Studies On The Activation Of Heparin On Astrocytes And Its Mechanism

Astrocytes play a supporting role in the central nervous system. In particular, under conditions of injury, such as trauma, ischemia and hypoxia, normal resting astrocytes are transformed into activated ones to participate in the regulation of synapse formation and neural plasticity. The reactive astrocytes play an important role in the acute injury period. They not only provide nutritional factors to promote nerve regeneration, but also separate the damage zone from normal tissue to prevent the increase in the size of damage. However, in the chronic phase, reactive astrocytes and other components form glial scar, which will become a mechanical barrier to nerve regeneration and do harm to the repair of nerve injury. Therefore, the specific mechanism of reactive astrocytes becomes a key element in the treatment of nerve injury.The secretion of heparin, which is one kind of the extracellular matrix, will increase after the astrocytes are activated. In this study, rat primary astrocytes were treated with different concentrations of heparin. Then we were going to prove that heparin was able to induce the activation of astrocytes from different aspects, such as morphology, proliferation, and migration. So heparin was used to prepare in vitro controllable models to simulate the activation in vivo. At last, the intrinsic molecular mechanisms in the induction of astrocytes activation were explored to provide the experimental basis for effective treatment of nerve injury. The main contents are as follows:1. The culture and purification of primary astrocytes. On the base of the classic method of McCarthy, the cell suspension was obtained from the dissociating cerebral cortex of newborn rats. After10days, the cell culture experienced the overnight shaking to remove the oligodendrocytes and microglia, which grew on the upper layer. In order to improve the purity of the astrocytes, we took the method of differential velocity adhesion to reduce the contamination of fibroblast. By the identification of immunofluorescence staining, the GFAP positive rate of cell cultures increased from80%to95%, meeting the requirements of following experiments on cell purity.2. Effects of heparin on the activation of astrocytes. First, the morphological changes of the astrocytes were observed by eosin staining and GFAP immunofluorescence staining. The results showed that heparin combined with FGF-2promoted the recombination of cytoskeleton in a dose-dependent manner and the cells changed from the polygonal resting state to the stellate activated state. Subsequent Western Blotting results showed that heparin increased the GFAP expression levels in astrocytes, and thus confirmed that heparin combined with FGF-2could induce the activation of astrocytes. Then, the proliferation of astrocytes was detected by BrdU incorporation assay and flow cytometry. The results showed that heparin combined with FGF-2increased the proportion of DNA replication of astrocytes in a dose-dependent manner and increased the percentage of S phase to improve the rate of cell proliferation. Finally, the result of transwell chamber assay showed that heparin combined with FGF-2promoted the migration of astrocytes.3. Possible mechanism leading to the activation of astrocytes. Inhibitor screening, Western Blotting and PCR were used to detect the signaling pathways involved in the regulation of reactive astrocytes. The results showed that ERK, PI3K/Akt signaling pathway and N-cadherin on the cell membrane were involved in the regulation of stellation induced by heparin and the ROCK pathway was associated with this effect.