Mechanism Of PNS Regulating Osteogenic Differentiation Of BMSCs By BMP-Smad Signaling Pathway

Objective1.To isolate and culture rabbit Bone Mesenchymal Stem Cells(BMSCs)in vitro,and to establish a stable rabbit BMSCs culture system after identification.And to investigate the osteogenic differentiation of BMSCs and the protein expression of bone morphogenetic protein 2 influenced by the traditional Chinese medicine Panax notoginseng saponins.2.To study the effect of PNS on the classical signaling pathway of BMP-Smad,and to explore the mechanism of PNS promoting osteogenic differentiation of BMSCs.Methods1.Isolation,culture and identification of rabbit BMSCs in vitro: under strict aseptic operation,long bones were harvested from the newborn New Zealand white rabbits and then washed bone marrow cavity with DMEM medium containing fetal bovine serum(Fetal Bovine Serum,FBS)and double antibody.Then collected bone marrow fluid and obtained BMSCs by density gradient centrifugation and whole bone marrow adherence method.(1)Observed morphological characteristics of BMSCs under an inverted microscope;(2)Detected surface marker antigens of BMSCs by flow cytometry;(3)Detected intracellular calcium nodules by alizarin red staining and identified osteogenic differentiation of rabbit BMSCs.2.Effect of PNS on osteogenic differentiation of rabbit BMSCs: Based on the previous study of the research group,rabbit BMSCs were cultured in the optimal concentration(100mg/l)PNS drug-containing medium,and the formation of l osteogenic index calcium ganglia was measured by alizarin red staining;Immunocytochemistry was used to detect the expression of BMP2 protein in culture medium,PNS medium and osteogenic induction medium at the time of 1 week.3.Rabbit BMSCs were cultured for 3 days,6 days and 12 days in the optimal concentration of PNS drug-containing medium.The osteogenesis-inducing group was set as the positive control group and the normal medium group was the negative control group.The positive expression of signal factor BMP2,receptor BMPRII and cytoplasmic signal transduction molecule Smad1 and nuclear transcription factor RunX2 were detected by immunofluorescence.Their protein expression and corresponding mRNA expression were detected by WB and qRT-PCR respectively.Results1.Isolation,cultivation,identification and osteogenic capacity test results of BMSCs in vitro.(1)Primary cells of rabbit BMSCs isolated and cultured by density gradient centrifugation are more pure.After 6 hours,the original generation of rabbit BMSCs with spindle shape fibroblast-like can be stick adherence at the bottom.After 48 hours,changed the liquid.When the cell colonies can be expanded and fused to each other after 7-9 days,reaching 80%-90% of the bottom area of the bottle,which can be passaged.After passage,the cells maintain a typical growth pattern and morphology,and the proliferation rateincreased.It can be seen that the typical morphology of rabbit BMSCs is long fusiform adherent fibroblast-like cells,which are arranged in a spiral shape and have a uniform morphology.(2)The positive rates of cell surface markers CD90 and CD105(adhesion molecules)were 83.21% and 89.48%,respectively,and the positive rate of HLA-DR(fibroblast surface markers)was 5.76%.(3)Under osteogenic induction culture,rabbit BMSCs were successfully induced into osteogenesis,and alizarin red staining showed positive calcium nodule staining.2.Under the optimal concentration of PNS medium,rabbit BMSCs could also be induced into osteogenesis.The alizarin red staining results showed positive staining of calcium nodules,the positive result was significantly stronger than the negative control group,but weaker than the positive control group;The results of immunocytochemistry showed that the staining results of BMP2 were strongly positive in the culture of PNS medium and osteogenic induction solution,while the staining results in the normal medium group were negative.3.The results of immunofluorescence showed that BMSCs express BMP2 in the cytoplasm,express BMPRII on the cell membrane,and express Smad1 and RunX2 in the cytoplasm and nucleus.The results of qRT-PCR of BMP2,BMPRII,Smad1 and RunX2 were basically the same.The results showed that there was no significant change in mRNA expression of the four indicators in the normal medium group at 3 days,6 days and 12 days(P > 0.05).The expression of mRNA in the PNS medium group and the osteogenic induction group increased gradually with time(P < 0.05).At 6 days and 12 days,the mRNA expression of the four indexes in the PNS medium group and the osteogenic induction group was significantly higher than that in the normalmedium group.At different time points,the mRNA expression of the four indexes in the PNS medium group was Lower than the osteogenic induction group(P < 0.05).The WB detection results of BMP2,BMPRII,Smad1 and RunX2 were basically consistent with the results of qRT-PCR.Conclusions1.PNS can promote osteogenic differentiation and the expression of BMP2 in BMSCs.2.PNS can achieve its osteogenic effect by promoting the expression of the signaling factor BMP2,the corresponding receptor of BMP2 and the activation of the classical signaling pathway of BMP-Smad.