The Screening?Cloning And Transgenic Study Of Mouse Fetal And Mouse Associated Scarless Wound Healing-associated Gene

OUTLINESkin is the largest organ in the human body,and its main function is to protcet the body to avoid the infection and injury.Scar caused by skin,soft tissue and organ damage lead to local morphology and histology and pathology change.Scar of of mammals is a repair and reconstruction process,the inevitable outcome of the morphology and the function of the normal tissue is not completely replaced.Scar not only affects the patient's beautiful and mental health,hyperplastic scar,scar contracture,and keloid is able to cause loss of function,the sensorimotor disorder and limit the growth of the infant patients.Scar cancer directly threatens the patient's life.Therefore,the prevention and treatment of scar formation and scar hyperplasia,reduce scar tissue on patients' appearance,function and psychological damage is one of the hotspots and difficulties of modern medicine.It was observed that the regeneration of the organs and the scarless wound healing tended to occur in the lower animals,and the skin,soft tissue injury of the higher mammals tended to be replaced by scar healing.However,in the 1970s,Burrington discovered that the embryo of the sheep that had undergone the surgery was put into the uterus,there was no scar formation on the skin of the lamb after birth.Then Harrisond founded that 17 cases of human embryos 18 to 28 weeks were surgeried and then put back into the womb,also have no scar on the skin after the baby was born,which suggests that human embryonic skin,soft tissue,and part of the organs capable without scar healing and reconstruction after trauma,and thus put forward the "scarless wound ranging" concept,Namely human and higher mammals early and mid embryonic skin,soft tissue and part of the organ has the capable of full reconstruction after trauma and no scar tissue to form.This kind of phenomenon shows that this scar is not ithe only way to heal after trauma of human and higher mammals skin,soft tissue,and organ.The main histological features of fetal skin and soft tissue repair and reconstruction in early pregnancy are summarized as follows:(1)There are not acute inflammatory response or inflammatory response was maintained at a low level after injury in the skin of the embryo;(2)The content of hyaluronic acid(HA)in the ECM of embryonic skin was significantly higher than that of the adult skin,while the synthesis and activity of hyaluronic acid was significantly reduced;(3)The collagen of embryonic skin derma after scarless wound healing is in normal mesh structure,and the collagen in the scar tissue chaotic and various proportion of collagen have obvious difference.According to the above conclusions,several factors related to scar healing in the embryonic skin of higher mammals are obtained:(1)Environmental factors in the uterine:there is a specific relationship between the absence of scar healing and the special physiological environment provided by amniotic fluid around the embryonic wound.Studies confirm that amniotic fluid contains more active ingredients such as prostaglandin(PG),hyaluronic acid can promote wound healing,the active substances in the embryonic tissue repair and reconstruction after trauma has played a positive role.Experiment has proved that the amniotic fluid itself play an important role of the activity of collagenase,and lower the activity of multiple degradation enzyme include hyaluronidase,elastase,cathepsin.Amniotic fluid regulate the the activity of enzymes of collagen and hyaluronic acid to influence the synthesis and degradation of collagen(?,?,?)and increase the content of hyaluronic acid.(2)The fibroblasts(FB)of the skin,soft tissues and organs are the main functional cells of repair,whether in embryos or adult mammals.The speed and timing of fibroblasts to the wound,the synthesis,secretion and proportion of collagen play a key role in the healing process of the wound.Experiments confirmed that fibroblasts from embryonic skin wound migration ability.enhanced obviously than adult skin fibroblasts.A lot of fibroblasts were found accumulation in embryonic skin with injury 3 to 5 days,and the fibroblast of adultsappear migration and secretion in the wound with injury 7 days.(3)There is a special point in the extracellular matrix of fetal skin e:the composition of extracellular matrix(ECM)is significantly different from that of adults,the hyaluronic acid and fibrin in the adult skin wound only have a small amount of secretion in the early trauma,and then rapidly decompose,and it was replaced by various types of collagen(including 75%of type I collagen and 25%of type ? collagen);and the composition and conten of ECM in fetal wounded skin is the same as the normal fetal skin,but hyaluronic acid get a significant rise in adult skin,collagen fibers(percentage of collagen type ? 60%)are arranged mesh.With the gestation time was extended,the healing mode of embryonic skin was gradually transformed from scaless healing to scar healing after birth.If scarless wound healing can be realized in adult skin,soft tissue and organs,it can fundamentally solve the scar damage caused by a series of problems.And he skin,soft tissue and organs with damage and surgery may get perfect reconstruction and even regeneration.Decades of research have shown that scar formation is mainly related to three factors as below:1.The imbalances in the synthesis and deconmposition of extracellular matrix.The research of Sato M,shows that in three dimensional culture system,the mRNA level of pre-alpha I(?)and pre-alpha 1(?)collagen in keloid keloid and hypertrophic scar maintains 20 times higher than normal levels.The experiment of Friedman,DW showed that the efficiency of translation of pre-alpha 1 collagen gene in keloid and HTS have increased,but pre-alpha 1 collagen collagen gene's mRNA level increased with type I collgen generation stablely only in keloid.Arakawa found that in HTS,the content and expression of collagenase mRNA were significantly decreased,while the activity of collagenase decreased significantly.That means the excessive.synthesis and lowering decomposition of collagen ? and collagen ? in wound fibroblasts play the key role in hyperplastic scar and keloid.2.Various types of cytokines have important bidirectional adjustment functions in scar healing.The growth factor? 