The Genetic Studies Of Heterotopic Ossification In Ligament Tissue Of Ankylosing Spondylitis

Part I Between Ankylosing spondying and normal control group ligament tissue osteogenesis mineralization degree, And distribution of mineralization genesOBJECTIVE:Comparing the AS patients and normal control group ligament osteogenesis mineralization of the differences between groups。To explore possible factors or promote AS ectopic osteogenesis。METHODS:Ankylosing Spondylitis (AS) patient’s spinal ligaments were taken for the experimental group, The intervertebral ligament tissue of spinal fractures as control group. With HE staining and alizarin red staining and Von kossa staining to observe the general form and degree of mineralization of the organization。At the same time, we detected using immunohistochemistry and RT-PCR technique ANKH gene expression whether there is a difference between the two groups.RESULT:Both two groups of HE staining contains relatively large Numbers of fibroblasts, and no obvious difference form the cell. Alizarin red staining and Von kossa staining results show that AS patient’s joint ligament attached side obvious mineralization, but not found that the mineralization in healthy people. By doing RT-PCR detection was found that ANKH gene between in the AS tissue and JK control tissue has significant difference (P<0.05)CONCLUSION:The heterotopic ossification of the AS patient’s ligament began in the joint ligament attachment points, and there are relatively large Numbers of fibroblasts in ligament ossification around the organization. The ligament fibroblasts of AS patients with less ANKH protein expression, and ANKH protein may play an important role with ectopic ossification process of the AS patients’ ligament tissue. staining and Von kossa staining results show that AS patient’s joint ligament attached side obvious mineralization, but not found that the mineralization in healthy people. By doing RT-PCR detection was found that ANKH gene between in the AS tissue and JK control tissue has significant difference (P<0.05)Part Ⅱ The cultivation of the original generation of fibroblasts and between the two groups of fibroblasts compared distribution of mineralization genesOBJECTIVE:To master the method of more simple and efficient cultured fibroblasts To study the genetic differences between the two composition of fibroblasts cells, explore cause or promote the AS possible factor in the development of heterotopic ossification occurred.METHODS:Tissue block adherent method to develop two groups of cells, and observed two groups of primary cell morphology with a microscope.The content of the third generation of fibroblasts with immunofluorescence technique identification. To groups of the third generation of fibroblasts were taken for mineralization induction training,and comparison of two fibroblasts cells differentiation and mineralization of development capability by detecting the ALP activity change and alizarin red staining.At the same time, we detected using RT-PCR technique ANKH gene expression whether there is a difference between the two groups.RESULT:Through the tissue block adherent culture method successfully cultivate ligament fibroblasts,and no obvious difference form the cell. Fibroblasts content in more than 99% with vimentin immune fluorescence detection. Alkaline phosphatase (ALP) activity detection and alizarin red staining results are shown that two groups of thin extracellular secretion of ALP were increased and And form a certain amount of mineralized nodules,but the ALP secretion and mineralized nodules number in AS the experimental group was higher than JK control(P<0.05).The ANKH gene expression of the AS patients ligament fibroblasts were significantly less than JK in the control group(P<0.05).CONCLUSION:Tissue adherent culture method can produce high purity ligament fibroblasts.Ligament fibroblasts with osteogenesis potential can be induced to differentiate into osteoblasts,and play ossification mineralization function.Ligament fibroblasts of the AS patients with osteogenesis trend is stronger than normal people under the condition of in vitro, namely the osteogenesis mineralization ability is stronger.and research shows that there is some relation between the AS patients heterotopic ossification and osteogenetic differentiation of fibroblasts. The ANKH gene expression of the AS patients ligament fibroblasts were significantly less than JK in the control group, so it may be involved in the formation of heterotopic ossification of ligament of the AS patients,but the specific mechanism still needs further research.