▎ 摘　　要

NOVELTY - Preparation of graphene oxide-nano silver/insulin-like growth factor-1 composite dressing comprises pure graphene oxide film (group A), graphene oxide film-nano silver (group B), graphene oxide film-insulin-like growth factor-1 (group C) and graphene oxide film-nanosilver/insulin-like growth factor-1 (group D). USE - Used as poreparation of graphene oxide-nano silver/insulin-like growth factor-1 composite dressing. ADVANTAGE - The preparation has antibacterial effects and growth-promoting properties, and wound healing effect. DETAILED DESCRIPTION - INDEPENDENT CLAIMS are also included for: (I) preparing graphene oxide-nano silver/insulin-like growth factor-1 composite dressing, comprising (i) preparing group A graphene oxide film using graphene oxide film with size of 9x 9 cm thickness: about 25 mu m, (ii) preparing graphene oxide-dopamine film by (ii.i) dissolving in 100 ml deionized water by adding 131.14 g tris hydrochloric acid, and adding 200 mg dopamine powder to the resulting tris solution to obtain tris-dopamine solution with a concentration of 2 mg/ml and a pH of 8.5, (ii.ii) soaking graphene oxide film obtained in step (i) in the above solution for 12 hours, transferring the mixture to a vibrating screen and vibrating at a speed of 100 rotations/minute at 37 degrees C, (iii) preparing group B graphene oxide-nano silver samples by first dissolving daptomycin powder in deionized water to prepare 10 mg/ml nano-silver aqueous solution, carefully cleaning graphene oxide sample in step (i) with deionized water, incubating in an aqueous solution of daptomycin, where incubation temperature is 37 degrees C and vibration speed is 100 rotations/minute and incubating for 12 hours, (iv) preparing group C graphene oxide film-insulin-like growth factor-1 comprises dissolving graphene oxide-dopamine membrane prepared in step (ii) in a 10 mu g/ml insulin-like growth factor-1 solution at 37 degrees C, vibrating at a speed of 100 rotations/minute and incubating for 12 hours, and (v) preparing group D oxidized graphene film-nano silver/insulin-like growth factor-1 comprises adding graphene oxide film-dopamine to step (iii) and same concentration of nano-silver/insulin-like growth factor-1 mixture in step (iv), incubating at 37 degrees C with a vibration speed of 100 rotations/minute and incubating for 12 hours; and (II) graphene oxide film-nano silver/insulin-like growth factor-1 composite dressing wound healing method, comprising (i) spraying gold on the samples of group A and group D and drying them thoroughly, then observing obtained thin film in vacuum with a scanning electron microscope instrument, and taking detailed picture of the pore structure, (ii) characterizing chemical structure of the prepared sample in the wavenumber range of 600-4000 cm-1 through Fourier infrared spectroscopy, (iii) placing four groups of ABCD samples horizontally, dropping 1 mu l deionized water on the surface of each material, and measuring contact angle with formed droplets for 12 hours, testing each sample three times to obtain average angle value, (iv) culturing and amplifying Escherichia coli and Staphylococcus aureus bacteria to a density of 1x 109 colony-forming unit (CFU/ml), diluting with lysogeny broth to a density of 1x 104 CFU/ml and extracting 100 ml bacterial solution, using 96-well plate, placing each ABCD sample in 3 wells, dropping 200 mu l bacterial solution into each well and incubating for 24 hours at 37 degrees C incubation temperature, using spectrophotometer to evaluate change of the bacterial solution, setting standard OD value of the bacterial stock solution to 0.7, testing OD value again after 24 hours to observe the influence of different materials on the bacterial stock solution, (v) deriving primary fibroblasts from normal newborn mice and passaging cells further to second and third generations, using 96-well plate for cell counting and culture, 2000 cells/well, culturing each group of samples and vascular endothelial cells together, each group has 3 wells and testing first, third, fifth and seventh day sequentially, incubating at 37 degrees C, adding lysogeny broth medium solution (150 mu l/well), performing above operation in triplicates, (vi) using Dulbecco's modified eagle's medium to culture after seeding the vascular endothelial cells in a 24-well plate (2x 104/well), using pipette tip to make a scratch, recording the time as 0 hour, co-culturing four groups of ABCD materials with cells and observing for 24 hours with a live cell workstation microscope, setting 6 repetitions for each group, and using ImageJ1.48V software (NIH, USA) for specific measurement and performing above operations in triplicate, (vii) anesthetizing mice by intraperitoneal injection of sodium pentobarbital (1%, 70 mu l/g), establishing full-thickness skin defect model with a hole punch, where defect area is 0.6 cm in diameter, adding bacterial solution (5 mu l/l 108/ml), adding dropwise to each wound to cultivate E. coli and Staphylococcus aureus, and disinfecting material with 75% alcohol, rinsing with phosphate-buffered saline solution to thoroughly remove impurities, applying prepared film on the wound, then fixing with viscose towel, taking photos 1, 3, 5, and 7 days after the injury to replace the material and using commercially available chitin dressing (CCD) as a positive control, (viii) comparing wound area before and after wound healing, calculating healing rate, using IPP6.0 software to assist, choosing target wound area according to the area of interest (AOI) function, using size counting method to measure the pixel area, calculating wound area by the formula of wound healing rate = (wound area-wound area after healing for a certain period of time)/wound areax 100%, (ix) detecting expression of PCNA and CD31 by Western Blot method on the 7th day after application, small square of about 10 mm x 10 mm, sampling from the defect wound including epidermis and granulation tissue of mouse full-thickness wound and immediately placing in liquid nitrogen to freeze and then lysing to extract the protein, diluting anti-CD31 antibody, anti-PCNA antibody at 1:1000, diluting anti-tubulin antibody 1:2000, maintaining all antibodies at 4 degrees C the night before use, diluting goat anti-rabbit secondary antibody labeled with horseradish peroxidase at 1:2000, incubating sample with the sample at 25 degrees C for 1 hour, washing in tris-buffered saline with 0.1% Tween 20 (RTM: Polysorbate 20) (TBST) 5 times, and sending the collected polyvinylidene fluoride (PVDF) membrane to chemiluminescence detection, and (x) using origin software to analyze the significant differences between the two groups and above by using one-way analysis of variance and two-way analysis of variance, respectively and expressing experimental data as mean plus minus standard deviation and considering P less than 0.05 statistically significant. DESCRIPTION OF DRAWING(S) - The drawing shows a schematic diagram of a graphene oxide film.