| In recent years,the number of related respiratory diseases in China has increased year by year,and the incidence of airway stenosis is also on the rise.According to the nature of the disease,it is divided into two categories:benign airway stenosis and malignant airway stenosis.Iatrogenic injury caused by airway intubation or airway incision is a major factor leading to benign airway stenosis.In addition,studies have shown that nearly 20%-30%of lung cancer patients will also develop symptoms of central airway stenosis as the disease progresses.Given the increasing trend of malignant tumors worldwide,it is not surprising that the incidence of malignant airway stenosis associated with cancer is higher than that of benign.However,regardless of the nature of the airway stenosis,its symptoms will lead to varying degrees of dyspnea,with mild symptoms affecting the quality of life,and severe symptoms being life-threatening.In the past,the treatment of airway stenosis was mostly surgical resection and reconstruction with end-to-end anastomosis.However,such surgical techniques are complex,the trauma is extensive,and the complication rate is relatively high.In recent years,the use of self-expandable metallic stent(SEMS)has gradually developed and matured in the treatment of airway stenosis,especially for patients with inoperable resection and elderly patients with poor cardiopulmonary conditions,the treatment effect is fast and efficient.Unfortunately,stent-related complications have always been a major problem in clinical treatment,such as airway restenosis(granulation tissue hyperplasia or tumor recurrence),respiratory infection,stent displacement,wall rupture,stent entrapment or incarceration,etc.These postoperative complications have forced clinicians to become more cautious when choosing airway stents.Although the currently used silicone-covered airway stents can prevent the proliferating granulation tissue from growing into the tracheal lumen through the stent mesh,the shear stress generated by the two ends of the stent and the tissue will continue to stimulate the tracheal wall and induce excessive granulation tissue proliferation.This results in airway restenosis.Additionlly,tumor compression in the airway and the recurrence of malignant tumor at the stent port are also the main factors leading to airway restenosis.A molecular investigation of bacterial biofilm(BBF)after placement of airway stents found that the adverse reaction state of long-term symbiosis between tracheal stents and microorganisms will accelerate the formation of bacterial biofilms.Besides,multiple studies have identified stented BBF as an independent risk factor for subsequent airway granulation tissue formation.Therefore,adding antibacterial materials to the surface of the covered stent may improve the airway microenvironment and inhibit the formation of BBF,thereby effectively controlling the proliferation of granulation tissue in the airway.For the two thorny problems of airway restenosis and BBF formation after airway stent placement,drug-eluting fiber-covered airway stents may be an effective way to solve these problems.Silver nanoparticles(AgNPs)have received extensive attention due to their powerful bactericidal,anti-inflammatory and anticancer properties.Due to the poor efficacy of commonly used antibiotic therapy on infections caused by bacterial biofilms,the selection of biomedical products containing AgNPs not only has strong antibacterial ability,avoids antibiotic resistance but also improves the inflammatory microenvironment.It is a high-quality antibacterial material for medical implant research and development.Additionally,given the large number of patients with tumor-derived airway stenosis,drugs that inhibit granulation tissue proliferation alone cannot reduce the incidence of airway restenosis after stent placement in these patients.Therefore,it is necessary to explore the effect of surface drugs on stent-grafts that can not only inhibit the excessive proliferation of granulation tissue,but also antagonize the proliferation of