The Expression Of Gap Junction Genes Connexin43 And His Protein In Rat Osteoblaste-liked Cell Line And The Role In Signal Transduction

The changes of the hard and soft tissues, get involved in the tooth movement during orthodontic. The bone tissue surrounds the teeth,any movement of the teeth affects the surrounding bone tissue centainly, brings a series of changes , such as bone resorption, bone formation.The reconstruction of bone, a series of complicated procedure involve in bone resorption and bone formation, which need the cooperation of osteoblast, osteoclast, and osteocytes. The cooperation of the cells depends on intercellular transmission. The transmission is accomplished via the gap junction of the cells.Gap junctions, composed by connexin ( Cx ) protein, permit the exchange of regulating molecules between contact cells which play a very impontant role in embryogenesis, cell proliferation and differetiation. Intercellular communication between contact cells is carried out by exchange ions and small molecules via gap junctional channels.The decreased expression of Cx genes, the gap junctional intercellular communication (GJIC)disorder and abnormal signal transduction pathway of Cx genes might contribute to the uncontrolled tumor cell growth. The loss of gap junctional intercellular communication due to the aberrant Cx genes expression appears to be an important event in cell transformation.The recent study discovered that the bone reconstruction has relationship with the gene expression of the gap junctional connexin; the alteration of mechanism of signal transduction and the adjustment of gap junctional intercellular communication. Meanwhile, the gap junctional connexin was regulated by some growth factors, neurotransmitters,hormone. These factors influence connexin, then adjust the amount of cells in bone tissue, affect the metabolism of cells, result in the corresponding alternation of bone tissue.Up to now, little work has been done of Cx gene expression, Cx genes regulation and their roles in signal transduction. In this study, we use rat osteoblast-liked cell line -ROS 17/2.8, learned the Cx43 gene expression, gene regulation, their roles in signal tranduction and their gap junctional intercellular communication.The results and conclusions are as followed.1. By immunohistochemical staining, detect the expression of Cx43 gene on ROS 17/2.8. We found that most immunoreaction is located on the cell membrane, a few around the nucleolus.2. Use the laser scanning confocal microscope to study the expression of Cx43 gene and quantitatively analyse the intensity of the Cx43 gene expression on the ROS 17/2.8 cells.3. Use different concentration bFGF to treated the ROS 17/2.8 cells. By immunohistochemical staining and laser scanning confocal microscope techique,we get the dose-depend and time-depend curve.4. By in situ hybridization (ISH), positive Cx43 mRNA in ROS 17/2.8 cells.5. Lucefer Yellow scrape loading and dye transfer (SLOT) showed the ROS 17/2.8 cells had ability of gap junctional intercellular communication.6. After Fluo-3 AM loading, laser scanning confocal microscope examined intercellular second messager[Ca2+]iin bFGF treated culture and non-bFGF treatedculture.7. Western blot showed that ROS 17/2.8 had Cx43 protein and had phosphorylated Cx43 protein.The results showed, Cx43 protein express on the cultured rat osteoblast-likedcell lineOS 17/2.8 . The growth factor梑FGF can influence the gap junctional protein Cx43 expression and affect the gap junction intercellular communication. BFGF treated the osteoblast cells, result in the exchange between the cells, may associated with it affectec the amount of Cx43, alter intercellular second messenger [Ca2+]j, and posttranslation phosphorylation on tyrosine of Cx protein.