| Objective: Distraction Osteogenesis(DO)is an endogenous bone tissue engineering technology that uses the callus healing mechanism to rapidly generate new bone tissue in the distraction gap.The new bone formation rate of DO not only leads infants and young children at the peak of growth and development,but also surpasses the growth rate of malignant tumors.However,the long course of DO treatment and complications limit the development of DO.How to improve the speed and quality of DO new bone formation,reduce complications and effectively shorten the course of DO treatment is an urgent problem to be solved.In the previous study,the research team confirmed that Panax Notoginseng Saponins(PNS)could upregulated transforming growth factor β1(Transforming Growth Factor-β1,TGF-β1)expression in rabbit bone marrow mesenchymal stem cells(Rabbit Bone Mesenchymal Stem Cells,r BMSCs)and distraction gap in DO,promoted the osteogenic function of r BMSCs and the quality of DO new bone formation.TGF-β1 signaling plays an important role in embryonic bone development and body bone homeostasis.Exploring the mechanism of PNS regulating TGF-β1 signaling will provide a theoretical basis for clinical application of PNS to promote DO treatment.Method:1.Regulatory mechanism of PNS promoting osteogenic function of r BMSCs via TGF-β1/Smads signal transduction: isolated and cultured primary r BMSCs.The identification of r BMSCs was accomplished by cytomorphological observation,flow cytometry detection of CD44,CD90,CD105,CD34,CD45 and HLA-DR surface marker positive rate and alizarin red staining.Three experimental groups were established,a normal control group(NC),a PNS induction group(P),and a positive control group(PC),and cells were treated according to the culture conditions of each group.Cell Counting Kit-8(CCK-8)method was used to draw the growth curve of each group of cells and observe the morphological changes of r BMSCs,and to detect the effect of PNS on the proliferation of r BMSCs.Each experimental group was induced by Alizarin Red Staining(ARS)after 21 days of induction to detect the effect of PNS on the osteogenic function of r BMSCs.Flow cytometry was used to detect the cell cycle changes after 8 days of induction in each experimental group,and the effect of PNS on the biological function of r BMSCs was detected.The expression and location of TβR-Ⅱ,Smad2,Smad3,PPM1 A,Ran BP3 in r BMSCs were detected by immunofluorescence.Quantitative Real-time PCR(q RT-PCR)and WesternBlot(WB)were used to detected the TβR-Ⅱ,Smad2,Smad3,PPM1 A,Ran BP3 expression by which PNS regulated TGF-β1/Smads signaling and promoteda the osteogenic function of r BMSCs after 3d,6d,and 12 d induction in each experimental group;2.Overexpressed TβR-Ⅱ in r BMSCs to explored the mechanism of PNS regulated osteogenic function of r BMSCs through TGF-β1/Smads signal transduction: constructed TβR-Ⅱ overexpression vector mediated by lentivirus.Selected the optimal multiplicity of infection(MOI)of r BMSCs.q RT-PCR and WB methods were used to verify the overexpression efficiency of TβR-Ⅱ.Four experimental groups were set up: normal control group(NC),PNS induction group(P),PNS induction negative overexpression virus control group(P+LvControl)and PNS induction overexpression virus group(P+Lv-TβRⅡ).CCK-8method was used to detected the effect of virus on the proliferation of r BMSCs.ARS method was used to detected the effect of overexpression of TβR-Ⅱ on the osteogenic function of r BMSCs.q RT-PCR and WB methods were used to detected the expression changes of TβR-Ⅱ,Smad2,Smad3,PPM1 A and Ran BP3 in each experimental group after 12 days cultured;3.Knocked-down TβR-Ⅱ in r BMSCs to explored the mechanism of PNS regulated osteogenic function of r BMSCs through TGF-β1/Smads signal transduction: constructed TβR-Ⅱ knock-down vector mediated by lentivirus.Selected the optimal MOI of r BMSCs.q RT-PCR and WB methods were used to verify the knock-down efficiency of