malignant tumors in the airway.Cisplatin(Diamminedichloroplatinum,DDP)is the first-line chemotherapy drug for many cancer treatments.Cell experiments found that DDP has inhibitory effects on the proliferation and fibrosis of human skin,liver and vocal cord fibroblasts.Therefore,the selection of AgNPs in combination with DDP and airway scaffolds may endow the stents with more effective antibacterial,anti-granulation tissue proliferation and anti-cancer multiple potentials.In this study,we combined the advantages of drug-eluting stent and coated metal stent,and designed a novely drug-eluting fiber coated airway stent PCLDDP-AgNPs through electrospinning technology.This stent capsule has the dual ability of carrying DDP and AgNPs,and can realize local drug delivery and release in the stent implantation area.It can inhibit BBF formation,granulation tissue proliferation and tumor recurrence in the treatment of benign and malignant airway stenosis.In the first part of this study,the mechanism of DDP inhibiting human embryonic lung fibroblasts from respiratory system was discussed through cell experiment,and the anti-proliferative property of DDP on fibroblasts was clarified.In the second part,four groups of drug-eluting fiber coated airway stent films PCL,PCL-AgNPs,PCL-DDP and PCL-DDP-AgNPs were designed by electrospinning technology,and the chemical properties,physical structure and antibacterial properties of the stent films were analyzed by various physical and chemical characterization.In the third part,drug-eluting fiber coated airway stents and commercial coated stents were implanted into the airway of New Zealand rabbits,and the safety and effectiveness of stent treatment were confirmed by comparative study.In the fourth part,the recurrence model of lung cancer was established by subcutaneous transplantation of tumor in immunodeficient mice to simulate the inhibitory effect of drug-eluting fiber coated airway stent on airway tumor,so as to evaluate the therapeutic effect on malignant airway stenosis.Part 1:Inhibition and mechanism of cisplatin on human embryonic lung fibroblastsObjectiveTo clarify the inhibitory effect of cisplatin(DDP)on respiratory system-derived human embryonic lung fibroblasts(CCC-HPF-1),and to further explore the mechanism of anti-proliferation and anti-fibrosis.Method1.Incubate CCC-HPF-1 cells 24H,48H and 72H with DDP gradient working solution at concentrations of 40ug/ml,20ug/ml,10ug/ml,5ug/ml and 0ug/ml by cytotoxicity assay(CCK-8).After the optical density(OD)value of the solution changed.2.Real-time quantitative PCR was used to detect the expression levels of collagen type Ⅰ(Collagen-Ⅰ)and collagen type Ⅲ(Collagen-Ⅲ)mRNA in CCCHPF-1 cells incubated with DDP gradient working solution for 24H,48H and 72H.3.The apoptosis data of CCC-HPF-1 cells incubated with DDP gradient working solution for 24 hours were detected by AnnexinV-FITC/PI double staining and flow cytometry,and the changes of live and dead cells were observed by Live-Dead staining reagent.4.After inducing the myofibroblast phenotype of CCC-HPF-1 with transforming growth factor(TGF-β1),and then continuing to incubate with DDP,the expression ofα-SMA,Collagen-Ⅰ and Collagen-Ⅲ in cells before and after DDP treatment was observed.Immunofluorescence expression was analyzed.5.After inducing the myofibroblast phenotype of CCC-HPF-1 with transforming growth factor(TGF-β1),the cells were incubated with DDP for 24H,48H and 72H,and the fibrosis-related proteins Collagen-Ⅰ,Expression levels of Collagen-Ⅲ,Smad-2,Smad-3,P-Smad-2,P-Smad-3 and apoptotic protein Casepase-3.6.Graphpad Prism7.0 software was used for statistical analysis,and measurement data were expressed as mean ± standard deviation.A two-way ANOVA or Student’s t-test was used for testing,and P<0.05 indicated a statistically significant difference.Result1.The results of CCK-8 showed that the OD value of CCC-HPF-1 cells gradually decreased with the increase of drug concentration.The same concentration of DDP solution treated the cells for a longer time,and the OD value of the cells also