The Effects Of Intergrin-linked Kinase In The Process Of Burn Wound Healing On SD Rats’ Skin

BackgroundWith the growth of the social trend of population aging, the incidence of diabetic ulcers,radiation ulcers, pressure ulcers and other chronic wounds increase significantly. Therefore,wound healing has become one of the focuses of modern medicine research. Wound healing is acomplex biological process that related to the structure of the epidermis and dermis tissueremodeling. The mechanisms of wound healing have not been elaborated clear. The result ofrepair of tissue has not been satisfactory. Therefore, Molecular mechanisms of wound healinghave become the focus for study. Further exploration for the molecular mechanisms of woundhealing will be beneficial for developing ideas and methods of wound treatment and improvingthe quality of wound healing.Interin-linked kinase (ILK) is a widely expressed serine/threonine kinase, which mediatedthe regulation of PKB/AKT, Wnt, LEF-1and β-catenin and other cell signaling pathways,biological functions of cell migration, proliferation, differentiation and apoptosis. Recent studieshave found that ILK played an important role in maintaining the skin dermis, and it was essentialfor normal cell response component in wound healing. Our research group also found that therewas a high correlation between wound healing and ILK, ILK may involve in the downstreamsignaling pathway through AKT, and affected the migration, differentiation, proliferation andapoptosis of the fibroblasts at the cellular level. However, the role of ILK on the mechanism ofwound healing need to still further explored.This study intends to transfect surrounding tissue of the deepⅡ°burn wound in rats withILK recombinant lentivirus to upregulate the expression of ILK, and use ILK inhibitorQLT0267inhibit the activity of ILK. The role of ILK and the relationship with Fn, collagen I,α-SMA and AKT phosphorylation in the process of wounds will be observed. Regulation onexpression of ILK in animal wound models, which will help to supplement the theoreticalmolecular biology of wound healing, to provide new ideas on wounds in gene therapy. PARTⅠ Preparation for animal model of burn woundObjective Stable model of burn wounds in rats was prepared.Methods20SD rats were randomly divided into four groups. The instrument withtemperature of80℃, weight0.5Kg and bottom area2.5cm2was placed on rat skin of two sides onback for4,6,8and10s respectively. Samples of the wound tissue were collected forhistopathological examination after24hours.Then, injured time was selected according to thepathological examination for deep Ⅱ°burn wound.Results The injured time for deep Ⅱ°wound model in rats was8seconds according topathological examination.Conclusion Application YLS-5Q type burns meter base area2.5cm2, mass0.5kg, hot headtemperature80℃, reaction time8s injury SD rats dorsal skin deep Ⅱ°burn model can beprepared.PARTⅡ ILK gene transfect the burn wound and its effect on the rate ofwound healingObjective To explore the effect of tranfection of ILK gene above the wound on rate ofwound healing.Methods The Lentivirus reconstructed with ILK were packed by using three plasmidsystem transfected into293T cells. The ILK expression of target gene was investigated withWestern Blot.75SD rats were divided into5groups randomly with15rats per treatment. Thedeep Ⅱ°burn wounds were built on rats’ backs.①Control group:100μL saline was injected perday;②LV-GFP group: Blank plasmid lentiviral100μL（2E+8TU/mL）was injected for one time;③LV-GFP-ILK group:100μL（2E+8TU/mL）over expressive ILK of lentivirus was injected forone time;④QLT0267group:100μL100μM QLT0267solution was injected daily;⑤DMSOgroup:100μL0.3%DMSO solution was injected daily. The experiment lasted for21d. The ratswere feed alone, water and food were given freely. Wound healing time and healing rate weremeasured for each group after21days. Lentivirus solution was injected subcutaneouslysurrounding in LV-GFP-ILK group the wound. The samples of five rats in each group were harvested after14days, and were done for frozen sections, GFP expression was observed underan inverted fluorescence microscope. The expression of ILK protein were detected with WesternBlot on day7, day10, day14and day21after transfection. The data was analyzed statistically.Results Expression of GFP in frozen sections of wound tissue were shown underfluorescence microscope in LV-GFP-ILK group, and its level of ILK expression was higher thanthat in LV-GFP group. The rate of wound healing was96.89%in LV-GFP-ILK group on day14.The rate of wound healing was56.32%in QLT0267group on day14, it was obviously lowerthan those in other groups（P<0.05）. There were no significance among Control group, LV-GFPgroup and DMSO group in each time point in rate of wound healing (P>0.05).Conclusion ILK recombinant lentivirus successfully infected the normal tissue surroundingthe wound by subcutaneous injection, and it could raise the level of ILK protein and maintainedthis effect for a relative persistent, stable period of time. The rate of wound healing increasedwhen the expression of ILK increased and the rate decreased when the activity of ILK wasinhibited. It indicated that ILK is a promoting factor of wound healing.PART Ⅲ The role of ILK in wound healing process and therelationship between ILK and Fn, collagen I, α-SMA, AKTphosphorylationObjective To investigate the effects of ILK on wound healing and its possible mechanisms,Fn, collagen I, and α-SMA were detected by increasing the expression of ILK or inhibiting theactivity of ILK activity and inhibition of ILK in vitro.Methods Grouping and management of animals was the same as Part II. SP and WesternBlot were applied to detect the expressions of ILK, AKT and PAKT in wound of rats after14days, fives rats were selected randomly from each group. Histological staining was applied forgranulation tissue of the wound to observe the changes of the wound and surrounding tissuestructures. PCR was applied to detect the expressions of Fn mRNA, collagen I mRNA, ILKmRNA and α-SMA mRNA.Results There was no significance in AKT protein expression among five groups (P>0.05), the expression of ILK protein in LV-GFP-ILK group was significantly higher than those in other4groups (P <0.05), and its PAKT protein was also higher significantly than other4groups (P<0.05). The new epidermis and collagen deposition were also higher in wound in this group; Theexpression of PAKT protein in QLT0267group was significantly lower than those in othergroups (P <0.05). PCR analysis showed that expressions of Fn mRNA, collagen I mRNA andα-SMAmRNAincreased significantly as the expression of ILK mRNAincreased.Conclusion Raise the level of ILK Burn wound tissue can significantly up-regulate the levelof Fn, collagen I and α-SMA; ILK activity and PAKT protein expression was positivelycorrelated. Therefore, ILK may promote wound healing by the way of PI3K-AKT-PAKT.