1,2,3(TGF? 1,2,3)is confirmed to be closely related to keloid and HTS formation,and is also one of the most studied cytokines currently.Occlestong added the corresponding antibodies to reduce the expression of PDGF and TGF?1 and TGF? 2 or exogenous TGF ? 3 in adult mice,rats and pigs of the wound(simulated within the embryo in the womb environment).The research obtained from similar embryonic skin scar healing results.It is indicated that the skin and soft tissues damaged in adult mammals can simulate the process of the repair of embryos without scar in certain conditions.TGF? 3 is a key factor in the non-scar healing of mammalian embryonic tissue.To add exogenous TGF? 3 In adult mammalian skin wound significantly decreased collagen deposition in the beginning of injury in the extracellular matrix,and the deposition of protein affected reshape and reconstruction of the dermal wound.Nerve growth factor(NGF)plays not only to promote nervous system development and differentiation,maintain the normal function of the nervous system,regulating neuropeptide secretion but also plays an important role in such aspects,as well as promoting wound repair and producing new capillaries.Rapala K'sresearch indicated that IL-1? and PGE2 can increase pre-alpha 1(?),pre-alpha I(?)mRNA level of collagen gene,but the collagen product ion decreased by 15%and 34%respectively.Cao PF confirmed HOXA9 gene could indtuc epidermal stem cells to secrete vascular endothelial growth factor(VEGF)which cause excessive proliferation of blood vessels in HTS and keloid.3.The genetic mechanism plays a major role in the formation of' scar,hypertrophic scar and keloid.Tsou R founded that 142 genes were overexpressed in normal scarring and HTS,and 71 gene expression decrease by gene chip detection technology,Stelnicki EJ,found that embryonic and adult skin have multiple homologous genes specifically expressed(such as:HoxB13,PRX-2,MSX-1,MSX-2,MOX-1,etc.)by comparing the gene expression.He bel ieved these genes differentially exprcessed in embryonic and adult skin may be the key factors of perfect reconstruction zand restoration in embryonic skin.The technology used in disease genomics research such as the difference of display technology(DD-PCR),northern hybridization,dot hybridization and gene chip technolgy(genechip)although can fully display mRNA difference between two samples,but is easy to missing trace expression of specific genes mRNA carrying large amounts of genetic information.Those technology may get high rate of false positives or can only comparing the difference between the known genes.Based on this,we designed this experiment with suppression subtractive dization(SSH),mRNA from the skin of BALB/C mice in 14 days of pregnancy and three-months mice were tested.By two-way suppression subtractive hybridization,inhibitory PCR and nested PCR technology,we screened and cloned 23 cDNA fragments and constructed two corresponding cDNA library whichspecific express in the two kinds of skin tissue.Among them,16 cDNA specific expression fragments were obtained from the skin of mice embryos and 7 cDNA specific expression fragments from the mice skin,and these cDNA fragments may be associated with scar formation or scarless wound healing.Subsequently,the cDNA fragments was cloned,gene sequencing and online searching.Finally,the homologous analysis was carried out by using the NCBI blast and DNAstar and other molecular biology software.Most of the cDNA fragments were found to be derived from known genes,and most of them were associated with cell proliferation and apoptosis,two of which were not reported from the mice embryos skin.The two unknown genes were cloned and associated with scarless wound healing genes(f32-5,f4-4),then the complex with the eukaryotic expression vector pcDNA3.1 were constructed.The fibroblasts of BALB/C mice were isolated and cultured in vitro.The two unknown genes were transfected by transgenic technology to cultured fibroblasts,and the proliferation activity of transfected cells was found by morphological observation and growth curve.Chapter I.THE SCREENING AND CLONING OF MOUSE FETAL AND MOUSE ASSOCIATED SCARLESS WOUND HEALING-ASSOCIATED GENE OBJECTIVE:The purpose of this study was to identify differentially expressed genes between the skin of mid-gestational(E14 day)and postnatal BALB/Cmouse which might be involved in wound healing process.METHODS:BALB/Cmouse with pregnancy underwent laparotomy and hysterotomy on embryonic day 14(E14),and skin from twenty fetuses were harvested.Full-thickness dorsal skin was also harvested from thirty three-months-old postnatal mouse.Total ribonucleic acid(RNA)and messenger RNA(mRNA)were purified side-by-side from the harvested skins.Differentially expressed cDNA fragments were screened by suppression subtractive hybridization(SSH).The cDNA fragments were then subsequently cloned,sequenced,and identified through Nucleotide Basic Local Alignment Search Tool(BLASTN)search.RWSULTS:Twenty three differentially expressed cDNA fragments were identified,including sixteen pieces uniquely expressed in fetal mice skin and seven pieces from postnatal mice skin.The known genes identified include RPS29,Nedd5,EndoA,TIEG?and eEF-1?,which may play an important role in scarless wound healing of embryonic mice.Two novel genes uniquely expressed in fetal mouse skin were also identified.CONCLUSION:Twenty three differentially expressed genes in the mid-gestational and postnatal mice skin were identified by SSH.Two novel genes were identified that were uniquely expressed in the mid-gestational mice skin.Chapter ?.TRANSGENIC STUDY OF SCARLESS WOUND HEALING-ASSOCIATED NOVEL GENES FROM THE SKIN OF MOUSE FETALOBJECTIVE:The transgenic technology was used to study the two novel genes specifically expressed in fetal mouse skin.METHODS:The eukaryotic expression vectors of fetal mouse skin specifically expressed genes were constructed.The plasmid vector with exogenous gene pcDNA3.1/F32-5?pcDNA3.1/F4-4 were transfected into the fibroblast in vitro.RWSULTS:The plasmid vector with the novel genes transfected into fibroblast successfully.And the novel genes were found to stimulate the proliferation of fibroblast in vitro culture.CONCLUSION:The authors suggest the two novel genes might play an important role in the scarless wound healing process of BALB/Cmice fetal skin.