| objective1 Clinical study: To observe the recurrence and adverse reactions of Epirubicin(EPI)and Gemcitabine(GEM)intravesical instillation after transurethral resection of bladder tumor in patients with NMIBC.2 experimental study: preparation of mmwcnt,detection and understanding of its physical properties.mMWCNT-PPⅡ was prepared by loading PPⅡonto mMWCNT.Objective to investigate the effect of mMWCNT-PPⅡ on apoptosis and proliferation of bladder cancer cells.method1 Clinical research methods A retrospective analysis was made on the newly diagnosed patients with NMIBC who underwent transurethral resection of bladder tumor(TURBT)in the Affiliated Hospital of Shandong University of traditional Chinese medicine from September2018 to September 2019 and started regular bladder perfusion with EPI and GEM within 24 hours after operation.The patients were divided into EPI group and GEM group according to the difference of intravesical instillation.By collecting clinical data and follow-up,the recurrence and adverse reactions of patients were counted.2 Experimental research methods mMWCNT was prepared by coprecipitation method and sterilized by60 Co irradiation.The mMWCNT was characterized by transmission electron microscope(TEM),field emission scanning electron microscope(SEM),X-ray diffraction(XRD),Fourier transform infrared(FTIR)spectrometer,vibrating sample magnetometer(VSM).The same mass of MWCNT was ultrasonically mixed into PPⅡ solution for30 minutes to prepare the mixture of MWCNT and PPⅡ.The mixture of MWCNT and PPⅡ was centrifuged at high speed for 10 minutes with a 50 k D ultrafiltration centrifuge tube,and the PPⅡ not bound to MWCNT was filtered out.The concentration of PPⅡ in the filtrate was determined by UV spectrophotometer.The drug loading of mMWCNT-PPⅡ and the concentration of PPⅡ in mMWCNT-PPⅡsuspension were calculated according to the formula.CCK8 method was used to determine the IC50 of mMWCNT-PPⅡ,PPⅡ and EPI.The logarithmic phase cells were divided into four groups:mMWCNT-PPⅡ group(culture medium with final concentration of IC50 value of mMWCNT-PPⅡ),PPⅡ group(culture medium with final concentration of IC50 value of PPⅡ),EPI group(culture medium with final concentration of IC50 value of EPI)and control group(culture medium without drug).Annexin V,propidium iodide(PI)staining and flow cytometry were used to detect the apoptosis of bladder tumor cells T24 and 5637.Ed U labeling and nuclear staining were carried out and then random photos were taken under the fluorescence microscope and counted by Image J software to calculate the Ed U positive rate(Ed U positive cells/total number of cells×100%).result1 clinical study results The recurrence rate was 27.03% in EPI group and 19.44% in GEM group,with no significant difference(P=0.443).The 1-year recurrence rate was 13.51% in EPI group and 16.67% in GEM group,with no significant difference(P=0.707).The recurrence free survival was(12.90±4.04)months in EPI group and(8.26±3.82)months in GEM group(P=0.032).There was no significant difference in Kaplan-Meier survival curve between the two groups(P=0.552).During the follow-up period,one patient who received bladder perfusion with EPI was excluded from the study because of severe bladder irritation.There was no significant difference in the total adverse reaction rate between the two groups(P=0.649),but there was significant difference in the incidence of bladder irritation(P=0.03)and digestive system reaction(P=0.046).There was no significant difference in the incidence of hematuria,fever and rash(P>0.05).2 experimental results2.1 Characterization of mMWCNT The physical morphology of mMWCNT was characterized by TEM and SEM.Compared with the smooth surface of MWCNT-COOH,the surface of mMWCNT is coated with many Fe3O4 particles.XRD shows that the surface of mMWCNT is surrounded by Fe3O4 particles,the diffraction peak of mMWCNT is not obvious compared with MWCNT-COOH.The FTIR spectrum of mMWCNT shows that the absorption peak near 582cm-1is Metal-Oxygen(M-O)bond.The hysteresis curve of mMWCNT determined by VSM is “s” shape,no hysteresis phenomenon,the saturation magnetization is more than 8.8 emu/g,the remanence and coercivity are 0.2.2 determination of drug loading The concentration of PPⅡ in mMWCNT-PPⅡ suspension(10ml)was about6.76mg/ml,and the drug loading rate was 67.61%.2.3 The IC50 of mMWCNT-PPⅡ,PPⅡ and EPI on T24 and 5637 cells were determined For T24 cells,IC50 ofmMWCNT-PPⅡ,PPⅡ and EPI were 2.22 μg/ml,1.88 μg/ml and 2.61 μg/ml,respectively.For 5637 cells,IC50 of mMWCNT-PPⅡ,PPⅡ and EPI were 1.68 μg/ml,2.43 μg/ml and 1.73 μg/ml,respectively.2.4 Effects of 2.4 mMWCNT-PPⅡ,PPⅡ and EPI on apoptosis of T24 and 5637 cells For T24 cells,the apoptosis rate of EPI group,PPⅡ group and mMWCNT-PPⅡgroup was significantly higher than that of the control group.The apoptosis rate of mMWCNT-PPⅡ group was higher than that of EPI group and PPⅡ group,and the difference was statistically significant(P<0.05).There was no significant difference in apoptosis rate between EPI group and PPⅡ group(P=0.969).For 5637 cells,the apoptosis rate of EPI group,PPⅡ group and mMWCNT-PPⅡ group was significantly higher than that of the control group.The apoptosis rate of mMWCNT-PPⅡ group was higher than that of EPI group and PPⅡ group,and the difference was statistically significant(P<0.05).The apoptosis rate of PPⅡ group was higher than that of EPI group(P=0.024).2.5 Effects of mMWCNT-PPⅡ,PPⅡ and EPI on the proliferation of T24 and 5637 cells For T24 cells,compared with the control group,the positive rate of Ed U in mMWCNT-PPⅡ group,PPⅡ group and EPI group was significantly lower(P<0.05).There was no significant difference in edu positive rate among the three groups(PPⅡ& EPI group P=0.913,mMWCNT-PPⅡ& PPⅡ group P=0.076,mMWCNT-PPⅡ&EPI group P=0.094).For 5637 cells,compared with the control group,the positive rates of edu in mMWCNT-PPⅡ group,PPⅡ group and EPI group were significantly lower(P<0.05).There was no significant difference in edu positive rate among three groups(PPⅡ Group & EPI group P=0.758,mMWCNT-PPⅡ Group & PPⅡ group P=0.784,mMWCNT-PPⅡ Group & EPI group P=0.974).conclusion1 Conclusion of clinical study:There is no significant difference between EPI and GEM in preventing recurrence of bladder cancer after TURBT.The recurrence free survival of patients with EPI was longer than that of patients with GEM.Severe bladder irritation may occur in patients with EPI.The incidence of bladder irritation symptoms of EPI was higher than that of GEM,but the incidence of digestive system reaction was lower than that of GEM.2 Conclusion of experimental study:PPⅡ was successfully loaded on mMWCNT to form mMWCNT-PPⅡ.Moreover,mMWCNT-PPⅡ could promote the apoptosis of bladder tumor cells T24 and 5637 and could also inhibit cell proliferation. |
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