| BackgroundHypertrophic scar is one healing result after trauma or severe burns,which is difficult to avoid.Continued scarring may lead to skin itching,limited activity,deformity and many other issues,which impacts the patient's physical and psychological health seriously.According to statistics,every year there are millions of people suffering from poor quality of life brought by hypertrophic scar.As the traditional treatments are difficult to produce satisfactory results,so there is an urgent need to find a new treatment to prevent hypertrophic scars.1991 Bradham firstly found human CTGF was existed in human umbilical vein endothelial cell culture medium.As a kind of stromal cell protein,CTGF was mainly expressed in fibroblasts,hepatic stellate cells,chondrocytes and other stromal cells.The expression of CTGF was increased with the body growth and development or damage.the pathophysiology of CTGF is mainly to stimulate cell mitosis,adhesion,apoptosis,ECM synthesis and secretion of other types of cell migration.Besides,it can also change the activity of other molecules.Recently,more studies have confirmed that CTGF was overexpressed in the dermis of pathological scar,but the specific mechanism is still not clear.Interference RNA(RNAi)is a short double-stranded RNA.As a gene silencing technique,RNAi can be complementary to m RNA and promotes degradation,and ultimately brings a specific gene blocking effect.siRNA is one kind of RNAi technology.Compared with antisense nucleic acid technology,siRNA consists of some advantages,such as high gene inhibition rate,specificity and low concentration,so it has been widely used in gene function research.However,due to the complexity of the environment in vivo and in vitro such as the presence of enzymes,bare siRNA can be easily degraded before entering the cell and nucleus,so the transfection efficiency is low.Although some chemical modification or virus carrier can delay its degradation,but they can bring cytotoxicity,poor results and other shortcomings.Thus,it is urgent to find a suitable vector to mediate siRNA into cell and exert gene blocking effect.Aa a non-viral vector,Chitosan have some advantages,such as low cytotoxicity,lowimmunogenicity,good biodegradability and good biocompatibility.In addition,as the only natural cationic material,chitosan can combine with negative charged nucleic acids such as DNA,siRNA and miRNA to form nanocomposites.Chitosan nanoparticles increase the efficiency of siRNA interference by enhancing the adhesion with negative charged cells and avoiding the siRNA degradation by endogenous nuclease.Recently,as a safe and economical non-viral vector,chitosan nanoparticles has gained more and more attention.Based on the above background,this study was carried out by applying siCTGF-loaded chitosan nanoparticles into the new healed wound of rabbit ear skin.By interfering CTGF expression and its biological function,we inhibited the excessive proliferation of fibroblasts and reduced the deposition of collagen.Ultimately,it reduced scar formation.Thus,siCTGF-loaded chitosan nanoparticles may become a safe and effective treatment for scar formation.Part? siRNA interfering the expression of CTGF gene in scar fibroblastsObjective: To isolate and identify primary scar fibroblasts;to confirm the effectiveness of chemical synthesis sequences and choose the optimal interference sequence.Methods: Combined tissue adherence culture with trypsin digestion,.scar fibroblasts were isolated and cultured rapidly.Then,scar fibroblasts were identified by flow cytometry.Based on the principle of siRNA design,three groups of RNA interference sequences were designed using RNA online design software.RT-PCR and Western blot were used to detect the the expression of CTGF mRNA and protein in the cells.Results: scar fibroblasts were isolated repaidly with high activity by modified method,and the positive rate of FSP was 94.37 ± 1.31% in flow cytometry.The expression of CTGF mRNA was reduced to 0.294±0.093 fold and the expression of CTGF protein was decreased to 0.33±0.065 fold,the difference was statistically significant(P <0.05).Conclusion: Through the modified method,we can isolate scar fibroblasts with hign activity,and the siCTGF-3 has the hignest blocking efficient in scar fibroblasts.Part ? Preparation and characterization of siCTGF chitosan nanoparticlesObjective: To study the preparation method of chitosan nanoparticles and its related