| Background:Peripheral nerve injury remains a high incidence of disabling factors. To some extent, peripheral nerve could repair and regenerate after injury. Nerve regeneration after peripheral nerve injury is an important indication of the whole repair process. On the first step, neuronal cytons largen, Nissl bodies decompose, the nucleus moves to the periphery of the cell and protein synthesis starts. Wallerian degeneration happens because of lack in nutrition and even distal nerve fracture, which is featured by cytoplasmic condensation, liquefaction, axon demyelination disintegration. Below the level of injury, myelin break down and phagocytosis axons wither. Similar changes can be seen above that level. Mostly the Wallerian degeneration do not appear immediately after the injury. While in this process, once activated, the axons can be completely disintegrated within a few hours. The Schwann cells in the local site of injury then raise the macrophages to phagocytosis, disintegrate axons and myelin into debris. Removal of the myelin debris for nerve repair could be very important, because the myelin contains inhibitory factor for axon growth, and is likely to impede nerve regeneration. After the removal, residual basement membrane and its associated Schwann cells (SCs) could form a complete endometrial pipeline structure, providing a channel for axonal regeneration. Once the axons start to regenerate, the distal nerve stumps would re-enter the endoneurial tube, then grow towards the target tissue. The palingenetic axons could still associate with former tissue and re-establish a functional synapsis accurately.The whole process of peripheral nerve regeneration involves the degeneration, proliferation and migration of Schwann cell; macrophages’chemotaxis towards the orientation of the damage zone; axoplasmic transport and axonal regeneration. A variety of cytokines are involved in the procedure. The influence of macrophages in peripheral nerve regeneration can be considered in two aspects:(1) macrophages serve the role of cleaners;(2) macrophages secrete active substances, promoting the whole process by stimulating SCs directly.Nerve growth factor (NGF) is one of the most important biological active molecules, and is one of the most typical the neurotrophic factor. NGF plays an important role in neuronal development, differentiation, and maintaining the normal function of neurons. Many studies have shown that when nerve cells are damaged, NGF can not only protect its associated neurons, reduce its degeneration and death, but also promote the growth of the regenerated nerve fiber, thus is conducive to the recovery of nerve function. The increase of NGF protein could guide nerve regeneration on one hand. On the other hand, with the growth of axons, the continuous antedisplacement of the growth cone, the surface receptors on axons and NGF combined through pinocytosis. The NGF is then transported to the neuron cell body through retrograde axoplasmic transport, to support the growth of neurons and promote nerve regeneration. In that way, the increase of NGF expression will benefit neural repair, and facilitate its regeneration after nerve damage. On physiological conditions, NGF is mostly secreted by its target organs (such as muscles, glands, etc.) and glial cells (peripherally, Schwann cells). While after peripheral nerve injury, the local proliferated Schwann cells become the main source of NGF synthesis and secretion. The NGF is carried to the neuron at the damage site through retrograde axonal transport and promotes the neuron survival and neurite extension, accelerate nerve repair and regeneration. Therefore, the capacity of the Schwann cells in synthesizing and secreting NGF is one of the most important influential factors to the quality of peripheral nerve regeneration.The extracellular matrix (ECM) is the macromolecular substances deposited between cells. It mainly present in the procedure of Schwann cells enveloping axons outside of the base film. Its main components are generated by the Schwann cells, which include laminin, fibronectin, collagen and heparan sulfate proteoglycan (HSPG), ECM components can provide appropriate adhesion for nerve growth, guide axonal growth along the matrix bridge to ensure the stability of the growth cone, and directional growth of nerve fibers. The influence of laminin in nerve regeneration has been widely recognized. Laminin mainly exists inside the tube of the base film, and could self-aggregated on its own. It can also be combined with other biological macromolecules, through the matrix of integrins, oligosaccharides and other receptors binding, thus to determine the cell differentiation, migration, and their structure, to maintain the phenotype of the organization, promoting their survival. In embryonic and neonatal period, laminin were expressed mostly around the inner surface of the