| The periodontal ligament stem cells(peridontal ligament stem cells,PDLSCs)isolated from human periodontal membrane with multi-directional differentiation potential of ectomesenchymal stem cells,a kind of belong to a kind of precursor cells,can differentiate for osteogenesis cells of bone cells,fibroblasts and into,in the teeth,periodontal tissue development and play an important role in tissue repair.People the periodontal ligament stem cells in the early 1980 s was the first to obtain from human periodontal membrane separation,Seo and others study confirms that the periodontal membrane is stem cells with multidirectional differentiation potential of stem cells,periodontal membrane,belongs to a kind of adult stem cells,can produce a certain phenotype and function of the mature cells,and play an important role in tissue injury and repair.In recent years,due to its ability to differentiate into osteoblasts,cementoblasts and fibroblasts under the stimulation of in vitro microenvironment,it has become a new stem cell in the oral field and is considered as an ideal seed cell for periodontal tissue regeneration.Akebia saponin D(Akebia saponin D,ASD)is a traditional Chinese medicinal herbs radix dipsaci,also implied active ingredient,also known as radix dipsaci saponins IV,ASD have clear pharmacological activities,in recent years is mainly used for the preparation of promoting fracture healing and treatment of osteoporosis drug,its clinical effect is safe and reliable,no obvious side effects to the human body.Cheng Zhian and Zhang Yunhui respectively by in vitro experiment,research has shown that also saponin D can obviously promote the osteoblast and rat bone marrow Mesenchymal stem cells,Mesenchymal stem cells,MSCs)proliferation and differentiation potential,ASD significantly increased the activity of osteoblasts,promote its calcification,so as to promote fracture healing effect of the prevention and treatment of osteoporosis.Yes-associated protein(Yes-associated protein,YAP)is a kind of collaborative transcription factor,YAP can move back and forth between the cytoplasm and nuclei,by itself does not include any DNA structure domain,but it is highly conservative in all kinds of biological ww domain structure,able to identify sequences that are rich in proline,enhance the effect of protein and protein between,YAP in promoting cell proliferation and inhibit apoptosis,and maintain dry cells play an important role in the process.YAP enters the nucleus and binds with corresponding proline-rich transcription factors to jointly start the transcription of its downstream target genes.When the downstream genes are activated,its main function is to inhibit cell apoptosis and promote cell proliferation.Studies have shown that overexpression of YAP gene in liver can significantly promote its proliferation and greatly increase the volume of liver.Recent studies have shown that YAP has a regulatory effect on the proliferation and differentiation of stem cells,and overexpression of YAP in embryonic stem cells and neural stem cells can promote their proliferation and enhance their self-renewal ability.In the abnormal pathological state,the weakening of the inhibitory factors on YAP will result in the abnormal increase of cell proliferation capacity and insufficient apoptosis,thus causing the occurrence of tumor.Purpose:In this study,on the basis of cultured human periodontal membrane stem cells in vitro,the effects of lignin D on biological behaviors of human periodontal membrane stem cells,such as cell cycle,phenotype maintenance and cell proliferation,were observed,and the related mechanisms were preliminarily explored,providing a new strategy and idea for periodontal tissue regeneration.Method:1.Isolated cultured human PDLSCs:human PDLSCs were isolated and cultured from healthy first and second premolar extracted for orthodontic treatment at the age of 12-18 years by tissue block method,and cells in good growth state for 3 to 6 generations were taken for follow-up experiments.2.Apoptosis detection:after PI staining with apoptosis kit,the effect of ASD on apoptosis of PDLSCs was detected by flow cytometry.3.Cell cycle detection:after PI staining with cell cycle kit,the influence of ASD on human PDLSCs cell cycle was detected by flow cytometry.4.Surface dryness detection:real-time quantitative PCR was used to detect the expression of Scleraxis and col-i,two factors related to the maintenance of surface dryness of human PDLSCs by ASD.5.Proliferation detection:real-time quantitative PCR was used to detect the effect of ASD on PCNA,Ki67 and CyclinDl,proliferation-related genes of PDLSCs.6.Study on the proliferation mechanism of human PDLSCs by ASD:the expression of YAP gene in human PDLSCs was targeted by small interfering RNA transfection,the transfection efficiency was detected by real-time quantitative PCR,and the effect of ASD on proliferation was observed by quantitative PCR.7.Statistical analysis;All data were analyzed using SPSS19.0 analysis software,and P<0.05 was considered statistically significant.Result:1.PDLSCs in good growth state were successfully isolated and cultured by tissue block method.2.Flow cytometry showed that ASD had no significant effect on the apoptosis of PDLSCs compared with the negative control group,and the results were statistically significant(P<0.05).3.Cell cycle detection by flow cytometry showed that ASD group significantly promoted the proliferation of human PDLSCs compared with the negative control group,with statistically significant results(P<0.05).4.Real-time quantitative PCR showed that the expression of Scleraxis,a surface dryness related factor,had been improved,but there was no significant change in Col-?.5.Real-time quantitative PCR showed that the expressions of proliferation-related factors Ki67 and CyclinDl increased significantly,while PCNA expression changed little,and the results were statistically significant(P<0.01).6.Transfection with shRNA significantly reduced the expression of YAP gene in PDLSCs,with statistically significant results(P<0.01).7.After reducing the expression of YAP gene in PDLSCs,ASD could no longer promote the proliferation of PDLSCs,and the results were statistically significant(P<0.01).Conclusions:1.Tissue block method can be used for the separation and culture of PDLSCs,and human PDLSCs in a good growth state can be obtained.2.Compared with the negative control group,ASD showed no significant cytotoxicity to human PDLSCs.3.ASD can promote PDLSCs to enter the M3 phase and promote cell division.4.ASD can promote the expression of Scleraxis,but its effect on Col-I is not obvious.5.ASD significantly promoted the proliferation of human PDLSCs,and significantly promoted the expressions of proliferation-related genes Ki67 and CyclinDl.With the addition of xylem saponin D for 24h,the expressions of the proliferation-related genes Ki67 and CyclinD1 reached the highest level,and the expressions decreased with time.The expression of PCNA was significantly promoted,and the change was less with the prolongation of dosing time.6.The si-RNA can effectively reduce the expression of YAP gene in PDLSCs,and the transfection efficiency is high.7.The proliferation of PDLSCs promoted by ASD may be related to YAP gene. |
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