An Experimental Study To Identify The Target Genes Of Transcript Factor Stat3 In Activated Schwann Cell

IntroductionIt remains as a clinical challenge in the treatment of peripheral nervedefects. The current gold standard for the bridging of significantperipheral nerve defects is autologous nerve grafting. This method hasbeen shown to be effective, but itself has some disadvantages which havelimited value in clinical practice. This has shifted the focus of thesearch to tissue engineering conduits. Following by the development ofconduits materials research and the cognition of mechanism of peripheralnerve pathology, tissue engineering has turned into a promising protocolin bridging peripheral nerve defects. Activated Schwann cell (ASC), asthe ideal seed cell of tissue engineering conduits, has the ability ofquick proliferation and to actively stimulate repair with neurotrophicfactors An active area of research is the systemic research of themechanism of its high bio-activity. The objective of this study is to findout the reason why the ASC could promote axonal regeneration across longerdefects prior to the normal Schwann cell (NSC) at the transcript and genelevel. Three parts involved in this research, following as:Part 1 Application of melanocyte growth medium in the cell culture ofSchwann cell from adult SD ratsObjective: To find out a effective approach to obtain high purifiedSchwann cells from the sciatic nerve of adult ratsmethods: 20 SD rats with 1 week predegeneration , weight: 150g, afterenzymatic digestion(0.25% Trypsin, 0.1% collagenase) of sciatic nerve,the suspension were cultured in group A (DMEM/F12 medium) and group B(melanocyte growth medium).cold jet as the passage method. S-100 stainfor detection of Schwann cell purity.Results: the cell yields and purity of group B was significant higher thanthat of group A. as the contrast, the control group, which has only 30%purity of Schwann cell.conclusions: the combination of melanocyte growth medium and cold jetprovides a convenient mean to obtain considerable Schwann cell with purity beyond 90%.Part 2 To identify the activated transcription factors of activatedSchwann cell using TranSignal^TM Protein/DNA Array techniquesObjective To identify the activated transcription factors ofactivated Schwann cell and normal Schwann cell. Methods TranSignal^TMProtein/DNA Array techniques were adopted to scan the activatedtranscription factors. Results Lattice of activated Schwann cellsshowed that there were 6 different transcription factors raised theirexpression 5 times than that of normal Schwann cells. Including PEPCKpromoter GR,NF-E2(2),Stat3,NFE-6/CP1,PRDâ…¡-BF1,NFkB(2).which may giveus the possible explanation of special condition of activated Schwanncells at transcript level. Conclusion Total 6 different transcriptionfactors correlated their level with activated Schwann cells' quickproliferation and vigorous secretion condition.Part3 Identifies the primary target genes of transcript factor stat3in activated Schwann cell/normal Schwann cell by the approach ofChromatin immunoprecipitation on chip assay(ChIP on chip)Objective to identify the primary target genes that directly under controlof transcript factor star3 in activated Schwann cell and normal Schwanncell, and to find out the possible differences between ASC and NSC tounderstand the mechanism that why ASC perform more bioactivity at the genelevel. Method scanning the whole genome of ASC/NSC by the application ofChromatin immunoprecipitation on chip assay(ChIP on chip),immunoprecipitated the target gene with specific antibody of transcriptfactor star3 Result 385,000 genes were scanned in ASC/NSC, 361 of whichwere bound to the stat3 both in ASC and NSC, we identified 122 kind of genes that only bind to star3 in ASC, some of these genes are "Atoh1,bex1,F2RL1,polg,Rcan2" responsible for the quick proliferation ofASC and has the protective effect from apoptosis. Conclusion the differenttarget gene of star3 in ASC/NSC may explain the mechanism of ASC highbioactivity.