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.

Early effectiveness of arthroscopic three-point suture technique in treatment of anterior cruciate ligament tibial eminence avulsion fracture / 中国修复重建外科杂志

Objective: To investigate the early effectiveness of three-point suture technique in treatment of anterior cruciate ligament (ACL) tibial eminence avulsion fracture by arthroscopy. Methods: Between January 2016 and December 2017, 12 patients with ACL tibial eminence avulsion fractures underwent arthroscopic fixation of avulsion fractures with Ethibon suture using three-point suture technique. There were 9 males and 3 females, with an average of 36.4 years (range, 18-50 years). The fracture caused by traffic accident in 10 cases and sports in 2 cases. Among them, 1 patient was old fracture and 11 was fresh fracture. According to the modified Meyers-McKeever classification criteria, the fractures were rated as type Ⅲ in 7 cases and type Ⅳ in 5 cases. There were 2 cases of medial collateral ligament injury and medial meniscus injury. The preoperative International Knee Documentation Committee (IKDC) score was 37.9±4.7 and Lysholm score was 46.0±3.7. Results: All operations completed smoothly. The operation time was 45-70 minutes (mean, 61.3 minutes). The incisions healed by first intention in all patients. The hospitalization stays ranged from 4 to 9 days (mean, 5 days). All patients were followed up 3-20 months (mean, 9.3 months). The anterior drawer test, Lachman test, and axis shift test in all patients were negative after operation. At last follow-up, the IKDC score was 89.7±2.5 and Lysholm score was 90.2±1.9, which were significantly higher than those before operation ( t=-30.94, P=0.00; t=-33.03, P=0.00). At last follow-up, the X-ray films showed 9 cases of fracture anatomical reduction and 3 cases of almost anatomical reduction, and 12 cases of fracture healing. Conclusion: For ACL tibial eminence avulsion fracture, arthroscopic three-point suture technique can effectively restore the stability of knee joint and obtain satisfactory early effectiveness.