Association Analysis Of Notch1-Hes Signaling Pathway On The Hip Joint Ligament Of Ankylosing Spondylitis

IntroductionThe incidence of ankylosing spondylitis in inflammatory arthritis ranked second,but theexact pathogenesis remains poorly understood.AS often occurs in young or middle-agedmen,and because of the high rate of disability and deformity,AS has played a crucial role inthe research of population and health both in China and abroad.The symbolic pathologicalcharacteristics in AS is different with other rheumatoid arthritis diseases.The latter mainlycauses erosion fracture of cartilage and bone.But AS mainly performs the ossification processof pathological formation of new bone.AS causes persistent formation of new cartilage andcapillaries in bone trabecula.At the same time,fibrous tissues Continuous generate.All resultin abnormal ossification, which eventually leds to the joint stiffness. This is the main reasonfor the disability.By the way,study shows that the ossification and inflammation of AS havedifferent pathogenesises,two relatively independent processes.Even if we block the process ofinflammation in patients with AS,we can’t prevent the process of joint stiffness.Notch1genecan keep stem cells in undifferentiated state, and also can adjust them to the differentiation ofosteoblast.Once directional differentiated into osteoblasts,Notch1signal can promote theproliferation of osteoblasts and restrain the maturity of osteoblasts.Therefore,study theNotch1-Hes signal pathway function in ossification among patients with AS,has importantbasic research value and potential clinical significance.PurposeWith the advanced Affymetrix GeneChip technology, we screened the ligaments of hipjoint in patient with AS and healthy people to get microarray gene expression profile data.And then we reviewed the domestic and overseas literatures, to choose the most possibleossification genes to do larger sample validation. Correlation analysis research with clinicaldata of AS patients was made to explore the relationship of these ossific genes with ASligament ossification.MethodsBased on the modified New York criteria of Ankylosing Spondylitis,3patients whoreceived THA in Changhai Hospital during2010-2012were choosen as the research object incase group.And another3patients with femoral neck fracture who also received THA inChanghai Hospital (excluding all kinds of other joint diseases) were selected as the healthy control group.We used the Affymetrix GeneChip technology to screen differential gene.According to the resullt of screening, combining with literature review, candidate ossificationgenes were selected to do real-time fluorescent quantitative PCR verification and WesternBlot. Enlarging the sample (22AS patients, the diagnosis standard as above), we usedimmunohistochemical technology to detect the manifestation of candidate genes in hipsamples, and to do correlation analysis research with patients’ clinical data.Results1. According to the difference of1.5times, the result of GeneChip between AS groupand control group showed there were652down-regulated genes and966up-regulated genes.The differences of functional genes were involved in a variety of signal pathways, the Notch1gene up-regulated1.53times, and the Hes gene up-regulated1.75times.2. In enlarged sample,real-time fluorescent quantitative PCR verification showed theexpression of Notch1gene and Hes gene were significantly higher than the normal controlgroup(P<0.05).Western Bolt results showed that Notch1protein in patients with AS groupwas significantly higher than the normal control group.3. In immunohistochemical slices,Notch1protein and Hes protein mainly manifested inperipheral of ligament and central of ligament near blood vessels.In AS group, positive ratesof both proteins were significantly higher than those in control group(P<0.05). And thepositive rate of Hes protein was positively correlated with disease duration in ASpatients(r=0.594，P <0.01).Conclusions1.Comparing the ligament tissue of AS patients with normal control group,somefunctional genes change,involving a variety of signal pathways.In all these genes,we find thatthe differences of Notch1gene and Hes gene are significant.2.Notch1protein and Hes protein mainly express in peripheral of ligament and central ofligament near blood vessels.The positive rate of Hes protein is positively correlated with theduration of disease in AS patients.3.Notch1-Hes signal pathway may play a key role in the process of hip ligamentossification in AS patients.