Detection And Analysis Of Differentially Expressed Genes Profiles Between Hypertrophic Scar And Normal Skin In Bama Minipig

OBJECTIVE: To establish a model of dorsal skin hypertrophic scar of Bama Minipig and screen out differentially expression genes between hypertrophic scar and normal skin though microarray analysis,and to explore the molecular mechanism underlying hypertrophic scar.Methods: Six healthy Bama Minipigs were selected to establish dorsal hypertrophic scar model of Bama Minipig.6-8 skin wound wells were made to the left and right side of the swine back.Each well was 3×3 cm in size.Burn wounds were created on the right side of dorsal skin at 100? for 20 S scalding by a digital thermodevice.Non-full-thickness skin grafts were cut on the left side of back to create incisional wound wells and the skin grafts were referred as normal skin tissue control group.All wounds were exposed heal after hemostasis.Wound healing and scar tissue specimens were collected at day 21 and day 56 after the dorsal hypertrophic scar model of Bama Minipig was established.The total RNA was extracted from the normal skin and the day 56 of incision and burns.The double-stranded cDNA was synthesized and the biotin-labeled cRNA was transcribed and synthesized.The gene was cloned with Affymetrix GeneChip? pig genome array.Gene Ontology(GO)Function Analysis and KEGG Pathway enrichment analysis method were carried out to category Representative genes.According to bioinformatics,KEGG pathway was used to analyze some of the representative genes in the Hippo signaling pathway were selected and verified by reverse transcription polymerase chain reaction(RT-PCR)and immunohistochemistry.Results: 1.Gross view: Day 56 after the dorsal hypertrophic scar model of Bama Minipig was established,tough cord-like hyperplasia tissue protruding pig skin surface could be observed in the central part of the wound.The vanishing of dermal papilla and increased collagen fibers could be observed by HE staining,which suggested that the model was successfully established.2.Microarray data analysis: Compared with the normal skin tissue of Bama Minipigs,897 differentially expressed genes in hypertrophic scar were screened out,of which 234 were up-regulated and 663 were down-regulated.3.The results of GO functional analysis showed that these differentially expressed genes were mainly involved in the biological processes such as skin development,epidermal cell differentiation and negative regulatory metabolicprocesses.Most of them constitute cell connections,ion channel complexes,polyribosomes and other cell components,and were involved in metal enzymes,amylase activity regulation and other molecular functions.4.The results of KEGG pathway analysis indicated that the differential genes were significantly enriched in 15 pathways.These genes were mainly involved in Hippo signaling pathway,TGF-? signal pathway,arachidonic acid metabolism,amino acid metabolism,et al.5.Real-time PCR results showed that compared with normal skin,the mRNA expressions of CTGF,BMP-7,ID1 and CDH1 in hypertrophic scar tissue were significantly different(P <0.05)at the day 56,and the expression of CTGF mRNA was up-regulated while BMP-7,ID1,CDH1 mRNA expression showed a downward trend,which were consistent to the microarray analysis.6.Immunohistochemistry results showed that the expressions of CTGF and BMP-7 in hypertrophic scar tissue were significantly different(P <0.05)from that of normal skin at the day 56.The expression of CTGF was up-regulated while the expression of BMP-7 was down-regulated,and this result was consistent to the microarray analysis.Conclusion: A model of dorsal skin hypertrophic scar of Bama Minipig was successfully established.Differentially expression genes between hypertrophic scar and normal skin were screened out and these genes were involved in multiple pathways.Hippo signaling pathway and TGF-? signalingpathway was found to be related to the formation of skin scars and CTGF and BMP-7 may play a role in the formation of skin scars.