decreased gradually.2.Real time quantitative PCR showed that the mRNA expression levels of Collagen-Ⅰ and Collagen-Ⅲ decreased gradually with the increase of DDP concentration,that is,DDP decreased the mRNA expression levels of Collagen-I and Collagen-Ⅲ in a dose and time-dependent manner.3.The results of apoptosis detection by flow cytometry showed that the early apoptosis rate and total apoptosis rate of CCC-HPF-1 cells decreased gradually with the increase of drug concentration.4.The Live-Dead staining analysis of cells incubated with DDP gradient working solution showed that when the drug concentration continued to rise to 10ug/ml,the green fluorescence representing living cells gradually decreased and the red fluorescence representing dead cells relatively increased.The cytoskeleton gradually changed,the long spindle cell shape became irregular,the nucleus began to become smaller and the chromatin concentrated.That is,with the increase of DDP concentration,the number of living cells decreased gradually.5.The results of immunofluorescence showed that DDP could reverse the activation and differentiation of TGF-β1 on CCC-HPF-1 cells,and at the same time down-regulated the immunofluorescence expression of α-SMA.In addition,DDP also possesses the ability to reduce TGF-β1-induced upregulation of Collagen-I and Collagen-Ⅲ expression.6.Western blot experiments showed that DDP can reduce the expression levels of TGF-β1-induced up-regulated fibrosis-related proteins Collagen-I,Collagen-Ⅲ,Smad-2,Smad-3,P-Smad-2,and P-Smad-3.And with the prolonged action time,the expression levels of fibrosis-related proteins gradually decreased.On the contrary,the expression level of Caspase-3 gradually increased with the prolonged incubation of CCC-HPF-1 cells with DDP.Conclusion1.DDP can reverse the TGF-β1-induced up-regulation of cellular fibrosis-related proteins,and the process of inhibiting the proliferation of lung embryonic fibroblasts is related to the TGF-β/Smad pathway.2.DDP-promoted apoptotic pathway may be one of the mechanisms of inhibiting lung embryonic fibroblast proliferation.3.DDP has potential application value in the preparation of drug-eluting fiber-coated airway stents and can be used as an alternative coating drug.Part 2:Design and antibacterial experiments of nanosilver and cisplatin-eluting electrospun fiber-covered scaffoldsObjectiveTo explore the feasibility of designing silver nanoparticles(AgNPs)synergistically with cisplatin(DDP)to elute fiber-coated airway stents by static spinning technology,and to analyze the drug stability and antibacterial properties of the obtained stent coating.Method1.A novel drug-eluting fiber-coated airway stent was prepared by electrospinning with polycaprolactone(PCL)as the carrier of AgNPs and DDP,and the obtained stent was coated with PCL,PCL-AgNPs,PCL-DDP,PCL-DDP-AgNPs by scanning electron microscopy(SEM),transmission electron microscopy(TEM),Fourier transform infrared spectroscopy(FTIR),X-ray photoelectron spectroscopy(XPS),mechanical analysis,contact angle and in vitro drug release Testing determines the drug stability and physical structure of its materials.2.The drug release behaviors of stent-coated PCL-AgNPs,PCL-DDP and PCL-DDP-AgNPs were evaluated using UV-Vis spectrophotometer and atomic absorption spectrophotometer.3.Measure the changes of OD value of the scaffold coating PCL-AgNPs,PCL-DDP and PCL-DDP-AgNPs after incubation with Staphylococcus aureus,Pseudomonas aeruginosa and Candida albicans for 24H by microplate reader and calculate the antibacterial activity.4.The effects of four groups of scaffold membranes on bacterial biofilm formation after incubation with Staphylococcus aureus,Pseudomonas aeruginosa and Candida albicans for 72H were analyzed by confocal laser scanning microscopy(CLSM).5.Graphpad Prism7.0 software was used for statistical analysis,and measurement data were expressed as mean±standard deviation.A two-way ANOVA or Student’s t-test was used for testing,and P<0.05 indicated a statistically significant difference.Result1.The drug-eluting fiber-coated