| background:Osteosarcoma is one of the most common malignant bone tumors in clinic.It accounts for about 20%of the primary malignant tumors.The incidence of the disease is more concentrated among the 3-18 year old teenagers.Because of the high degree deterioration of the primary lesion and the low survival rate at the later stage,it not only causes catastrophic psychological trauma to the patient and the family,but also adds heavy burden to the society.The occurrence of osteosarcoma is related to the growth of bone and the change of hormone.Osteosarcoma,as a systemic malignancy,is common in younger patients,where it grows rapidly on the metaphysis(femur,humerus).Modern medical studies have shown that osteosarcoma is developing rapidly and can lead to high risk of death.Therefore,clinical medical research on this kind of malignant tumor more and more attention.As early as the 60s of last century,the treatment of osteosarcoma mainly depended on surgical intervention abroad.At the time of medical equipment,environment,technology and other factors,the actual treatment of osteosarcoma is very little.Until the last century,the early 70s,scientists first adriamycin(Doxorubicin)applied to advanced osteosarcoma patients with distal metastasis patients,the treatment effect is increased significantly,which also extends the new value treatment of chemotherapy of osteosarcoma.By the middle of 70s of last century,combined with the characteristics of osteosarcoma and chemotherapy drugs,methotrexate was used,and its treatment effect was satisfactory.And the concept of combined chemotherapy was formed.The survival rate of osteosarcoma patients at the later stage of treatment is further improved.By late 70s,some scholars in order to further improve the effect of retention in patients with osteosarcoma limb removed tumor,inhibit local tumour response,reduce patients with distal metastasis risk,thus put forward the new idea of preoperative chemotherapy.Finally,Professor Rosen has established a new adjuvant chemotherapy mode with preoperative chemotherapy + surgical operation + postoperative chemotherapy in combination with the pathological study of osteosarcoma for many years.Especially in recent years,many scholars at home and abroad for osteosarcoma patients immune and genetic characteristics of intervention,coupled with biological target intervention progress,but also for patients with a new dawn.However,affected by the pathological features of osteosarcoma progression,with the development of the metastatic process,there is likely to be caused by secondary bacterial infection,to the treatment of patients with advanced or metastatic patients with distal great difficulty.So has a long way for the system effective treatment of osteosarcoma,found in a recent study of osteosarcoma,MAPK signal transduction pathway have a close contact with the development of osteosarcoma.MAPK pathway is a serine/threonine protein kinase which is widely expressed in cells.It is activated by phosphorylation and participates in a series of cellular physiological activities such as cell growth,development,differentiation and apoptosis.including extracellular regulated protein kinase(ERK)pathway,P38 mitogen activated protein kinase MAPK signaling pathway,C-jun N-terminal kinase pathway(JNK).ERK transduction pathway is the main pathway to transfer extracellular signals to the nucleus,and plays a major regulatory role in the process of cell proliferation,differentiation and apoptosis.It is a key signaling molecule that determines cell proliferation,differentiation and apoptosis.ERK signaling pathway is divided into ERK1 and ERK2,ERK1 and ERX2 are the key proteins that transmit signals from surface receptors to the nucleus,and their molecular weights are 44kDa and 42kDa.ERK1/2 signaling pathways in after activation,affect T cells,promote its proliferation significantly increased,and impact on a variety of cytokines,leading to significant increase in the level of its secretion;And the above factors will affect the macrophages and monocytes,lead to the aggregation and activation,macrophages and monocytes after being activated,can be in osteosarcoma tissues secrete a large number of different types of inflammatory cytokines,and induced by osteosarcoma cells,producing a large number of biological small molecules,lead to the increase of the malignant degree,the clinical symptoms in patients with osteosarcoma also rising,resulting in further deterioration of condition,and intracellular toxin-lipopolysaccharide(LPS)is the important bacterial virulence factors.LPS is the specific structure of cell wall of gram negative bacteria.It is the main component of endotoxin.It is composed of core polysaccharide,O polysaccharide,side chain and lipid A.The extracellular regulated acts on osteosarcoma cells via mitogen activated protein kinase(MAPK)signaling pathway,which can induce the development of osteosarcoma.The concentration of LPS is positively related to the expression of inducible enzymes in osteosarcoma cells,which interact with inflammatory factors,induce different inflammatory reactions and infections,and aggravate the malignant degree of osteosarcoma.This role is achieved by the presence of MAPK signaling pathways in synovial tissue of osteosarcoma.Objective:The study on osteosarcoma cell MG-63.The effect of LPS on osteosarcoma cell line MG-63 was shown.The mechanism of MAPK pathway to LPS on the growth of osteosarcoma cell line MG-63 was explored.The important role for further study of MAPK pathway proteins exist in the occurrence and development of osteosarcoma,in order to find the therapeutic targets new,lay the foundation for the development of effective treatment for osteosarcoma.Methods:Human osteosarcoma