| Therapy of peripheral nerve injury is continuously studied and the methods are improved constantly for many years. Cooperating with Dalian Institute of Chemical Physics,Hu zhaohua in our department make a good progress of studies on transplanting microencapsulated nerve tissue/cells in stimulating peripheral nerve regeneration in 2001. This research is focus on cell survival, secretion of nerve growth factor (NGF) and affection on regeneration of peripheral nerve after transplanting the microencapsulated NIH3T3 cells transfected with NGF for searching better methods in therapy of peripheral nerve injury.Methods: LA recombinant NGF (pcDNA3.1+/NGF) was constructed in which the DNA sequence coding the human IL-2 signal peptide was linked to the leader sequence of NGF cDNA;2. pcDNA3.1+/NGF was trsnsfected into cultured NIH 3T3 cells using FuGENE? transfection reagent in vitro,proved its expression and its biological activity of protein by Western Blot immunohistochemistry and the neurite outgrowth of PC 12 cells stimulated by culture supernatant;3.The genetically modified cells expressing NGF gene were enclosed wihin the microcapsules which were made from alginate-polylysine-alginate (APA) in vitro before implantation,and the growth and NGF secretion of the cells were measured periodically by means of MTT and ELISA;4. The microcapsulated NGF-expressing cells was transplanted into theinjured sciatic nerve.Explore the effects of NGF secreted by the microcapsulated cells on the regenerating axons by means of methylamine blue stain, immunohistochemistry and electronic microscope. Explore the effects of NGF on the functional recovery of the regenerating nerve by means of electrophysiological assessment,SFI(sciatic nerve function index) and morphometric analysis.Resultes: 1 .The eukaryotic expression plasmid pcDNA3.1+/NGF has been constructed successfully;2. pcDNA3.1+/NGF has been transfected into NIH 3T3 cells successfully .The culture supernatant could stimulate the neurite outgrowth of PC12 cells, indicated the stable expression of NGF protein with good biological activity was observed;3.Over the 70-day observation period, the genetically modified cells were able to grow and survive in the microcapsules. The secreting of NGF coincide with the cell viability,the higher the cell activity the more the amount of NGF secreted at the initial stages .The microcapsules remained well after the long-time culture and the changes of NGF secretion and the cell viability were in a same manner at the metaphase;4.The microcapsulated NGF-expressing cells could survive,grow and secret NGF positively in vivo for a long time(70 days).There was no apparent inflammatory response from the host tissue. The amount of regenerating axons was larger in the group transplanted with microcapsulated NGF-expressing cells, and the myelin sheath was thicker as well. The regenerating nerve fibers were parallel, and were arranged orderly. The recovery time of the regenerating axons'ultrastructure last shorter .The SFI, electrophysiologic parameters mean NGF secreted by the microcapsule can significantly enhance the recovery of the function of the injured sciatic nerve. Conclusion: By constructing pcDNA3.1+/NGF successfully, we transfectedpcDNA3.1+/NGF into NIH 3T3 cells and the transfected cells could express NGF stablely. The transfected cells were then microcapsulated and were transplanted into the injured sciatic nerve .With a series of tests such as MTT, ELISA, immunohistochemistry ,electronic microscope,SFI and morphometric analysis,we found: 1. Alginate-polylysine-alginate (APA)biomicrocapsule is a simple and safe way in protecting living cells from immune rejection for transplantation therapies;2. The microcapsulated gene altered cells could survive,grow and secret active NGF continuously in vivo;3.NGF can improve the regenerating peripheral nerve both in its structure and functional recovery definitely; 4.It was an effective and applicable way for peripheral nerve injury repaire by using microcapsulation of NGF gene altered cells.
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