Expression Change Of CNTFR In Ankylosing Spondylitis And Its Influence On The Activity Of Osteoblasts

Ankylosing spondylitis(Ankylosing spondylitis(AS) is a complex disease which caused by many factors, may be associated with a variety of factors such as genes and environments.Most of Ankylosing spondylitis patients are men, the feature of the disease marked by arthritis and ankylosis, mainly involving the spine and peripheral joints.HLA B27 as the gene which is the strongest correlation with ankylosing spondylitis has been studied for more than 40 years, though in the past studies we have made a certain of progress, but in the search for AS pathogenesis is very slow.Compared with other complex diseases such as inflammatory bowel disease, little of the AS susceptibility genes have been identified. The findings of ERAP1 and IL23 R suggest that these genes may be associated with the onset of ankylosis.In genome-wide association study, identify and prove whether related genes and their variation with the AS diseases is a difficult challenge.Osteoblasts play an important role in bone formation and bone calcification, as well as regulates osteoclast differentiation.Runx2 transcription factors in bone marrow stem cell with highly expression in result that prompting them to differentiate into osteoblasts.Osteoblast regulates osteoclast differentiation by macrophage colony-stimulating factor(M- GSF), receptor activatior of nuclear factor kappa-B ligand(RANKL) and osteoprotegerin(OPG). M- GSF expands sosteoclast precursor cells pool, and promotes the survival rate of them. Receptor activatior of nuclear factor kappa-B ligand(RANKL) is the essential molecular of osteoclast formation and interact with RANK receptor of osteoclast percursor cell, thus promote the osteoclast differentiation.Bone protection element and receptor activatior of nuclear factor kappa-B ligand interaction between ligands, inhibit the formation of osteoclasts.Other effects factor involved in osteoblast-osteoclast include parathyroid hormone(parathyroid hormone PTH) and prostaglandin E2(PGE2). Bone formation process of osteoblasts contain osteoblast maturity,proliferation,and mineralization of extracellular matrix, one of the important symbol of osteoblast and extracellular matrix maturity is the activity of alkaline phosphatase(ALP),so ALP can be used to test the activity in osteoblasts.Blocking CNTF can lead to the lost of the motor neuron and movement syndromes and resulet in a light damage which compared with the lost of VEGF. The area of the central nervous system damage in the AS patients, the expression of CNTF reduced relativity. In addition, the serum levels of CNTF gradually rise of the AS patients, especially the lumbar be violationed.In motor neuron injury in mice model, subcutaneous injections of CNTF can protect motor neurons.CNTFR widely exists in the skeletal muscles and nervous system and periperal organization.In addition, skeletal muscle, skin, lung, intestine, kidney, liver, spleen and thymus are expressing CNTFR, of which the highest content expressed in skeletal muscle.In the central nervous system, CNTFR expression in the cerebellum, hindbrain, midbrain, thalamus, hypothalamus, striatum, hippocampus and cortex, among them the highest expression parts is the cerebellum.CNTFR be composed of three elements include CNTFRa, glycoprotein(gpl30) and LIFRβ. CNTFRa is the core of this structure, molecular weight of which is about 52 kd.Glycoprotein(gpl30) on IL- 6 family play a role in signal transduction. LIFRβis the binding protein of LIF. In cytoplasm of the LIFRβdo not have function of protein kinase and binding setes of GTP, but the parallel signal transmission body of gpl30 associated with Jak-Tyk activation of LIFRβ,so the gpl30 and LIFR LIFRβassociated with signal transduction.We use gene chip to detect the difference gene expression beteen ankylosing spondylitis patients and normal patients, found that the gene of C7, C3, ENTPD3, HBA2, FAM70 A, DSG2, ANGPT1, ACVR1 C, CNTFR, FMO2, RERGL, SLPI, IL7 R and HP are increased expressed in ankylosing spondylitis patients in hip joint ligament compared with normal