physical characterization.Methods: Chitosan nanoparticles were prepared by the modified ionic gelation method,which was carried out by exploring the conditions such as molecular weight,nitrogen and phosphorus ratio.Particle size analyzer and transmission electron microscope were used to detect the average particle size and potential of the nanoparticles.loading efficiency was calculated by UV spectrophotometer.At the same time,the controlled release period and cytotoxicity test were carried out in vitro.Finally,we used siCy3 to trace whether they can enter into cells.Result: TEM showed that chitosan nanoparticles were round particles with uniform size and uniform distribution,and the average particle size was about 98.3±2.7 nm.The zeta potential was measured by particle size analyzer,which was 15.3 ± 4.2mV.The entrapment efficiency of siCTGF was 96.5 ± 2.4%.In PBS solution,controlled release cycle can be up to 7 days.Compared to the blank control group,there was no significant cytotoxicity in chitosan nanoparticles(P> 0.05).Conclusion: Our results showed that chitosan nanoparticles prepared by modified method have the characteristics of smaller particle size,low cytotoxicity and long release cycle,which are a good gene transfection vector.Part? Evaluation the fibrosis genes interfering effect of siCTGF-loaded chitosan nanoparticles on scar fibroblasts in vitroObjective: To evaluate the inhibiting effect of siCTGF-loaded chitosan nanoparticles on fibrosis gene in vitro.Methods: The experimental group was divided into the blank control group,the siNC-loaded chitosan nanoparticles group and the three concentrations of siCTGF-loaded chitosan nanoparticles(50nmol/ml,100nmol/ml,200nmol/ml).The expression of CTGF,type ? collagen and cell differentiation marker ?-SMA was determined by RT-PCR and Western-blot.We use CCK8 to test cell proliferation of chitisan particles.Result: Compared with the blank group and the negative control group,the cell proliferation of siCTGF-loaded chitosan nanoparticles were significantly decreased(P<0.05).The results of RT-PCR and Western-blot showed that the expression levels ofthree genes of siCTGF-loaded chitosan nanoparticles were significantly reduced,compared with blank group and negative control group(P <0.05),and 200nmol/ml group had the biggest blocking effect.Conclusion: The application of siCTGF-loaded chitosan nanoparticles can effectively inhibit fibroblast proliferation,type ? collagen synthesis and differentiation into myofibroblasts.Part IV The application of siCTGF-loaded chitosan nanoparticles on hypertrophic scar of rabbit earObjective: To evaluate the inhibiting effect of local application of siCTGF-loaded chitosan nanoparticles on scar.Methods:The experimental animals were divided into blank control group,siNC-loaded chitosan nanoparticles group and the three concentrations of siCTGF-loaded chitosan nanoparticles.we used In Vivo Image System to monitor the release of siCy3 from nanoparticles on nude mice skin.The new healed wounds were injected with different concentrations of siCTGF-loaded chitosan nanoparticles.General images of local scar were observed by camera,and we used ultrasonic to achieve the dynamic monitoring of scar thickness during treatment.The expression of CTGF?type ? collagen and ?-SMA of local dermis was detected by immunohistochemical staining.Masson trichrome staining was used to observe the accumulation and arrangement of collagen.At the same time,the expression of CTGF,type ? collagen and ?-SMA protein in the skin were detected by Western-blot.Result: Red fluorescence was observed in the nude mice skin and continued until day5.Compared with the blank control group and the siNC-loaded chitosan nanoparticles group,the ultrasonic measurements(scar thickness)of si CTGF-loaded chitosan nanoparticles were significantly decreased(P<0.05).Histological and western-blot results showed that the expression of CTGF protein in the dermis of the treatment group was decreased,and the expression of other fibrosis-related factors were also reduced.The results of Masson staining showed that the accumulation and arrangement of collagen in siCTGF-loaded chitosan nanoparticles group were improved.Conclusion: siCTGF-loaded chitosan nanoparticles can effectively reduce the scarformation of rabbit ear,and have a great application prospect on the prevention and treatment of hypertrophic scar. |
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