The Study Of The Depressant Effect Of ODN-PSsXT-1 On Chondroitin Sulphate Proteoglycans And Its Affect On Repair Of Spinal Cord Injury

Spinal Cord Injury (SCI) is divided into the primary and the secondary, as the former is irreversible, so the focal piont of the treatment is to prevent secondary lesion,remain the remant nerves's function, promote nerve regeneration, It has been thought that after the injury of the CNS(central nervous system),its recovery is impossible.however with the development of molecular biology and cell biology in recent years,providing new means in foundament researches for regeneration of CNS.It has been certificated that the regeneration of CNS is possible. It is believed that the main causes to defect neuraxis regeneration is there are inhibitors in the local surroundings.It has been confirmed that CSPGs (chondroitin sulphate proteoglycans) is the main inhibitor in the collagen cicatricle,which distributed mainly in extracelluar matrix (ECM) and collagen cicatricle.with the further researched with GSPGs , it is possible that the GSPGs come from horizontal cells (HCs), the former bodies of oligodendrocyte (OG) and meninges cells (MC), however the later two is most important. The depresent element of GSPGs is connected with the corn protein chains (GAG).There are few effective antagonism,the Chondroitinase ABC (ChABC)is the early and effective enzyme which can depress proteoglycans(PGs), ChABC can promote the neuraxis's regeneration and recover the nerves'function by the menas to degradate the chains of GAG. Many experiments within or without the bodies have got above simmilar consequences.it is the better means till now.But ChABC can't completely dissolve GAG from the core proteins. The remaining lateral chains can, though have weaker effect, lower the neuraxis's regeneration.so we can not get the expected result. So we must to look for an improved method which has a complete depressive effect on the glycosylation of PGs,which can completely dissolve the chain of GAG,and to resist the depressive effect of GSPGs ,promote the growth of neuraxis.In our experiment we synthesize an enzym: oligodeoxyribonucleotide phosphorothioate nucleotide sequences (ODN-PSs), it can cut inside xylosyltransferase-1's (XT-1) mRNA's, and XT-1 is a primary enzym in PGs glycosylation. So in this way thoroughly reducing special GAG chain's depressant effect, promoting the neuraxis's regeneration and recover the nerves'function, to provide a new strategy aiming at RNA for SCI.I. Methods:Outside body using primitive AS induced and cultured technology, applying transforming growth factor-β(TGF-β) stimulates GSPGs'expression, getting the experimengtal cell model,and then identified through immunohistochemistry. At the same time,we synthesized OND-PSsXT-1(Recorded:RNO295748)and OND-Psscontrol。The cell models are divided into four groups: a.Normal group; b.OND-PSsXT-1+TGF -β; c.TGF-?+OND-Psscontrol; d.TGF-? control. Analyze by menas of CS-56Immunocytochemical stain.,RT-PCR, Western blot. Using cell apoptosis technology to evaluate the expression level of XT-1, GFAP, CSPGs,proving the effect of OND-PSsXT-1.Inside body the SCI Model is founded by improved AllenWD, and then randomly divided into experimental group treated with OND-PSsXT-1 and control group treated with normal sodium (NS), with 30 rats in each group. On the first, fourth, seventh, tenth, fouteenth, twenty-first day, executed five rats randomly, geting injury cord sample to analyze CSPGs, GFAP with RT-PCR, CS-56 Immunocytochemical stain.In the SCI model inject intrathecal directly with XT-IODN-PSs, using Western-blot to kinesisly detect CAP-43, which is a special protein associated with neuraxis'regeneration.II. Resuils:Outside body experiment, using this experimental method, the HC'purity from rat brain is above 98%, fit finely to the requirement of the experiment. Under this condition the cultured HC: by the means of Western blot, RT-PCR,the levels of GFAP, GSPGs are generally the same; In the TGF-βgroup the level is the highest; and the next is the normal, ODN-PSs control group; the ODN-PSsXT-I group is the lowest. The expression of XT-I CSPGs's mRNA, protein decreased in the ODN-PSsXT-I group, statistical significance is clear. With flow cytometry PI stain we find ODN-PSsXT-I can reduce AS apoptosis.Inside body experiment ,the SCI model, after giving intervention of OND-PSs, the protein of GFAP, CSPGs downregulated, statistical significance is clear. So the outside body experiment is furtherly proved truly.it is the synthesized ODN-PSsXT-I can effectively,specificly reduce the expression of GSPGs. The protein of GAP-43 upregulated, statistical significance is clear too.III. Conclusions:The synthetized ODN-PSs which aims at the mRNA of XT-I, can efficiently, specificly reduce the chain of GAG. By this means to depress the expression of GSPGs,the effect is high effective,specific:In the SCI model: ODN-PSs can successfully depress the expression of CSPGs, upregulate CAP-43, these are excellent evidence to prove the generation of neuraxis,it will possiblely become a new treatment for SCI.Prospect:According to the depressing theory of GSPGs,we will advance our researches further, combining other resear methods to exploit genic treatment which can efficiently, specificly inhibit GSPGs.providing new methods to treat SCI in clinic..