Function And Mechanism Research Of Recombinant Adenovirus Expressed CCN1 In Healing Process Of Combined Radiation And Wound Injury

ObjectiveCombined radiation and wound injury (CRWI) often occurs in nuclear accidents and attacks. Prolonged wound healing process is its primary character. Cysteine-rich 61(Cyr61/CCN1) is a member of immediate-early gene family CCN, it appears to be important regulator of diverse cellular functions such as cell cycle progression, division, adhesion and proliferation. It has been implicated as important regulator of development, angiogenesis, tumor growth and wound healing. Thus, its role in healing process of CRWI is indefinite. In this study, we firstly establish a rat model of CRWI, and observe CCN1 expression in this model. After that, we construct adenovirus AdCCN1 and observe its infection efficiency to CHO cell lines and granulation tissue cells, to confirm the appropriate expression and increased secretion of CCN1. And then investigate the expression of some cytokines and proteases involved in wound healing in granulation tissue infected AdCCN1 by subcutaneous injection, to approach function and mechanism of CCN1 in CRWI.Methods and Results1.Establishment of rat CRWI models and expression detection of CCN1 in granulation tissue:SD rats were divided randomly into simple wound group and CRWI group. CRWI group was exposed in 5 Gy 60Coγ-ray full-bodies and a dorsum full-thickness wound was created in one hour. On day 0, 3, 6, 9, 15, 21 after injury, wounds and surrounding skin areas were surgically obtained.CCN1 mRNA and protein were measured by RT-PCR and immunohistochemical staining. Compared to simple injury control, CCN1 protein and mRNA expression were down-regulated in CRWI granulation tissue before day 15, however up-regulated on day 21. 2.Construction of recombinant adenovirus expressed CCN1pCMV-CCN1 was digested by restriction enzyme and inserted into digested pAdTrace-TO4 shuttle vector. Linearized recombinant vector pAdTrace-CCN1 underwent homogenous recombination in AdEasy-1 strain of E.coli. Then 293 cell lines were transfected to pack viral particles. After propagation in 293 cell lines, viral particles infected CHO cell lines and granulation tissue cells. Western blot and immunohistochemical staining were used to affirm the secretory expression of CCN1 in CHO cell and increased secretion in granulation tissue cells.3.Function and mechanism research of AdCCN1 in healing process of CRWI:AdCCN1 was administrated through subcutaneous injection after injury. Equivalent control virus AdRFP was applied at the same time. 7 days later, wound surface tissue samples were obtained to make paraffin section. Compared to control, quantity of fibroblasts and micro vessel density in granulation tissue infected AdCCN1 in HE staining sections and proteins involved in wound healing- MMP1, TIMP1, bFGF were up-regulated expression in IHC sections.Conclusion1.CRWI models of rat were established efficaciously. Compared to simple wound injury control, CCN1 protein and mRNA expression were down-regulated in CRWI granulation tissue before day 15, however up-regulated on day 21. Decreased expression of CCN1 is a possible cause to prolonged healing process of CRWI.2.Utilizing the AdEasy system, recombinant adenovirus AdCCN1 was prepared. Restriction enzyme digestion identification confirmed its correctness. It could efficiently infect CHO cell line and granulation tissue cells; CCN1 was expressed appropriately and secreted increase.3.Possible mechanism of AdCCN1 in healing process of CRWI: Increased secretion of CCN1 after AdCCN1 infected granulation cells leads to elevated expression of cytokines and proteases involved in wound healing, including MMP1, TIMP1 and bFGF, thereby leading to ECM remodeling and wound healing.