摘要: Objectives: To investigate the mechanism of mechanical stimulation in bone formation and regeneration during distraction osteogenesis. Materials and methods: In this study, microarray technology was used to investigate the time course of bone-related molecular changes in distraction osteogenesis in rats. Real-time PCR and Western-blot analyses were used to confirm the expression of genes identified in microarrays. Meanwhile, we used a lentivirus vector to inhibit Fak expression, in order to identify the osteogenic effect of Fak and Fak-Mapk pathway during distraction osteogenesis. Results: Several components of the Wnt and Hippo pathways were found to be up- or down-regulated during distraction osteogenesis by microarray. Meanwhile, it was found that Fak, Src, Raf-1, Erk1, Jnk and p38-Mapk were up-regulated during gradual distraction, compared with consolidation. To further determine whether Fak-Mapk pathway played an important role in distraction osteogenesis, Fak was disrupted with a lentivirus vector. The expressions levels of p-Fak, p-Erk1/2, p-JNK and p-p38Mapk were decreased. Meanwhile, a poor early and late osteogenesis effect was found in the shRNA-Fak group. Conclusion: It was inferred that the mechanical stimulus induces increased expression of Fak and activates Fak-Mapk pathway, by activation of Erk, Jnk and p38-Mapk pathway, and that Fak at least, in part, plays an important role in maintaining osteogenic effect by activating Fak-Mapk pathway during distraction osteogenesis.

摘要: Objectives The sonic hedgehog (Shh) signalling pathway has an important role in the maintenance of various stem cells and organogenesis during development. However, the effect of Shh in skin-derived precursors (SKPs), which have the capacity for multipotency and self-renewal, is not yet clear. The present study investigated the effects of the Shh signalling pathway on the proliferation and self-renewal of murine SKPs (mSKPs). Methods The Shh signalling pathway was activated by treatment with purmorphamine (Shh agonist) or recombinant Shh in mSKPs. Cyclopamine (Shh antagonist) or GANT-61 (Gli inhibitor) was used to inhibit the pathway. Western blot, qPCR, and immunofluorescence were used to analyse the expression of genes related to self-renewal, stemness, epithelial-mesenchymal transition (EMT) and the Shh signalling pathway. In addition, cell proliferation and apoptosis were examined. Results Inhibiting the Shh signalling pathway reduced mSKP proliferation and sphere formation, but increased apoptosis. Activating this signalling pathway produced opposite results. The Shh signalling pathway also controlled the EMT phenotype in mSKPs. Moreover, purmorphamine recovered the self-renewal and proliferation of aged mSKPs. Conclusion Our results suggest that the Shh signalling pathway has an important role in the proliferation, self-renewal and apoptosis of mSKPs. These findings also provide a better understanding of the cellular mechanisms underlying SKP self-renewal and apoptosis that allow more efficient expansion of SKPs.

摘要: Objectives: Glucagon-like peptide 2 (GLP2) is involved in the regulation of energy absorption and metabolism. Despite the importance of the GLP2 signalling mechanisms on osteoclast, little has been studied on how GLP2 works during osteoclastogenesis. Materials and Methods: RAW264.7 cells were infected with rLV-Green-GLP2. The induction of osteoclasts was performed by RANKL. TRAP were detected by RT-PCR, Western blotting and staining. Total nitric oxide and total NOS activity were measured. Cells apoptosis was detected by Hoest33258 and Annix V staining. Animal test, chromatin immunoprecipitation (CHIP), co-immunoprecipitation(IP) and luciferase reporter assay were also performed. Results: We indicate that GLP2 is associated with osteoporosis-related factors in aged rats, including BALP, TRAP, IL6, TNF alpha, Nitric Oxide (NO), iNOS, calcitonin and occludin. Moreover, GLP2 is demonstrated to result in negative action during proliferation of tartrate-resistant acid phosphatase-positive (TRAP+) osteoclasts. Furthermore, GLP2 decreases osteoclasts induced from monocyte/macrophage cells RAW264.7 as well as the serum TRAP activity in aged rats. Mechanistic investigations reveal GLP2 enhances the expression of iNOS through stimulating the activity of TGF beta-Smad2/3 signalling in osteoclasts. In particular, inhibition of TGF beta fully abrogates this function of GLP2 in osteoclasts. Strikingly, overexpression of GLP2 significantly increases the product of nitric oxide via iNOS which promotes apoptosis of osteoclasts by decreasing bcl2 or increasing caspase3. Thereby, the ability of GLP2 to regulate apoptosis depends on TGF-Smad2/3-iNOS-NO signalling pathway since total NOS inhibitor L-NMMA specifically inhibits the actions by GLP2. Conclusions: GLP2 induces apoptosis via TGF beta-Smad2/3 signalling, which contributes to the inhibition of the proliferation of osteoclasts and which may provide potential therapeutic targets for the treatment of osteoporosis.