airway stent coating PCL-DDP-AgNPs and the other three stent coatings PCL,PCL-AgNPs,PCL-DDP can be successfully prepared using electrospinning technology.SEM showed that all fibrous membranes exhibited randomly interconnected structures with uniform fiber size.The diameters of AgNPs in the fiber membrane PCL-AgNPs and PCL-DDP-AgNPs ranged from 15 to 20 nm,and the frequency of 16 to 17 nm was the highest.2.FTIR and XPS data showed that AgNPs and DDP were successfully introduced into the fiber membrane PCL-AgNPs,PCL-DDP and PCL-DDP-AgNPs,and the physical and chemical properties were stable.The water contact angles of the four groups of scaffold films all exceeded 100°,indicating strong hydrophobic properties.3.In the first eight days,DDP in the PBS solution of the stent-coated PCL-AgNPs,PCL-DDP and PCL-DDP-AgNPs showed explosive drug release behavior,and then DDP and AgNPs in the fibrous membrane were released slowly and uniformly in the PBS solution within 25 days.4.During the co-incubation of the four groups of scaffold-coated PCL,PCL-DDP,PCL-AgNPs and PCL-DDP-AgNPs with the medium containing Staphylococcus aureus,Pseudomonas aeruginosa and Candida albicans,respectively,the fibrous membrane PCL-DDP The bacteriostatic rate of-AgNPs was significantly higher than that of PCL and PCL-DDP,and the difference was statistically significant(P<0.0005).There was no significant difference between the antibacterial rate of PCL-AgNPs and PCL-DDP-AgNPs in fiber membrane(P>0.05).5.The bacterial biofilm formation experiment showed that the microbial activity of most of Staphylococcus aureus,Pseudomonas aeruginosa and Candida albicans in PCL group was higher.With the addition of AgNPs,the intensity of green fluorescence representing surviving microorganisms gradually decreased,while the red fluorescence representing dead bacteria was relatively enhanced.Compared with fibrous membrane PCL and PCL-DDP,the bacterial biofilm thickness and total microbial load on the surface of AgNPs-containing fibrous membrane were less,and the difference was statistically significant(P<0.0005).Compared with PCL-DDPAgNPs group,there was no significant difference in bacterial biofilm thickness and total microbial load between PCL-AgNPs group and PCL-DDP-AgNPs group(P>0.05).Conclusion1.PCL,PCL-DDP,PCL-AgNPs and PCL-DDP-AgNPs of drug-eluting covered airway stents can be successfully prepared by electrospinning technology.2.Silver containing nano scaffold coated PCL-AgNPs and PCL-DDP-AgNPs can significantly inhibit the growth of common pathogenic bacteria Staphylococcus aureus,Pseudomonas aeruginosa and Candida albicans,inhibit the formation of bacterial biofilm,and have the ability to improve microbial colonization after airway stent implantation.Part 3:Synergistic effects of silver nanoparticles and cisplatin in combating inflammation and hyperplasia of airway stentsObjectiveTo investigate the efficacy and safety of nano-silver(AgNPs)synergistically with cisplatin(DDP)eluting fiber-coated airway stents in inhibiting the proliferation of granulation tissue in the airway.Method1.The in vitro cytotoxicity of four groups of scaffold coated PCL,PCL-AgNPs,PCL-DDP and PCL-DDP-AgNPs on human embryonic lung fibroblasts(CCC-HPF-1)was determined by cytotoxicity test(CCK-8).2.Incubate CCC-HPF-1 cells on the surface of the four sets of scaffolds for 24H and 48H respectively,and use the Live-Dead kit for live/dead cell staining analysis.3.CCC-HPF-1 cells were treated with four groups of scaffold membrane slow-release solution for 24H and 48H,and the percentage of apoptosis was detected by flow cytometry.4.Set the commercial covered airway stent as the control group,and the drug-eluting fiber covered stents PCL-AgNPs group,PCL-DDP group and PCL-DDP-AgNPs group were placed in the airway under fluoroscopic guidance.Four weeks later,animals were sacrificed and airway tissues were subjected to HE staining,Masson staining,immunohistochemistry(a-SMA,CD68),immunofluorescence(Casepase-3,PCNA)and western blotting(TGF-β1,Smad-2,Smad)-3,α-SMA).5.The expression levels of inflammatory