| Nowadays,the oral implantation of the patients with alveolar defects or bone deficiency,especially those with bone metabolism diseases such as T2 DM,has become one of the most worrisome parts in dental care,considering the poor osteointegration and the low success rate.Fortunately,stem cells working as the seed cells in bone tissue engineering turn out to be a promising solution for the problem,thus becoming the research focus in recent years.Among the various candidate seed cells,ADSCs has been regarded as the most hopeful one for its unique advantages,such as abundant resources,easy accesses and few ethics limitations.However,affected by its intrinsic property,ADSCs tend to differentiate towards adipocytes rather than osteoblast,and,consequently,it's how to improve the osteogenesis of ADSCs that become the key point to address the clinical issue in term of implantation patients with poor alveolar conditions.The new osteo-protection factor Sema3 A participates in the regulation of the bone metabolism and promotes the osteogenesis process of osteoblast and its progenitor cells(such as ADSCs).This study treated the mouse ADSCs with Sema3 A recombinant protein,observed the timeeffect characteristics,and analyze the role of Notch signaling pathways in the process to explore the underlying mechanism and thus provide a scientific basis for the clinical application of Sema3A-modified ADSCs in the oral implantation field.ObjectiveThe aim of this study is to explore the potential mechanism of the osteogenesis promotion effect of Sema3 A on ADSCs and thus provide an experimental basis for its application in clinical dental care,by observing traits and characteristics in terms of time-related effects of Sema3 A on the osteoblast differentiation process of ADSCs and analyzing the role of Notch signaling pathway in the process with GSI.Methods1.The ADSCs were harvested from adipose tissues of C57BL/6 mouse by enzyme digestion with type?collagenase,and were identified through morphological observation,multi-lineage differentiation induction and surface markers detection.2.The expression profiles of Sema3 A and its receptor Nrp1 were measured by qPCR,Western Blot and fluorescent immunocytochemistry.And while the effect of Sema3 A on the osteoblast differentiation of ADSCs was determined by qPCR for osteogenesis related genes,Alizarin Red staining for calcium deposits and ALP staining for ALP activity,the priority was put on the traits and characteristics in terms of time-related effects and the initiation effects.3.The effect of the Notch signaling pathway on the stem cell property of ADSCs was analyzed by qPCR and Western Blot for stem property-related genes after the block of Notch signaling with GSI.The expression profiles of the genes involved in the Notch signaling was also detected by qPCR.And the osteogenesis of ADSCs with Notch signaling blocked by GSI was observed via qPCR for bone-related genes and ALP for ALP activity,Sirius Red staining for collagen secretion,Alizarin Red Staining for calcium deposits respectively after osteogenesis inducing for 7,14,21 days.The effect of Sema3 A recombinant protein on the expression of Notch-targeted genes and NICD was measured by qPCR and fluorescent immunocytochemistry.And the osteogenesis promotion effect of Sema3 A with blocked Notch signaling was observed by determining the osteoblastdifferentiation of the ADSCs treated with Sema3 A or GSI or both Sema3 A and GSI.Results1.The ADSCs with good multiple differential potential were obtained.2.Both Sema3 A and Nrp1 were expressed by ADSCs.And the expression level of Sema3 A increased significantly in initial stage of osteogenic differentiation,while the expression of Nrp1 remained a constant level.The longer and the earlier Sema3 A interfered in,the better ADSCs differentiated into osteoblast.Indeed,Sema3 A decreased the expression of stemness-related genes and launched the osteogenic process of ADSCs cultured in growth medium directly.3.The expression level of stemness-related genes decreased notably once the Notch signaling in ADSCs was blocked by GSI.The main genes involved in Notch signaling in ADSCs include Notch2,Jag1 and Hes1,and all these genes presented a lower level at the early stage of ADSCs' osteogenic differentiation.The osteogenic potential differed when ADSCs was treated with GSI for different time,that is to say an enhanced osteogenesis presented when GSI intervened for the first 7 days,while impaired osteogenesis was more likely to be induced by longer treatment.4.The treatment with Sema3 A recombinant protein decreased the Notch signaling in ADSCs.Compared with the treatment of either Sema3 A or GSI,the recombination of the both didn't result in an enhanced osteogenesis.Conclusion1.Sema3 A plays an active role throughout the whole process of osteogenic differentiation of ADSCs,while it's in the initial stage where Sema3 A exerts its effect majorly.And the Sema3 A can even start the osteogenic differentiation of ADSCs directly.2.Sema3 A plays its role during osteogenic process of ADSCs by inhibiting Notch signaling,and thus lowering the stemness property of ADSCs. |
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