Study On The Expression And Effect Of Integrin-linked Kinase In The Course Of Burned Wounds Healing

BackgroundWound healing is an ultimate goal of various acute and chronic wound treatments. There are a variety of cells, extracellular matrix components and regulatory factors in this complex biological process. However, the mechanism of wound healing has not been understood completely. The result of wound healing is still unsatisfactory at the present time. The study of molecular mechanism in wound healing is a focus topic in recent years. We may find some new therapies to improve the speed and quality of wound healing if the basic theory about molecular mechanisms of wound healing is explored clearly.The roles played by collagen, fibronectin and other extracellular matrix (ECM) in wound healing have attracted scholars'attention extensively. On the one hand, ECM plays an important role in filling up the wounds; On the other hand, ECM can also regulate the proliferation, migration, apoptosis and other cell functions through integrin signaling pathway. Integrin-linked kinase (ILK) is a protein kinase discovered recently, which is the key kinase in integrin pathway. In addition to mediates integrin signaling pathway, ILK also mediates the interactions between growth factor signaling pathway and integrin signaling pathway by PINCH. Moreover, ILK plays a crucial role in cytoskeletal protein synthesis and assembly as well.At present, the research about ILK mainly focused on cancer and fibrosis diseases. Some scholars found that ILK had participated in liver, heart, nerves and other tissues and organs repairing after damage, but there were no reports about the effects and mechanisms of ILK in skin wounds healing before. Thus, the purpose of this study is to explore the effects and mechanisms of ILK in the course of skin wounds healing. First of all, we will observe the expression of ILK in the process of skin wounds healing to reveal the relationship between ILK and wound healing. Further more, we are going to investigate the regulation of TGF-β1 on ILK, the influence of ILK on wound contraction and extracellular matrix synthesis. The effects and mechanisms of ILK in skin wounds healing may be initially revealed through this study.PART I Experimental study on the expression of integrin-linked kinase and beta-1 integrin during burned wound healing in ratsObjective To investigate the expression of integrin-linked kinase and beta-1 integrin, as well as their effects, during burned wound healing in rats.Methods Fifteen SD rats were prepared as burned models with deep partial thickness burned wounds by scalding. Three rats were selected to detect the expression of integrin-linked kinase and beta-1 integrin at random in day 1, day 5, day 10, day 15 and day 20 by immunohistochemistry respectively. Normal skins from another 3 SD rats were as control.Results The expressions of beta-1 integrin was significantly higher than those in normal skins and in the healed wounds in day 5, day 10 and day 15. In the other hand, the level of beta-1 integrin was significantly increased in day 10 and day 15, compared with normal skins and healed wounds.Conclusions The expression of integrin-linked kinase and beta-1 integrin were significantly increased in the course of wound healing. ILK plays an important role possibly in the regulation of wound cell proliferation and cell interaction with extracellular matrix via integrin signaling pathway.PART II Study on the regulation of transforming growth factor beta-1 on integrin-linked kinase in human skin fibroblastsObjective To investigate the relationship between ILK and transforming growth factor beta-1 (TGF-β1) in human skin fibroblasts.Methods The human normal skin specimens were obtained from six patients during plastic surgery. Fibroblasts isolated from the skin specimens were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% fetal calf serum (FCS) and TGF-β1(5ng/ml) in TGF-β1 group. Identical fibroblasts were cultured under the same conditions without TGF-β1 in control group.Immunofluorescence cytochemistry were employed to detect the expression of ILK in fibroblasts for 24 hours,48 hours and 72 hours after given TGF-β1, respectively.Results The expressions of ILK in TGF-β1 group increased gradually with time passing. Its value was significantly higher than that in control group at 48 hours and 72 hours.Conclusion The expression of ILK induced by TGF-β1 increased in time-relied manner. TGF-β1 can regulate the expression of ILK to some extent in human skin fibroblasts.PART III Study on the influences and mechanisms of integrin-linked kinase on fibroblast-populated collagen lattice (FPCL) contractionObjective To investigate the influences and mechanisms of integrin-linked kinase on FPCL contraction.Methods The human skin fibroblasts from 4th-6th generation in seven cases of plastic patients were used to construct FPCLs. The fibroblasts of each case were divided into 4 groups. FPCLs were cultured in Dulbecco's modified Eagle's medium(DMEM) containing 10% fetal calf serum (FCS) and LY2940029 (dissolved in DMSO) 50mmol/L in LY294002 group. In DMSO group, FPCLs were cultured in DMEM supplemented with DMSO and 10% FCS. Control group's FPCLs were cultured in DMEM only with 10% FCS. Collagen gels which didn't include fibroblasts were as blank group. The photograpies of FPCL were taken at 24h,48h,72h,96h and 120h respectively. Image pro plus software was adopted to calculate the size of area and contraction rate of each FPCL. The same fibroblasts were transfected with ILK siRNA 100pm and 200pm. Cells transfected with ILK con-siRNA were as a negative control, untransfected as control. The expression of ILK and a-SMA were detected by western blot at 48h after transfection.Results FPCLs in three groups had various extents of contraction, but collagen gels which didn't include fibroblasts appeared no contraction. The contraction rate of FPCLs in LY294002 group was significantly lower than that in control group at 24h and 48h (P<0.05). It was also significantly lower than that in control group and DMSO group at 72h,96h,120h (P<0.05). The expression of ILK and a-SMA in ILK siRNA 200pm transfected group was significantly lower than other groups (P<0.05).Conclusion LY294002 can significantly inhibit the FPCL contraction via blocking ILK-AKT pathway; ILK and a-SMA expression has a positive correlation in human skin fibroblasts.PARTⅣStudy on the influence of integrin-linked kinase on collagen I, collagenⅢand fibronectin expression in human skin fibroblastsObjective To investigate the influence of integrin-linked kinase on collagen I, collagenⅢand fibronectin expression in human skin fibroblastsMethods The human skin fibroblasts from 4th-6th generation in senven cases of plastic patients were divided into 3 groups. Cells were transfected with plasmid expressing ILK cDNA in ILK cDNA transfected group, and with empty plasmid in empty plasmid transfected group. The identical fibroblasts were cultured only in DMEM containing 10% FCS in control group. Real-time quantitative PCR were used to detect the expression of collagen I, collagenⅢand fibronectin mRNA at 48h after transfection in each group.Results The expression of ILK mRNA in ILK cDNA transfected group was remarkably increased comparing with the other two groups (P<0.01); Fn and collagen I mRNA expressions of fibroblasts transfected with ILK cDNA was significantly higher than those in the other two groups (P<0.05); The expression of collagen III in ILK cDNA transfected group was increased, but there was no statistically significant difference (P> 0.05).Conclusions ILK is able to up-regulate the mRNA expression of collagenⅠ,Ⅲand fibronectin of human skin fibroblasts.