factors IL-1α,IL-8 and TNF-α in the bronchoalveolar lavage fluid of the four groups of experimental animals were analyzed by Elisa reagent,and the corresponding lung tissues were stained with HE.Additionally,a linear regression model was used to analyze the exact relationship between intratracheal microbial content and granulation tissue content.6.Graphpad Prism7.0 software was used for statistical analysis,and measurement data were expressed as mean±standard deviation.A two-way ANOVA or Student’s t-test was used for testing,and P<0.05 indicated a statistically significant difference.Result1.The CCK8 results of four groups of scaffold coated sustained-release solution treated cells showed that the scaffold coated PCL AgNPs,PCL DDP and PCL DDP AgNPs could control the proliferation of CCC-HPF-1 cells,and the cell activity decreased gradually with the extension of time.2.The Live-Dead fluorescence staining of cells incubated on the surface of fibrous membrane showed that with the prolongation of the incubation time of cells on the scaffold membrane PCL-AgNPs,PCL-DDP and PCL-DDP-AgNPs,the living cells containing green fluorescence gradually decreased,and the red fluorescence representing dead cells increased relatively.The inhibition of fibrous membrane on cells was time-dependent.PCL-DDP and PCL-DDP-AgNPs are the main factors affecting the living and dead state of cells.3.Compared with the PCL-AgNPs group and the PCL group,the PCL-DDP group had significantly higher rates of early apoptosis and late apoptosis,and the difference was statistically significant(P<0.0005).The late apoptosis rate in the PCL-AgNPs group was higher than that in the PCL group,and the difference was statistically significant(P<0.005).The rates of early apoptosis and late apoptosis in PCL-DDP-AgNPs group were higher than those in PCL-DDP group,and the difference was statistically significant(P<0.005).4.The degree of inflammatory cell infiltration in airway tissue showed that the inflammation score in the PCL-AgNPs group was lower than that in the control group,and the difference was statistically significant(P<0.05).The inflammation score in the PCL-DDP group was slightly higher than that in the PCL-AgNPs group,and the difference was not statistically significant(P>0.05).Compared with the PCL-DDP group,the PCL-DDP-AgNPs group had a lower tracheal inflammation score,and the difference was statistically significant(P<0.05).5.The results of collagen fiber deposition score showed that the tracheal collagen fiber deposition score of the control group was higher than that of the PCL-AgNPs group,and the difference was statistically significant(P<0.05).The collagen deposition score in the PCL-AgNPs group was higher than that in the PCL-DDP group,and the difference was not statistically significant(P>0.05).Compared with PCL-AgNPs group,the collagen deposition score was significantly lower in PCL-DDP-AgNPs group,and the difference was statistically significant(P<0.0005).The collagen deposition score in the PCL-DDP-AgNPs group was slightly lower than that in the PCL-DDP group,and the difference was not statistically significant(P>0.05).6.The results of the average percentage of granulation tissue area showed that the area of tracheal granulation tissue in the control group was higher than that in the PCL-AgNPs group,and the difference was statistically significant(P<0.0005).The area of granulation tissue in the PCL-AgNPs group was higher than that in the PCL-DDP group,and the difference was statistically significant(P<0.0005).Compared with the PCL-DDP group,the area of granulation tissue in the PCL-DDP-AgNPs group was slightly decreased,and the difference was statistically significant(P<0.05).7.The average thickness of the tracheal submucosa showed that the thickness of the tracheal submucosa in the control group was significantly higher than that in the PCL-AgNPs group,and the difference was statistically significant(P<0.0005).The thickness of the tracheal submucosa in the PCL-AgNPs group was greater than that in the PCL-DDP group,and the difference was