TβR-Ⅱ.Four experimental groups were set up: normal control group(NC),PNS induction group(P),PNS induction negative knock-down virus control group(P+Lv-sh Control)and PNS induction knockdown virus group(P+Lv-sh TβRⅡ).CCK-8 method was used to detected the effect of virus on the proliferation of r BMSCs.ARS method was used to detected the effect of overexpression of TβR-Ⅱ on the osteogenic function of r BMSCs.q RTPCR and WB methods were used to detected the expression changes of TβR-Ⅱ,Smad2,Smad3,PPM1 A and Ran BP3 in each experimental group after 12 days cultured;4.Osteogenesis promoting mechanism of PNS in rabbit mandibular DO model via TGF-β1/Smads signal transduction: 36 healthy adult rabbits were randomly divided into six experimental groups after established DO model:normal control group(NC);PNS intramuscular group(P);matrigel control group(P+Matrigel);BMSCs control group(P+BMSCs);TβR-Ⅱ negative virus control group(P+Lv-Control);TβR-Ⅱ overexpression virus group(P+Lv-TβRⅡ).Each experimental group was divided into 0-week consolidation group and 2-weeks consolidation group according to consolidation time.During DO period,the experimental rabbits were treated according to the treatment methods of each group,and specimens of the distraction gap were obtained.CBCT was used to detected whether the distraction gap was opened.Scanning electron microscopy was used to examined the histological changes of the dsitraction gap under the action of PNS and overexpression of TβR-Ⅱ.Energy spectrometer analysis was used to detect the mass ratio of calcium to phosphorus in the distraction gap of each group.q RT-PCR and WB methods were used to detected the changes of TβR-Ⅱ,Smad2,Smad3,PPM1 A,and Ran BP3 expression in the distraction gap of each group,and explored the mechanism of PNS promoting the formation of new bone via TGF-β1/Smads signal transduction.Result: 1.The mechanism of PNS regulating the osteogenic function of r BMSCs through TGF-β1/Smads signaling: the morphology and proliferation characteristics of isolated cultured cells are consistent with r BMSCs.The cells highly expressed CD44,CD90,CD105,and lowly expressed CD34,CD45,and HLA-DR,which was consistent with the surface marker characteristics of r BMSCs.The cells showed calcium nodules via ARS,indicating that they had osteogenic differentiation ability.It was confirmed that the obtained cells were r BMSCs.The results of CCK-8 method showed that PNS had no significant effect on the proliferation of r BMSCs(P>0.05).After 8 days of induction of r BMSCs by PNS,osteoblast-like morphological changes appeared in r BMSCs.At the same time,the cell cycle results suggested that PNS blocked r BMSCs in G1 phase at 8days and promoted BMSCs differentiation.21 days after PNS induction,ARS showed that the number of calcium nodules was PC>P>NC(P<0.05),and the osteogenic differentiation function of r BMSCs was improved.Immunofluorescence results showed that r BMSCs expressed TβR-Ⅱ on the cell membrane,Smad2 and Smad3 in the cytoplasm and nucleus,and PPM1 A and Ran BP3 in the nucleus.The results of q RT-PCR and WB were consistent,showing that the expression levels of TβR-Ⅱ,Smad2,and Smad3 at three time points of 3d,6d,and 12 d were PC>P>NC(P<0.05),and increased with the induction time The expression level gradually increased(P<0.05),while the expression levels of PPM1 A and Ran BP3 were PC<P<NC(P<0.05),and the expression level gradually decreased with time(P<0.05);2.Overexpressed TβR-Ⅱ in r BMSCs to explored the mechanism of PNS regulated osteogenic function of r BMSCs through TGF-β1/Smads signal transduction: constructed the lentiviral overexpression vector Lv-TβRⅡ.Selected MOI=10 as the optimal MOI.q RT-PCR and WB experiments confirmed the overexpression efficiency of TβR-Ⅱ.CCK-8 method showed that the virus had no significant effect on r BMSCs proliferation(P>0.05).ARS results showed that overexpression of TβR-Ⅱ did not affected the osteogenic differentiation function