb distal calvicular fractures.They were 39 men and 26 women,aged from 18 to 58 years(average,42.6 years).Open reduction plus anatonical locking compression plating augmented with anchor nailing was performed in 33 patients (group A) and open reduction plus calvicular hook plating was conducted in 32 (group B).The internal fixation was removed oue year after surgery in all.Constant Scale was used to evaluate shoulder function at 1,3 and 6 months postoperatively.X-ray examination on the shoulder was also conducted to observe fracture healing.Complications and percentage of the patients who resumed their job 3 months postoperatively were documented.Results The 2 groups were comparable because they showed no significant differences in general clinical information preoperatively (P ＞ 0.05).All the 65 patients were followed up tor 12 to 18 months (average,15.2 months).The fracture clinical healing time in group A (23.9 ± 2.3 weeks) was significantly shorter than in group B (26.1 ± 3.0 weeks) (P ＜ 0.05).The mean Constant-Murley scores at 1,3,6 months and the last follow-up postoperatively in group A (91.2 ±3.6,95.2 ±2.4,96.1 ±5.1 and 97.3 ± 1.6) were significantly higher thau those in group B (89.2 ± 6.1,91.1 ± 1.1,91.2 ± 6.2 and 92.1 ± 3.1) (P ＜ 0.05).The rate of total complications in group A (6.1％,2/33) was significantly lower than in group B (25.0％,8/32) (P ＜ 0.05).At postoperative 3 months,31 patients (93.9％) in group A resumed their job while 23 (71.9％) ones did in group B,showing a significant difference (P ＜ 0.05).Conclusions For Neer lⅡb distal calvicular fractures,anatomical locking compression plate augmented with anchor nail is obviously superior to calvicular hook plate,because the former can avoid damage to the soft tissue surrounding the acromion,leading to satisfactory functional recovery of the affected shoulder.