statistically significant(P<0.005).Compared with the PCL-DDP group,the thickness of the tracheal submucosa in the PCL-DDP-AgNPs group was not statistically significant(P>0.05).8.The test results of inflammatory factor IL-1α showed that the expression level of IL-1α in the control group was significantly higher than that in the PCL-AgNPs group,and the difference was statistically significant(P<0.0005).Compared with the PCL-DDP-AgNPs group,the expression level of IL-1α in the control group was also significantly increased,and the difference was statistically significant(P<0.0005).There was no significant difference in the expression level of IL-1α between the PCL-AgNPs group and the PCL-DDP group(P>0.05).Compared with the PCL-DDP group,the expression level of IL-1α in the PCL-DDP-AgNPs group was not statistically significant(P>0.05).9.The test results of inflammatory factor IL-8 showed that the expression level of IL-8 in the control group was significantly higher than that in the PCL-AgNPs group,and the difference was statistically significant(P<0.0005).The expression level of IL-8 in PCL-AgNPs group was also lower than that in PCL-DDP group,and the difference was statistically significant(P<0.05).The expression level of IL-8 in PCL-DDP-AgNPs group was lower than that in PCL-DDP group,and the difference was statistically significant(P<0.05).Compared with the PCL-DDP-AgNPs group,the expression level of IL-8 in the PCL-AgNPs group was not statistically significant.10.The test results of inflammatory factor TNF-α showed that the expression level of TNF-α in the control group was higher than that in the PCL-AgNPs group,and the difference was statistically significant(P<0.0005).Compared with the PCL-DDP group,the expression of TNF-α in the PCL-AgNPs group continued to decrease,and the difference was statistically significant(P<0.005).Compared with the PCL-DDP group,the expression level of TNF-α in the PCL-DDP-AgNPs group was further decreased,and the difference was statistically significant(P<0.005).There was no significant difference between the PCL-AgNPs group and the PCL-DDP-AgNPs group(P>0.05).11.In vivo antibacterial experiment showed that the microbial content on the surface of scaffold membrane in the control group was higher than that in the PCL AgNPs group,and the difference was statistically significant(P<0.005).The microbial content of pcl-ddp scaffold membrane was significantly higher than that of PCL AgNPs group(P<0.0005).The microbial content on the scaffold surface of PCL DDP AgNPs group was significantly lower than that of PCL DDP group(P<0.0005).There was no significant change in microbial content between PCL AgNPs group and PCL DDP AgNPs group(P>0.05).The linear regression analysis between the microbial content and the thickness of tracheal submucosa in the control group and PCL AgNPs group showed that the microbial content on the surface of airway stent was positively correlated with the thickness of tracheal submucosa(R=0.947,P=0.0004).12.The immunofluorescence results of Casepase-3 showed that the positive expression of the control group was lower than that of the PCL-AgNPs group,and the difference was statistically significant(P<0.0005).The positive expression of PCL-DDP group was significantly higher than that of PCL-AgNPs group,and the difference was statistically significant(P<0.0005).Compared with the PCL-DDP group,the positive expression of the PCL-DDP-AgNPs group was further increased,and the difference was statistically significant(P<0.0005).13.The results of immunofluorescence PCNA showed that the positive expression in the control group was higher than that in the PCL-AgNPs group,and the difference was statistically significant(P<0.005).The positive expression of PCL-DDP group was lower than that of PCL-AgNPs group,and the difference was statistically significant(P<0.0005).The positive expression level of PCL-DDP-AgNPs group was still lower than that of PCL-DDP group,and the difference was statistically significant(P<0.05).14.The results of immunohistochemical α-SMA showed that the positive expression