The Effects Of LPS On The Proliferation And Osteogenic Differentiation Of Human Bone Marrow Mesenchymal Stem Cells And The Study On The Related Mechanisms

In recent years,with more and more research on adult stem cells,it has found that MSCs play a very important role in the field of tissue regeneration and wound repair.Bone marrow mesenchymal stem cells,as a kind of adult stem cells in bone marrow tissue,own high ability of self-renewal and multi-directional differentiation potential.AS BMSCs exist widely in the body and have small damage to the body,be easy to osteoblast differentiation and osteogenesis characteristics of stable performance,BMSCs are the ideal seed cells for the treatment of bone nonunion.AS BMSCs are widely used in clinical treatment,it had found that the proliferation and osteogenesis ability are the important factors influencing the clinical curative effect.The inflammatory microenvironment of the parts which stem cell transplant is the key factors affecting the effect of transplantation and bone regeneration.Microenvironment of inflammatory molecules are often divided into two categories,a class of exogenous molecules such as LPS,wear particles,another kind is endogenous inflammatory mediators such as TNF-a,PGE2,etc.The process of fracture healing,especially for open fractures,low immunity,fixation,used to encounter infection.as infection can not get effective control for a long time,it can be infectious bone nonunion.Infectious bone bone nonunion is not even the most difficult of all types of processing category.Fracture end necrosis infection as well as the nutrient vessels occlusion,the callus formation of the normal process,bone absorption or dead bone formation,leading to bone nonunion.Gram-negative bacteria for infectious bone is not even one of the most common pathogenic bacteria.Bacterial endotoxin lipopolysaccharide(LPS)is the main pathogenic factor for gram-negative bacteria,gram-negative bacteria release large amounts of LPS during the process of proliferation or collapse.Clinical infectious bone nonunion surrounding tissues can be detected a certain amount of LPS.Transplanted BMSCs into bone nonunion site,will inevitably contact with LPS and works.Therefore,in the process of bone regeneration,endotoxin environment influence on the proliferation and osteogenesis differentiation of BMSCs and its mechanism need further research.This study adopts the method of whole bone marrow adherent and limited dilution method and purified BMSCs were isolated and cultured,LPS stimulation at different concentration of BMSCs,observe the LPS impact on the proliferation and osteogenesis ability of differentiation of BMSCs,and preliminary exploration of the effect of signaling pathways,to observe the LPS effect on biological characteristics of BMSCs,in order to better BMSCs was applied to clinical treatment of bone nonunion.Our research results are as follows:1.The isolation,culture,purification and identification of h BMSCsWe cultivate primary cell by the whole bone marrow adherent method and then the limited dilution method is adopted to get the primary cell purification,as to get relatively homogenous BMSCs cell line.Then,we have adopted a series of method for the identification of BMSCs.By flow cytometry instrument to test the cells characteristic of phenotypic analysis,at the same time detection ability of multi-directional differentiation of BMSCs,the results are in accordance with a recognized BMSCs biology characteristics,prove that we have successfully constructed BMSCs cell line.2.The effect of LPS on hBMSCs ability of proliferation.We stimulate BMSCs to detect its effect on the proliferation ability of BMSCs by different concentration of LPS in different time.Different concentrations of LPS(0.01-100ug/ml),respectively,to stimulate BMSCs after 12 h,24 h,48 h to join determined by MTT train 4 h,test results show: 12 h after stimulation,no obvious difference was found between each cell proliferation ability;Stimulus after 24 h,48 h,compared with the control group,0.01,0.1,1 ug/ml proliferation ability of LPS stimulation group was obviously enhanced,of which 1 ug/ml LPS stimulation group increases the most obvious;10 ug/ml LPS stimulation of cell proliferation ability was reduced,the difference has no obvious statistical significance;100 ug/ml LPS stimulation of cell proliferation ability decreased significantly,it has the obvious statistical significance difference.The above shows that low concentration of LPS to stimulate the proliferation of BMSCs,with the increase of concentration,LPS could inhibit the proliferation ability of BMSCs.3.The effect of LPS on hBMSCs osteogenesis ability of differentiationWe use different concentration of LPS(0.01-100 ug/ml)to stimulate BMSCs to detect their effects on BMSCs osteogenesis ability of differentiation.Compared with the control group(0 ug/ml),to incorporate the osteogenetic differentiation of different concentration of LPS induced liquid after 7 d,the results showed that at low concentrations(0.01,0.1,1ug/ml),the osteogenetic differentiation related genes OCN,Runx2,OPN,ALP mRNA expression increases,the results have statistical significance;High concentrations(10,100ug/ml),the osteogenetic differentiation related genes OCN,Runx2,OPN,ALP mRNA expression decreased,the results have statistical significance.Then,we using alizarin redstaining detection of different concentration of LPS in the process of the differentiation of BMSCs development’s influence on the mineralized nodule formation,the results show that low concentration of LPS(0.01,0.1,1 ug/ml)can significantly promote the formation of BMSCs mineralized nodules,but with the increase of concentration of LPS(10,100ug/ml),BMSCs mineralized nodule formation.Alizarin red staining results consistent with the real time quantitative PCR results.These results confirmed that the low concentration of LPS can promote BMSCs differentiation into osteoblast,but with the increase of concentration of LPS,the showed obvious inhibition to the differentiation of the cells.4.The role of MAPK signaling pathways in LPS regulated the differentiation of hBMSCs.In order to explore the molecular mechanism of bone regeneration,we also has carried on the experimental analysis by the effect of the LPS in the process of regulating the differentiation of BMSCs.By alizarin red mineralized nodules staining and real-time quantitative PCR analysis,we found that blocking ERK1/2 and p38 MAPK signaling pathway can reduce LPS control the amount of BMSCs mineralized nodules and osteogenetic differentiation related gene expression,but blocking JNK signaling pathway but had no obvious effect on both.Westernblot test results also showed that LPS can activate p38 lightning,ERK1/2 kinase,and enzyme activity was positively correlated with LPS effect time.ERK1/2 and p38 MAPK signaling pathways involved in the LPS of regulation of osteogenetic differentiation of BMSCs,and play the role of a positive adjustment,and JNK signaling pathway may not be involved.The result laid a certain foundation to explore the mechanism of bone regeneration.