| The regeneration and function recovery of peripheral nerve is one of the hottest issues in the field of neuroscience. Because of the anatomical and functional characteristics of peripheral nerve, anastomosis can not be achieved ideally in most cases. It is necessary to search for nerve substitutes for the nerve repair. Over the past ten years, with the development of material science and molecular biology, more and more kinds of materials have come out. At present, the emphases of nerve repair research shift from nerve repair technology to nerve regeneration. There are four phrases in the repair of peripheral nerve defects: autologous nerve graft, homogeneous nerve graft, autologous non-nerve graft, and biomaterial substitute.NT-3 plays an essential role in the development of both the neural-crest-derived peripheral nervous system and the central nervous system. Neurotrophin-3(NT-3), composed of 774 amino acid, is belonged to a protein family closely related to nerval structure and fuction. NT-3 regulates development, differentiation and survival of neurons, so it's important in mentaining normal function of nerval system. Studiesshowed NT-3 is important material to survival of peripheral Schwann cells and neurons.In this experiment, tissue engineering nerve was constructed to repair peripheral nerve defects by applying the method of tissue engineering and exogenous growth factor. We have studied the culture of schwanncells in vitro, the complex of cells and biomaterials and the repair of sciatic nerve defects, and established a method to construct tissue engineering peripheral nerve. It is expected for a alternative method for repair peripheral nerve defects. There are four parts in this experiment as follows:1. Culture schwann cells(SCs) in vitro: the aim is to optimize culture condition of schwann cells(SCs) in vitro and obtain more purified SCs to repair peripheral nerve. Methods: Five to seven-day-old SD rat's sciatic nerve were digested by trypsin and collagenase by many times. Cytosine arabinoside and geneticin were used to eliminate fibroblast, and forskolin can promote the proliferation of SCs. 0.125% typsin was used to purify SCs in the course of passage. Results: Large amount of SCs were harvested by this method, then these cells were identified with rabbit anti-Si 00 protein antibody by SABC immunohistochemistry(IHC) methods. The percentage of SCs purified was over 97%. Conclusion: The method used in this paper is an optimal means to culture and purify SCs which can meet the needs of nerve repair by tissue engineering method.2. Cloning human neurotrophin-3 (NT-3) cDNA and constructing its eukaryon vector: The total RNA was isolated by single-step (AGPC) from human liver tissue and its cDNA was gained by PT-PCR. The purified large fragment of pUC19 was digested with double-enzyme and the purified RT-PCR products were ligated and transformed into E. coli XL 10. The white clones were selected and the plasmid was purified, which was further identified by double-enzyme digestion and sequenced. pUC19-NT-3 and pIRES2-EGFP were digested by double-enzyme respectively. The purified 774bp NT-3 DNA fragment of the former and the purified large fragment of the latter were ligated and transferred into E. coli XL 10. The clones were randomly selected and recombinant plasmid was purified, which was identified by enzyme digestion and PCR reaction again. The human NT-3-cDNA was successfully cloned and its eukaryotic expression vector (pIRES2-EGFP-NT-3) was constructed.3. Study of biological effect of NT-3 on the schwann cells in vitro: SCs were transfected with vector pIRES2-EGFP-NT-3. SC proliferation activity was assessed by MTT and flow cytometry(FCM) motheds. NT-3 can promote proliferation of SC significantly.4. Co-culturing the DRG ganglion cells with Schwann cells transfected with NT-3: We estained Schwann cells which stablely expressed NT-3 protein, then co-culture the DRG ganglion cells with these cells, to observe the growth of DGCs in different condition. Re...
|
PDF Full Text Request