in the control group was higher than that in the PCL-AgNPs group,and the difference was statistically significant(P<0.005).Compared with PCL-AgNPs group,PCL-DDP group had lower positive expression,and the difference was statistically significant(P<0.0005).The positive expression of PCL-DDP-AgNPs group was lower than that of PCL-DDP group,and the difference was statistically significant(P<0.005).15.The immunohistochemical results of CD-68 showed that the positive expression of the control group was slightly higher than that of the PCL-AgNPs group,and the difference was statistically significant(P<0.05).The expression of PCL-DDP group was lower than that of PCL-AgNPs group,and the difference was statistically significant(P<0.0005).The positive expression level of PCL-DDP-AgNPs group was lower than that of PCL-DDP group,and the difference was statistically significant(P<0.005).16.The results of TGF-β1 immunoblotting experiment showed that the expression of TGF-β1 in PCL-AgNPs group was lower than that in control group,and the difference was statistically significant(P<0.05).Compared with the PCL-AgNPs group,the expression of TGF-β1 in the trachea was significantly decreased in the PCL-DDP group,and the difference was statistically significant(P<0.0005).Compared with the PCL-DDP group,the expression of TGF-β1 in the PCL-DDP-AgNPs group was further decreased,and the difference was statistically significant(P<0.05).17.The results of Smad-2 western blotting experiment showed that the expression level of Smad-2 in PCL-AgNPs group was significantly lower than that in control group,and the difference was statistically significant(P<0.0005).Compared with the PCL-AgNPs group,the expression of Smad-2 in the trachea was decreased in the PCL-DDP group,and the difference was statistically significant(P<0.0005).The expression level of Smad-2 in PCL-DDP-AgNPs group was slightly lower than that in PCL-DDP group,and the difference was statistically significant(P<0.05).18.The results of Smad-3 western blotting experiment showed that the expression of Smad-3 in PCL-AgNPs group was lower than that in control group,the difference was statistically significant(P<0.0005).Compared with the PCL-AgNPs group,the expression of Smad-3 in the PCL-DDP group was significantly decreased,and the difference was statistically significant(P<0.0005).The expression of Smad-3 in PCL-DDP-AgNPs group was slightly lower than that in PCL-DDP group,and the difference was statistically significant(P<0.05).19.The results of α-SMA immunoblotting experiment showed that the expression of α-SMA in the PCL-AgNPs group was slightly lower than that in the control group,and the difference was statistically significant(P<0.05).Compared with the PCLAgNPs group,the expression of α-SMA in the trachea was significantly decreased in the PCL-DDP group,and the difference was statistically significant(P<0.0005).Compared with the PCL-DDP group,the expression of α-SMA in the PCL-DDP-AgNPs group continued to decrease,and the difference was statistically significant(P<0.05).Conclusion1.Electrospinning drug-eluting fiber stent-graft coated PCL-AgNPs can reduce the proliferation of granulation tissue by inhibiting the proliferation of microorganisms in the airway.2.The scaffold coating PCL-DDP can up-regulate Casepase-3,down-regulate the level of TGF-β1 and reduce the expression of α-SMA to control the formation of granulation tissue in the airway.3.The stent-coated PCL-DDP-AgNPs can utilize AgNPs and DDP to synergistically have antibacterial,anti-inflammatory and anti-proliferative properties,improve the airway microenvironment and inhibit the formation of granulation tissue.This will help address clinical complications associated with airway inflammation and granulation tissue hyperplasia,thereby inhibiting tracheal restenosis.Part 4:Nanosilver synergistic cisplatin-eluting electrospun fiber-covered stent in tumor recurrence modelObjectiveThe feasibility and safety of silver nanoparticles(AgNPs)combined with cisplatin(DDP)eluting fiber-covered airway stent in the treatment of malignant