endoneurial tube of peripheral nerve. The directional growth of nerve cells and nerve fibers is guided through the concentration gradient of laminin. On adults laminin express less. After sciatic nerve injury, the expression of Laminin resumes and even increase may be related to the Schwann cell proliferation and secretion. Schwann cell could help to guide axon growth and play a role in the neurotrophic system. RSC96cell line is naturally transformed from rat Schwann cells after primary cell culture. It could perform as a ideal class of Schwann cell analogues.Our study is based on previous ones and the RSC96cell model. The aim of this experiment is to explore the effect of macrophages on RSC96cells, the expression of NGF, and to explore the mimicry of topological structure of nerve axon on the expression of NGF and Laminin of RSC96cells.Objective1. Compare primary cultured Schwann cells and RSC96cells detect the expression of NGF and Laminin in vitro RSC96cells. 2. Study the expression of NGF and Laminin with different activation state of macrophages co-cultured with RSC96cells.3. To explore the mimicry of topological structure of nerve axon on the expression of NGF and Laminin of RSC96cells.Methods1. The bilateral ischial nerves and brachial plexus nerves were taken from SD rats(3to5days). D-Hanks solution wash several times and strip the epineurium carefully under a dissecting microscope, then washed3times with D-Hanks solution. We cut the nerve tissue into small blocks in size of1mm3and Isolate the tissue blocks with the dual-enzyme digestion. Cytarabine was used inhibit fibroblast growth, low concentration of trypsin was used to quick-digest and pass further purified Schwann cells. Morphology was observed under light microscopy. Culture medium containing10%FBS,100U/mL penicillin,100μg/mL streptomycin, DMEM/F12was used to culture the RSC96cells.Then the culture dishes were placed in the CO2incubator at37℃. The medium was changed changed every2day. After the cell fusion rate got80%, digest and pass the cell culture with0.25%trypsin0.02%EDTA.The morphology was also observed under light microscopy. Trypsin digest and collect the two types of cells, extracted total protein. Western blotting analysis of NGF and LN was done to assess protein expression levels.2. Two1-month-old SD rats, one injected with DMEM/F12culture fluid into the peritoneal cavity. Macrophages were extracted from abdominal liquid culture after10minutes. While the other, sciatic nerve distal cut, pre-degenerated2d, made of the nerve homogenate was reinjected autologous intraperitoneal3d. Also extract the abdominal liquid to culture macrophages (autologous nerve homogenate activated).3. A Transwell system was established to co-culture the rat peritoneal cavity macrophages with RSC96cells. We divided the cells into five groups:simple non-activated macrophage, activated macrophages and without co-cultured macrophages RSC96cells as A, B, C as control groups, non-activate macrophages RSC96of cells co-cultured group and activation state of macrophages and Schwann cell lines were co-culture groups D, E,as experimental group.4. On the3rd day of cell culture, macrophages in Group A and B were collected. RNA of RSC96cells of other groups were extracted by Trizol method following the protocol. Reverse transcriptase Oligo (dT) was used to synthesize cDNA. Then RNA was expanded by designed primer (Table) and used for q-PCR. The same amount of transcript was used for q-PCR, with SYBR Green/ROX qPCR MasterMix (2X) by following the protocol and repeated for3times. The relative expression levels of genes were analyzed using the2-ΔΔCT method by normalizing with β-actin house-keeping gene expression.5. The culture fluid was collected on the3rd day. Rat Laminin ELISA kit and Rat P-NGF ELISA kit were used by following the protocols. The supernatant was collected from each group and added to96-well plates pre-coated with NGF or Laminin polyclonal antibody for24hour of incubation at4℃. After extra antibodies were rinsed, rabbit anti mouse IgG with biotin labeling was added for incubation for another2hours. Then stop buffer was added to incubate25min at37℃. Bio-Rad680was used for the detection of light absorption value at OD450nm, with the accuracy of l.Ong/ml. Samples from each group were repeated twice.6. Macrophages from Group A and B were collected on the3rd day of culture. Proteins of RSC96cells were extracted from other groups by following the protocols of Total Cellular Protein Isolation Kit. Bradford Protein Assay Kit was used to detect the protein level. Equal quantities of total proteins from samples were mixed with equal volume of2×SDS gel loading buffer. After boiling at100℃for10min, samples were separated on SDS-PAGE and transferred onto PVDF membrane. After blocked by primary antibodies of anti-NGF, anti-Laminin and anti-β-actin for2hours, membranes were rinsed3times and incubated