cell line LPS was treated with different concentrations of MG-63.The cell growth status was detected by MTT.And the optimum time and concentration were obtained.MG-63 was used to collect the cells at different time by LPS,and the protein was extracted.The phosphorylation status of key proteins ERK1/2,JNK and p38 in MAPK pathway were detected by Western blot method.the activation protein is involved in the proliferation of human osteosarcoma cell line MG-63.The variation of MG-63 was detected on phosphorylation key proteins inhibited by U0126.MAPK pathway was detected by western blot.The cell growth status was detected by MTT.The role of LPS in human osteosarcoma cell MG-63 is further determined through the key protein pathway.Using RNAi method to inhibit TLR-4,we studied the effect of LPS on human osteosarcoma cell line MG-63.MAPK pathway was detected by western blot.The cell growth status was detected by MTT.Results:(1)The growth rate of MTT was calculated at zeroth,24,48 and 72h after the action of LPS on human osteosarcoma cells MG-63 by 0,10,20 and 40ng/ml.When 24h was added to LPS,the growth rate of the osteosarcoma cells increased gradually.In group 10ng/ml,the rate of cell growth also increased with time.But,the rate of cell growth not significantly increased in 20 and 40ng/ml groups.The growth was similar in the 20ng/ml and 40ng/ml groups.At 48 h,the rate of cell growth reached a maximum of 94.03 + 0.211%.When compared with the growth rate of 48 h,the growth rate of 72h was 97.53 + 0.20,and there was no obvious change(P<0.05).This shows that the growth rate of human osteosarcoma cell MG-63 is the best when the 20ng/ml of LPS is 48h,and it is 94.03 + 0.211%.(2)In order to study the effects of LPS on proliferation of human osteosarcoma cell line MG-63 further.With a final concentration of LPS 20ng/ml in human osteosarcoma cell line MG-63 was added.And in 0,2,4 and 8h,the cells were collected and the total proteins were extracted.The phosphorylation status of ERK1/2,JNK and p38 in MAPK pathway were detected and analyzed.Phosphorylated ERK1/2 increased as time went on,and the band became deeper at 4h,the deepest and then almost the same as 4H at 8h.Other ERK1/2,JNK,p38,and phosphorylated JNK,p38 expression bands did not change with time.In order to further study the phosphorylation of protein ERK1/2,JNK and p38,we analyzed the protein bands of Western and blot.Phosphorylation of ERK1/2 increased as time went on,and phosphorylation increased gradually to 4h(P<0.05).The phosphorylation of JNK and p38 did not change significantly during the whole process(P>0.05).(3)U0126 with 0,1 0,20,30,40 and 50ng/ml acting on human osteosarcoma cells MG-63,the growth rate of U0126 was slowed down.And the rate of 40ng/ml was the slowest.The rate of cell growth dropped from 87.32%to 22.87%.At 40ng/ml,the growth rate of cells was 23.24%,which was significantly lower than that of 30ng/ml.To further demonstrate the role of LPS through the ERK1/2 pathway,U0126,an inhibitor of phosphorylated ERK1/2,was added to LPS to detect the growth of 0,24,48 and 72h.In the U0126 group,the cells grew slowly,from 34%in the beginning to 41.5%in 72h,and the rate of cell growth did not change significantly during that time.When U0126 was added,the growth rate of human osteosarcoma cell MG-63 slowed down and the change was not obvious.Phosphorylation of ERK1/2,JNK,and P38 did not change.Phosphorylation of ERK1/2 did not change significantly with time(P>0.05),whereas phosphorylation of JNK and p38 did not change significantly during the whole process(P>0.05).(4)The cDNA was obtained by reverse transcription.Drawn under different concentrations of quantitative standard curve of PCR and reference gene fluorescence.The regression equation of target gene TLR-4 amplification curve was y=-1.199x+27.52,and the regression coefficient was r2=0.994.Reference gene amplification regression equation was y=-0.2951 x+3.02,regression coefficient r2=0.992.Quantitative PCR method can be used to detect gene expression and can be applied to 2-Ct formula of relative gene expression calculation.After plasmid transfection,the cells were collected at 0,12h,24h,36h,48h and 60h,respectively,and quantitative PCR detection of TLR-4 was performed.The expression of TLR-4 decreased with the increase of action time.The expression levels of 12h,24h,36h,48h and 60h were lower than 0.After plasmid transfection,36h was observed under fluorescence microscope,most of the cells produced green fluorescent protein.But in blank control group,there was nothing.The expression of TLR-4 in the interference group was very low,but in the undisturbed group,the expression of TLR-4 did not change.After TLR-4 inhibited,LPS acted 0,2,4 and 8h,the phosphorylation of ERK1/2,JNK,and P38 did not change.Phosphorylation of ERK1/2 was low.Phosphorylation of ERK1/2 did not change significantly with increasing time(P>0.05),whereas phosphorylation of JNK and p38 did not change significantly during the whole process(P>0.05).In the TLR-4 inhibited group,cells grew slowly,from 34.09%at the beginning to 49.5%of 72h.There was no obvious change in cell growth rate(P>0.05).When TLR-4 inhibited the growth of osteosarcoma cells,the growth rate of MG-63 was slower and the change was not obvious.Conclusions:In summary,our results show that the LPS suppress the growth of bone sarcoma cells with MG-63 mainly by the MAPK pathway of ERK1/2 ways,in order to further study the MAPK pathway proteins in bone sarcoma cells exist in the process of the occurrence and development of the important role of found new therapeutic targets,for clinical effective scheme laid a solid foundation for the treatment of osteosarcoma. |
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