airway stenosis were investigated through in vitro experiments and tumor recurrence models.Methods1.The in vitro cytotoxicity of scaffold-coated PCL,PCL-AgNPs,PCL-DDP and PCL-DDP-AgNPs to human non-small cell lung cancer cells(A549)was detected by cytotoxicity assay.2.Using cell Live-Dead staining to analyze the live and dead cells of tumor cells A549 by PCL,PCL-AgNPs,PCL-DDP and PCL-DDP-AgNPs in scaffolds.3.A549 cells were treated with scaffold-coated PCL,PCL-AgNPs,PCL-DDP and PCL-DDP-AgNPs for apoptosis and cycle experiments.In addition,the mRNA expressions of Bcl-2 and Bax were detected by real-time quantitative PCR.4.The lung cancer recurrence model was established subcutaneously in nude mice,and the blank control group,PCL group,PCL-AgNPs group,PCL-DDP group and PCL-DDP-AgNPs group were set to observe the subcutaneous tumor recurrence,and the tumor volume was measured and stained by HE.Immunohistochemistry(Ki67,Bax,Bcl-2),fluorescent staining(TUNEL,PCNA,Casepase-3)and Western blotting experiments(Bax,Bcl-2,Casepase-3)were used to analyze tumors.5.The pathological results of the heart,liver,spleen,lung and kidney of five groups of mice were analyzed by HE staining.In addition,ELISA reagents for animal cardiac index contain creatine kinase(CK),creatine kinase-MB(CK-MB),liver function indicators alanine aminotransferase(ALT)and aspartate aminotransferase(AST)and renal function indicators blood urea nitrogen(BUN)and creatinine(Cr)were detected.6.Graphpad Prism7.0 software was used for statistical analysis,and measurement data were expressed as mean ± standard deviation.A two-way ANOVA or Student’s t-test was used for testing,and P<0.05 indicated a statistically significant difference.Result1.The results of CCK8 showed that the scaffold sustained-release solution PCL-AgNPs,PCL-DDP and PCL-DDP-AgNPs could inhibit the proliferation of A549 cells.DDP-containing fibrous membrane PCL-DDP and PCL-DDP-AgNPs were the main factors affecting cell proliferation,and the OD value of cells decreased significantly with the prolongation of incubation time.2.The fluorescence results of live and dead cells showed that with the prolonged incubation time of cells on the surface of fibrous membrane PCL-AgNPs,PCL-DDP and PCL-DDP-AgNPs,the number of live cells gradually decreased and the red fluorescence representing dead cells increased relatively.The number of dead cells in PCL-DDP-AgNPs group was the most.3.The results of cell cycle showed that the proportion of cells in G0/G1 phase in PCL-AgNPs group was slightly lower than that in PCL group,and the difference was not statistically significant(P>0.05).The proportion of cells in G0/G1 phase in PCL-DDP and PCL-DDP-AgNPs groups was lower than that in PCL-AgNPs group,and the difference was statistically significant(P<0.0005).The proportion of cells in G0/G1 phase in the PCL-DDP group was slightly higher than that in the PCL-DDP-AgNPs group,but the difference was not statistically significant(P>0.05).The proportion of S-phase cells in the PCL-AgNPs group was slightly higher than that in the PCL group,and the difference was not statistically significant(P>0.05).The proportion of cells in S phase in the PCL-DDP and PCL-DDP-AgNPs groups was higher than that in the PCL-AgNPs group,and the difference was statistically significant(P<0.0005).The proportion of S-phase cells in the PCL-DDP group was slightly lower than that in the PCL-DDP-AgNPs group,and the difference was not statistically significant(P>0.05).The proportion of cells in G2/M phase in PCL group was slightly higher than that in PCL-AgNPs group,and the difference was not statistically significant(P>0.05).The proportion of cells in G2/M phase in PCL-DDP group was higher than that in PCL group,and the difference was statistically significant(P<0.05).The proportion of cells in G2/M phase in PCL-DDP-AgNPs group was higher than that in PCL-DDP group,and the difference was not statistically significant(P>0.05).4.The results of cell apoptosis showed that the total cell apoptosis rate in PCL-AgNPs group was higher th... |
PDF Full Text Request