with horseradish peroxidase-conjugated secondary antibodies. Films were digitally imaged using Image J software.7. After the conventional recovery of RSC96cells, proliferation was done for2-3generations. Group A (control):RSC96were seeded on the plain cell culture plate directly. Group B (experimental):Cells were seeded on the random arranged electrospinning-fibers. Group C (experimental):Seeded on the parallel electrospinning-fibers. Each group containing the equal amount of cells was then placed in the incubator under37℃,5%CO2for3days (medium changed once ad int.). The protein an RNA was then extracted for qPCR test ans Western blot analysis (as mentioned before).Results1. The shape of the Schwann cells were bipolar spindle with bright edge, oval or oblong nucleis, protuberances around cell biopolar with multi-length ranges, cells growed side by side and arrayed in radial or nests under the light microscope, while RSC96cells were typically bipolar spindle or polygon shape and mostly extended long processes, and the shape was much similar with the original generation of Schwann cells. The results of western blotting analysis showed that RSC96cells cultured in vitro can also express NGF and Laminin protein as similar with original generation of Schwann cells.2. Changes were slightly on cell quantity but were not on cell morphology after co-culture; Unactivated macrophages were round shape in same size with no protuberances while activated macrophages were in irregular shape and size and the nerve fragments phagocyted by cell in the body can be observed.3. The gene expression of quantitative polymerase chain reaction assay showed no amplified expression on NGF and Laminin gene among control Groups A and B. Statistical significant in NGF expression differences were found among the three remaining group of RSC96cells (p<0.05). NGF expression of Group E was the highest among groups at the same time, and Group D was higher than Group C, the difference has statistical significance (p<0.05). As the Laminin gene expression among three RSC96cell groups, Group D was statistically significant compared with Group C and E(P<0.05),but no significance was found between Group C and Group E(p>0.05).4. On day2of culture, there is very little NGF and LN in the culture fluid of Group A and B. The relative concentrations of NGF in the other3groups are noticed with significant difference. To be detailed, NGF content from culture fluid of Group5is significantly higher at the same time point than the other groups (P<0.05). it is higher in Group D than in Group C, with significant difference (P<0.05). With regard to Laminin content of the culture fluid, there is no significant difference among the3groups (P>0.05).5. We further tested the protein levels on the day2of cell culture by west blotting. The results show no NGF and Laminin expression in Group A and B. There is significant difference among the other3groups. The NGF expression in Group E is significantly higher than the other groups (P<0.05), and it is significantly higher in Group D than in Group C (P<0.05). The Laminin grey value shows no significant difference among the3groups (P>0.05).6. Detecting mRNA expression by qPCR and protein expression by western blot: According to different culture conditions, cultured RSC96were divided into three groups, group A, group B and group C, and all the three groups were cultured for three days. NGF mRNA expression of RSC96was lower in group B and group C, compared to group A (P<0.05). There was no statistical differences between group B and group C (P>0.05). The laminin mRNA expressed in all the groups, increasing from group A to group C (P<0.05). What’s more, Laminin and NGF protein expressed in all the groups. NGF protein expression had no statistical differences in all the groups(P>0.05). The laminin protein expression increased from group A to group C (P<0.05).Conclusion1. The growth pattern of RSC96cells cultured in vitro was much similar with the original generation of Schwann cells, the cell also expressed neurotrophic factors and laminin and was a kind of ideal substitutes for the original generation of Schwann cells, which avoided the long cycle and complicated primary schwann cells cultivation and long-term poor schwann cell function, etc2. Macrophage cells were capable of stimulating NGF expression for RSC96 cells, and activated macrophages expressed more obviously affected by the activation state; while Laminin expression, which affecting cell migration function of RSC96, was not obvious and without any statistical significance, which needed further study.3. By simulating the surface topology of the axon form (physical factors) to influence RSC96cells, we detect the difference between the expression of NGF and Laminin, found that it promoted the expression of Laminin, and parallel arrangement structure promotion significantly. |
PDF Full Text Request