摘要: Objectives Dysregulation of YAP by the Hippo signalling is associated with intervertebral disc degeneration (IDD). However, the relationship between the F-actin and Hippo pathway in IDD, and their effects on YAP remain poorly understood. Methods The characteristics of Hippo pathway and F-actin the in the NP (nucleus pulposus) and annulus fibrosus of immature, mature, ageing and disc degeneration model rats were observed by immunofluorescence, western blot and qPCR. Nucleus pulposus cells (NPCs) were transfected with lentivirus Sh-LATS A, Sh-LATS B and harvested for SA-beta-gal staining, qPCR, western blotting and immunofluorescence staining to investigate the mechanism of Hippo pathway and F-actin interact in NPCs. Results We observed moderate decreases in F-actin and YAP expression with age in healthy intervertebral discs (IVDs). F-actin stress fibres distributed throughout the cytoplasm disappeared following treatment with latrunculin B (Lat B), resulting in a punctate distribution. Depletion of large tumour suppressor homologues 1/2 (LATS1/2) did not decrease the rate of cellular senescence, and YAP remained in the cytoplasm following Lat B treatment. Furthermore, angiomotin 130 (AMOT130) was associated with F-actin through a conserved actin-binding domain to retain YAP in the cytoplasm. Conclusions This study showed that a mechanism by which Hippo pathway and F-actin synergize to modulate YAP activation and localization in the context of IDD and help to control NPCs proliferation.

摘要: ObjectivesThe purpose of this study was to explore the effectiveness of concurrent GRP78 overexpression combined with Cripto on hMSC proliferation and migration both in vitro and in vivo. Specifically, we explored whether the treatment enhances effectiveness of hMSC transplantation in ischaemic tissue. Materials and methodsHuman MSCs obtained from human adipose tissue were cultured in -minimum essential medium (Hyclone, Logan, UT, USA) supplemented with 10% (v/v) foetal bovine serum (Hyclone), 100UmL(-1) penicillin and 100gmL(-1) streptomycin. Murine hindlimb ischaemic model was generated with 8-week-old male nude BALB/c mice (Biogenomics, Seoul, Korea) maintained under a 12-h light/dark cycle following the established protocol with minor modification. Cellular injection was performed no later than 3hour after surgery. Lipofectamine transfection, single-cell cultivation assay, transwell assay, scratch wound-healing migration assay, immunohistochemistry and western blotting assays were performed. ResultsOverexpression of GRP78 along with Cripto enhanced hMSC proliferation, migration and invasion. It increased interaction of surface GRP78 receptor with Cripto via JAK2/STAT3 pathway. We confirmed our proposed mechanism by showing that treatment with GRP78 antibody blocks the enhancement in vitro. In vivo, we observed that Cripto induced by the hypoxic environment in hindlimb ischaemic model interacts with the overexpressed GRP78 and increases hMSC proliferation, migration and invasion potentials as well as angiogenesis around transplanted ischaemic site via cytokine secretions. ConclusionsThese results demonstrate supporting evidences that GRP78-Cripto combination technique offers novel strategy to enhance MSC proliferation, migration and invasion potentials as well as angiogenesis around ischaemic site, ultimately facilitating MSC-based transplantation therapy in ischaemic conditions.