Experimental Study Of The Impact Of Fusion Protein Of Neural Cell Adhesion Molecule L1-Ig(1-4) On The Injured Spinal Cord

The functional recovery of the injured spinal cord remains a difficult problem for neurologists. So far, there has no breakthrough in theory and no effective treatment in clinic. The major reason might lay in the complicated mechanisms of primary and secondly reactions following the spinal cord injury about which a little is known. One possible reason is that the ability of the regeneration of the injured neuron is very limited and cannot be improved by modern methods. Another is that the regenerated neural axon lacks the ability to transform effective impulses. However, after years of hard working, neurologists had made a great progress in neurological area and found many neurotrophilic fectors such asNGR BDNF, NT-3, LI, etc. Neural cell adhesion molecule LI (LI), discovered in the 1980's and widely existed in neuron, Schwann cells, epithelium of intestine and some tumor cells, can promote the rebirth of neural axon and mediate the interaction of cell-to-cell and cell-to-matrix. It plays an important role in promoting the growth and conglomeration of neuritis, the migration of neural cells, and the formation of the myelin, in restructuring neural signaling ways and transducing trans-membrane signals, and in inhibiting apoptosis of neural cells. It can even work on immune system and formation of the tumor. Studies found that specific domains of the complete structure had some certain bioactivities like promoting the growth and adhesion of neurons, and the fragments can be resolved and transferred to far distance. Because of this, it becomes a possibility for treating injured spinal cord and other neural diseases. So research of the function and mechanism of fragmental protein has become a major subject for many neurologists. Foreign studies have been trying to understand the functions of LI fragments, but functions of the fragment of Ll-Ig(l-4) are seldom reported. So we designed to get this fragment and further to investigate its bioactivity in promoting growth of neuron, which will provide a sounding foundation for clinical use and a further study of its mechanisms.The PC.12-cell line has the character of multi-differentiation under differentconditions, and has been extensively used as a model for testing the activity of neural trophic factors and observing neuron growth. In natural serum-containing medium, PC-12 cells undergo mitosis. But when exposed to NGF or FGF, PC-12 cells could stop splitting and transform to neuron-like cells. Previous studies found that LI can stimulate neural axon-like growth of PC-12 under the help of NGF. This study will utilize the character to detect the bioactivity of fusion protein in promoting the growth of ner\e cells. And another experiment hi neurons of the spinal cad, the most important organ in bodies, was also made to determine the biological activity of fusion protein cultured in vivo.In this study, hippocampi of a four-day old rat was used as the origin for acquiring LI gene. LI was amplified by RT-PCR and cloned into a pET28a(-H)vector. GFRa 1 was high expressed in E.coli after IPTG induction. By Ni2+chelation affinity chromatography, the purified recombinant Ll-Ig(l-4) protein were obtained. The biological activity of the recombinant protein was tested by above methods. The experiment was got along by using a moderate contusive SCI model and puting catheter infradura to give durg periodically. The results were evaluated first through behavioral test of Basso, Beattie, Bresnahan locomotor rating scale and inclined plane test, then through electrophys (logical MEPtest, and measuring remained spinal cord area, forth NF immunohistochemical staining and Western blot test, finally through the retrograde transport of the horsradish peroxidase (HRP). The capital results of the experiment are as follows: Firstly, Ll-Ig(l-4) gene and reconstruct expression vector The cDNA encoding Ll-Ig(l-4) of the rat was isolated using RT-PCR with total RNA extracted from newborn SD-rat hippocampus tissue. cDNA was amplified by RT-PCR, then the expression plasmid pET-Ll-Ig(l-4) was co...