| Objective:To explore whether mi RNAs and their predicted target genes with significant expression differences in the early stages of distraction osteogenesis and fracture have a regulatory effect on the osteogenic differentiation of canine BMSCs,and to provide a theoretical basis for elucidating the rapid osteogenesis mechanism of distraction osteogenesis.Methods:1.In vitro isolation,culture and identification of canine BMSCs:Under strict aseptic operation,the tibia bone marrow blood of puppies was drawn,and BMSCs were obtained by density gradient centrifugation.Identification methods:(1)Observe the morphological characteristics of BMSCs under the microscope(2)Detect the surface marker antigen of BMSCs by flow cytometry(3)Use alizarin red staining test to detect the formation of intracellular calcium nodules,and to detect the osteogenic differentiation ability of the cells.2.Screening of mi RNA and target genes:2.1 The mi RNAs in the healing tissues of the dog distraction group and dog fracture group were extracted,and the top 10 mi RNAs with down-regulated expression in the previous high-throughput sequencing results of the research group were screened to verify the differences in relative expression levels in the tissues.BMSCs were cultured in vitro and divided into normal group(N)and osteogenic induction group(B).After cultured 1,3,5 and 7 days,each gruop‘s mi RNAs were extracted.Further screening of mi RNAs related to osteogenic differentiation was conducted to study the mechanism of osteogenic differentiation.2.2 Combined with bioinformatics analysis and literature review,the target genes of mi R-505 involved in osteogenesis were predicted.The mi RNA was overexpressed or suppressed by lentivirus transfection,and divided into 4groups:(1)overexpression group(505+),(2)overexpression’s lentiviral empty vector group(505+NC),(3)suppression group(505-)(4)suppression’s lentivirus empty vector group(505-NC).Whether the expression of the above predicted target genes was affected by the mi RNA was detected.Finally,dual luciferase reporter gene assay was used to verify the results.3.The effect of mi R-505 targeting CRTC1 on the osteogenic differentiation of BMSCs:The mi RNA was overexpressed or suppressed by lentivirus transfection,and divided into four groups:(1)overexpression group(505+),(2)overexpression’s lentiviral empty vector group(505+NC),(3)suppression group(505-)(4)suppression’s lentivirus empty vector group(505-NC).q RT-PCR,WB and alizarin red staining were used to detect the expression differences of target genes and OCN in each group,and the effect of mi RNA on osteogenic differentiation ability of canine BMSCs.CCK8 and Transwell experiments were used to detect the effects of mi RNA on the proliferation and migration of BMSCs.Results:1.In vitro isolation,culture and identification results of BMSCs:1.1 The medium of primary cells of the canine BMSCs cultured by density gradient centrifugation were changed for the first time after 24 hours.After 48 hours,the bottles were adhered to the wall and grew in the form of short spindle colonies.After 5-7 days,the cell colonies expanded and merged.Mutual fusion,reaching 80%-90% of the bottom area of the bottle,then it can be passaged.After passage,the cells maintain the typical growth mode and morphology.The typical morphology of BMSCs is long spindle-shaped cells,which grow adherently,and the cells as a whole were arranged in a spiral shape with uniform morphology.1.2 The positive rates of cell surface markers CD90 and CD44 detected by flow cytometry were 97.9% and 98.0%,respectively,while the positive rates of CD45 and CD34 were 0.39% and 2.11%,respectively.It shows that the purity of canine BMSCs isolated in this experiment is relatively high.1.3 The canine BMSCs can be successfully induced to the osteogenic direction under the culture of osteogenic induction medium.Alizarin red staining test shows positive calcium nodule staining.2 Screening results of mi RNA and target genes:2.1 Among the top 10 mi RNAs whose expression was down-regulated in the results of the research group’s early high-throughput sequencing results,the expression differences of mi R-505 and mi R-551 b were the most significant.2.2 Divide BMSCs into normal group(N)and osteogenic induction group(B).They were cultured for 1,3,5 and 7 days.The expression of mi R-505 had the most significant difference.2.3Using bioinformatics methods and combining literatures review,predict that CRTC1,E2F2,and WNT10 B are possible osteogenic target genes of mi RNA-505.2.4 Using lentiviral transfection to overexpress or inhibit mi R-505,divided into 4 groups:(1)overexpression group(505+),(2)overexpression’s lentiviral empty vector group(505+NC),(3)suppression group(505-)(4)suppression’s lentivirus empty vector group(505-NC).The results of q RT-PCR showed that overexpression of mi R-505 decreased the expression of CRTC1,and inhibition of mi R-505 increased the expression of CRTC1,with statistical differences.The expression of E2F2 and WNT10 B was not statistically different or had no significant correlation with mi R-505.The dual luciferase reporter gene detection experiment showed that mi R-505 can target CRTC1.3 The results of mi R-505 targeting CRTC1 on the osteogenic differentiation of BMSCs:3.1 Overexpression or inhibition of mi R-505 using lentiviral transfection,divided into 4 groups:(1)overexpression group(505+),(2)overexpression lentiviral empty vector group(505+NC),(3)inhibition group(505-)(4)Suppress the lentivirus empty vector group(505-NC).The results of q RT-PCR showed that the expression of osteogenic differentiation-related gene OCN decreased after overexpression of mi RNA-505,and the expression of osteogenic differentiation-related gene OCN increased after mi RNA-505 was inhibited(P<0.05).WB results showed that when mi R-505 was overexpressed,the expression of CRTC1 and OCN protein decreased;on the contrary,when mi R-505 was inhibited,the expression of CRTC1 and OCN protein increased.The results of Alizarin Red showed that when mi R-505 was inhibited,calcium nodules were more numerous and distributed widely.In summary,after inhibiting the expression of mi R-505,the osteogenic differentiation ability of BMSCs was significantly improved.3.2 The results of CCK8 and Transwell showed that mi R-505 had no significant effect on the proliferation and migration of canine BMSCs.Conclusions:1 Among the top 10 mi RNAs that were down-regulated in the early stage of mandibular distraction compared with early stage of fracture in dogs,mi R-505 proved to be significantly associated with osteogenic differentiation at the tissue and cell levels.At the same time,CRTC1 is predicted to be a target gene related to mi R-505 osteogenesis.2 mi R-505 can negatively regulate the expression of CRTC1 at the gene and protein level,while inhibiting mi R-505 can improve the osteogenic differentiation of canine BMSCs.mi R-505 may affect the osteogenic differentiation of canine BMSCs by targeting CRTC1.3 mi R-505 has no significant effect on the proliferation and migration of canine BMSCs. |
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