of r BMSCs,and the number of calcium nodules was NC<P=P+Lv-Control<P+Lv-TβRⅡ(P<0.05).The results of q RT-PCR and WB showed that after overexpression of TβR-Ⅱ,the expression levels of TβR-Ⅱ,Smad2 and Smad3 in r BMSCs were up-regulated(P<0.05),while the expression levels of PPM1 A and Ran BP3 were down-regulated(P<0.05);3.Knock-down TβR-Ⅱ in r BMSCs to explored the mechanism of PNS regulated osteogenic function of r BMSCs through TGF-β1/Smads signal transduction: constructed the lentiviral overexpression vector Lv-sh TβRⅡ.Selected MOI=10 as the optimal MOI.q RT-PCR and WB experiments confirmed the knock-down efficiency of TβR-Ⅱ.CCK-8 method showed that the virus had no significant effect on r BMSCs proliferation(P>0.05).ARS results showed that knock-down of TβR-Ⅱ did not affected the osteogenic differentiation function of r BMSCs,and the number of calcium nodules was P+Lv-sh TβRⅡ<NC<P=P+Lvsh Control(P<0.05).The results of q RT-PCR and WB showed that after knockdown TβR-Ⅱ,the expression levels of TβR-Ⅱ,Smad2 and Smad3 in r BMSCs were down-regulated(P<0.05),while the expression levels of PPM1 A and Ran BP3 were up-regulated(P<0.05);4.Osteogenesis promoting mechanism of PNS in rabbit mandibular DO model via TGF-β1/Smads signal transduction: constructed rabbits right mandible DO models in each group.CBCT showed that the distraction gap of each group had been opened.Gross specimens and X-ray examination showed that the new bone-like texture in the distraction gap of each group was soft and filled with connective tissue at 0 week of consolidation,and the connective tissue was replaced by new bone tissue in the distraction gap at 2 weeks of consolidation.Scanning electron microscopy and energy spectrometer examination results showed that PNS could promoted the calcium deposition of new bone tissue and ultimately improved the quality of osteogenesis in the distraction gap.Overexpressed TβR-Ⅱ in the distraction gap could further promoted the new bone formation effects of PNS on the distraction gap.q RT-PCR and WB experiments showed the expression of TβR-Ⅱ,Smad2,Smad3,PPM1 A,and Ran BP3 in the new bone tissue in the distraction gap of each group(P<0.05),and the highest in P+Lv TβRⅡ group(P<0.05),the expression levels of PPM1 A and Ran BP3 were P<NC(P<0.05),and the lowest in P+Lv TβRⅡ group(P<0.05).Moreover,the expressions of TβR-Ⅱ,Smad2 and Smad3 gradually decreased with the extension of fixed time(P<0.05),and the expressions of PPM1 A and Ran BP3 gradually increased with the extension of fixed time(P<0.05).Conclusion:1.PNS could up-regulated the expression of TβR-Ⅱ in r BMSCs,activated the TGF-β1/Smads signal pathway,up-regulated the expression of Smad2 and Smad3,promoted TGF-β1/Smads signal transduction,inhibited the expression of PPM1 A and Ran BP3,and reduced the termination of TGF-β1/Smads signaling.Thereby promoted the osteogenic differentiation of r BMSCs and enhanced the osteogenic function of r BMSCs;2.Overexpressed TβR-Ⅱ without affected the function of r BMSCs,could upregulated TβR-Ⅱ expression,further activated TGF-β1/Smads signal pathway,upregulated Smad2 and Smad3 and down-regulated the expression of PPM1 A and Ran BP3,further promoted TGF-β1/Smads signal transduction enhanced the osteogenic differentiation of r BMSCs by PNS;3.Knocked-down TβR-Ⅱ without affected the function of r BMSCs,could down-regulated the expression of TβR-Ⅱ,inhibited the TGF-β1/Smads signal pathway,down-regulated Smad2 and Smad3 and up-regulated the expressions of PPM1 A and Ran BP3,further inhibited TGF-β1/Smads signal transduction and reduced the osteogenic differentiation of r BMSCs by PNS;4.PNS could up-regulated the expression of TβR-Ⅱ,Smad2 and Smad3,down-regulated the expression of PPM1 A,Ran BP3 in DO distraction gap,activated TGF-β1/Smads signal pathway,and improved the rates and qualities of new bone formation by promoted TGF-β1/Smads signal transduction. |
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