patients.The gene of PAMR1, KCNA1, FGFBP2, PPP4R4, CRTAC1, MAMDC2, GPR64, GALNT13, F5, NOV, PIEZO2, LPAR4, CDH13, ITGA11, Q6ZUQ1, PIEZO2, ARNTL2, TNXB, NRK, GPSM2 and HTR2 A expression lowerly in the hip ligament of ankylosing spondylitis patients than normal patients.As CNTFR widely exists in the skeletal muscles and nervous system, and expressed in ankylosing spondylitis patients also highly, in addition, blocking CNTF can lead to the lost of motor neurons and motor syndrome, serum levels of CNTF rising in ankylosing spondylitis patients especially the lumbar has been involoved. In mice model with motor neuron injury, subcutaneous injections of CNTF can protect motor neurons.So we speculated that CNTFR may be associated with ankylosing spondylitis.Osteoblasts in bone formation and bone calcification plays an important role and regulates osteoclast differentiation, in addition,have an important effects in incidence and development of ankylosing spondylitis. The maturity of osteoblast and extracellular matrix can be estimated by the activity of alkaline phosphatase.So to use CNTF to handle with the CNTFR activated and to use alkaline phosphatase activity of osteoblast detection in order to observe CNTFR whether associated with ankylosing spondylitis is feasible in theory.Part 1 Gene chip detection of hip joint ligament in AnkylosingspongdylitispatientsObjectiveTo explore the genes associated with anlylosing spondylitesMethodsWe selected 3 cases of ankylosing spondylitis patients and 3 cases of normal patients,take their hip ligament,then send to company to detect with gene chip.In order to make it clear that the defferences of gene expression between ankylosing spondylitis patients and normal patients. ResultWe use gene chip to detect the difference gene expression beteen ankylosing spondylitis patients and normal patients, found that the gene of C7, C3, ENTPD3, HBA2, FAM70 A, DSG2, ANGPT1, ACVR1 C, CNTFR, FMO2, RERGL, SLPI, IL7 R and HP are increased expressed in ankylosing spondylitis patients in hip joint ligament compared with normal patients.The gene of PAMR1, KCNA1, FGFBP2, PPP4R4, CRTAC1, MAMDC2, GPR64, GALNT13, F5, NOV, PIEZO2, LPAR4, CDH13, ITGA11, Q6ZUQ1, PIEZO2, ARNTL2, TNXB, NRK, GPSM2 and HTR2 A expression lowerly in the hip ligament of ankylosing spondylitis patients than normal patients.PART2 Verify the gene expression of CNTFR inankylosing spondylitis patientsObjectiveVerify the gene expression in ankylosing spondylitis patients.MethodsTake 2 cases of anlylosing spondylitis patients’ ligament of hip joint as experimental group and 2cases of normal patients’ ligament of hip joint either as control group,using the methods of western blot to test the difference of protein levels which expressed by CNTFR between the two groups.ResultFrom western blot testing we found that the colour of the strips stand for ligament of hip joint from ankylosing spondylitis patients are deeply than normal patients’.ConclusionThe protein levels which expressed by CNTFR gene increased significantly in anlylosing spondylitis patients.Part 3 CNTF treatment effects on osteoblast activity of alkalinephosphataseObjectiveTo make clear that CNTF treatment effects on osteoblast activity of alkaline phosphatase.MethodsTo cultivate the osteoblast then ceiling the cells when.the degree of fusion up to 50%-70%, Experiment can be divided into two groups, one group is CNTF treatment group, the other group is as control group, the number of cases in each group of 6. The cells in each group be handled with CNTF within 24 h and then according to the instructions on the activity of alkaline phospatase detection kits, to detect the activity of alkaline phosphatase.Resulthe cells in each group be handled with CNTF within 24 h and then according to the instructions on the activity of alkaline phospatase detection kits, to detect the activity of alkaline phosphatase.We found that alkaline phosphatase activity of CNTF treatment group was obviously higher than control groupConclusionCNTF can decrease the activity of osteoblast alkaline phosphatase, which involved in the occurrence of developing ankylosing spondylitis.