| BackgroundGlioma is a kind of tumor derived from neuroepithelial tissue and is the most common primary intracranial tumor[1].Patients at any age can be affected,mainly in the 45-70 years old,the average age of this disease is about 53 years old,young patients are very rare.According to the world health organization(WHO),I-II grade of glioma belongs for low grade gliomas,Ⅲ-Ⅳ level belongs for high grade glioma,accounting for 77.5%of the total number of glioma.The main pathological type of grade Ⅲ glioma is anaplastic glioma(AA),and grade Ⅳglioblastoma multiforme(GBM),also known as glioblastoma.High-grade glioma is characterized by low differentiation,high malignancy,strong invasiveness,high recurrence rate and high mortality,which has a serious impact on the prognosis and quality of life of patients.Unfortunately,the effectiveness of GBM radiotherapy is controversial.With the continuous in-depth study of GBM,scholars realized that the application of nano-drugs in high-efficiency radiotherapy for high-grade glioma could help solve this problem,and made in-depth exploration.PurposeThis study aims to construct a nanometer drug delivery system that can enhance the radiotherapy sensitivity of human glioma,and to explore the sensitization effect and safety analysis of AU@GA on human glioma U251 in vitro radiotherapy.Materials and Methods1.Under certain reaction conditions,trisodium citrate and tetrachloroaurate trihydrate reacted in proper proportion to prepare gold nanoparticles of nano level.At the same time,the gold nanoparticles were characterized by transmission electron microscope and dynamic light scattering instrument,and the concentration of the gold nanoparticles was measured by icp-aes.The gallic acid was loaded onto the gold nanoparticles by physical stirring adsorption method.2.Human glioma cells U251 were cultured,cells were given different radiation doses and different drug concentrations,and the effects of radiation doses and drugs on the toxicity and cell survival of glioma cells were detected by MTT method.The sensitization mechanism of AU@GA radiotherapy was studied by flow cytometry and western blot.Result1.It was found that the prepared gold nanoparticles were circular,relatively uniform in size and shape,with an average diameter of 2010.65 nm,with good dispersibility and no obvious fusion and adhesion between the particles.2.Under DLS,it was found that the average diameter of gold nanoparticles was 23±0.34nm.Considering that water molecules were coated with gold nanoparticles,the diameter of gold nanoparticles was slightly larger than that under tem.At the same time,it can be seen from the particle size distribution image that the particle size distribution is uniform,and the two detection results are basically consistent.3.The actual concentration of gold nanoparticles(gold elements)detected by icp-aes was 44.80 mug/ml,so the actual recovery of gold elements was 91.24%.4.The gallic acid solution was scanned by uv spectrophotometer,and the maximum absorption peak was found at 265nm.GA standard curve was drawn at 265nm.The linear equation was obtained:Y=0.04193X+0.00552,correlation coefficient R2=0.99981,and the optimal feeding ratio was 1:1.5.The PH values of the negative control group,the lowest dose group,the medium dose group and the highest dose group were respectively determined.It was found that the PH values of the negative control group were 7.39±0.056,while the PH values of the lowest dose group,the medium dose group and the highest dose group were 7.34±0.041,7.20±0.0251 and 7.11 ±0.055,respectively.6.Normal glial cells are grown under the condition of the gold nanoparticles,first day to the control group as the reference standard(98%vs.95.25%vs.94.25%vs.94.75%),the second day(96.5%vs.95%vs.93.25%vs.93.75%)and on the third day(95.5%vs.93.25%vs.93.75%vs.92.75%)in different concentrations of gold nanoparticles experimental cell survival rate is slightly lower than the control group,but no statistical significance(P>0.05).Normal glial cells under the condition of different concentrations of gallic acid,first day to the control group as the reference standard(97.5%vs.83%vs.76.25%vs.64.5%),the second day(95.25%vs.63.25%vs.56.25%vs.51.6%)and on the third day(97.75%vs.47%vs.45.5%vs.43.25%),the different concentration of gallic acid group cell survival rate significantly lower than the control group,with statistical significance(P<0.05).Normal glial cells in culture under the conditions of different concentration of Au@GA,first day to the control group as the reference standard(98.25%vs.92%vs.86%vs.83.5%),the second day(87.75%vs.83.75%vs.87.5%vs.75.5%)and on the third day(97.25%vs.70.25%vs.70%vs.68.75%),the different concentration of gallic acid group cell survival rate lower than the control group,with statistical significance(P<0.05),However,the reduction degree was smaller than that of the GA group alone,and the difference between the two groups was statistically significant(P<0.05).7.By analyzing the survival rate of normal glial cells after 6MV radiotherapy and combined therapy,it can be seen that when the radiation dose is 8Gy,radiotherapy combined with 100 mug gold nanoparticles has a significant difference compared with radiotherapy alone(P=0.0237<0.05).There was a significant difference between radiotherapy and radiotherapy alone(P=0.0201<0.05).There was a significant difference between radiotherapy combined with 100 mu gAu@GA and radiotherapy alone(P=0.0002<0.01).8.By analyzing the survival rate of U251 cells of human glioma after 6MV radiotherapy and combined therapy,it can be seen that when the radiation dose is 8Gy,radiotherapy combined with 100 mug gold nanoparticles has a significant difference compared with radiotherapy alone(P=0.006<0.01).There was a significant difference between radiotherapy and radiotherapy alone(P=0.001<0.01).There was a significant difference between radiotherapy combined with 100 mu gAu@GA and radiotherapy alone(P=0.003<0.01).9.After treatment with 150 mug of different drugs combined with radiotherapy,the U251 cells of human glioma were found to have different proportion of cell cycle in each group.In the control group,the G2/M phase was 9.89%,and the G0/G1 phase was 47.65.The G2/M phase and G0/G1 phase were 12.23%and 36.87%respectively.The G2/M cell cycle was 19.32%and the G0/G1 cell cycle was 29.43%.In Au@GA group,the G2/M phase was 25.65%and the G0/G1 phase was 29.43%.It can be seen from cell cycle changes in each group that,compared with the control group,the addition of drug gold nano,gallic acid and Au@GA can increase the proportion of G2/M phase and decrease the proportion of G0/G1 phase.Compared with the control group,the G2/M phase and G0/G1 phase had statistical differences(P<0.05).10.Human glioma U251 cells were treated with different 150 mug drugs combined with radiotherapy,and apoptosis kit was used to analyze the apoptosis of different groups of cells.The percentage of apoptosis in the control group was 5.23± 1.68%,the percentage of apoptosis in the gold nanoparticles group was 6.12± 1.22%,the percentage of apoptosis in gallic acid group was 14.43±3.46%,and the percentage of apoptosis in Au@GA group was 13.22±2.12%.The apoptotic fraction of 6mw 8Gy cells was 19.35±2.56%,the percentage of gold nanoparticles combined with radiotherapy was 27.72±1.84%,the percentage of gallic acid combined with radiotherapy was 38.52±3.15%,and the percentage of Au@GA combined with radiotherapy was 45.32±2.87%.Compared with the control group,there was no significant difference in apoptosis in the gold nanoparticles group.There was a statistical difference between GA group and Au@GA group.There were significant statistical differences between Au@GA combined radiotherapy group and radiotherapy alone group,gold nanoparticles or GA combined radiotherapy group.Conclusion1.Gold nanoparticles with an average diameter of 20±0.65 nm,good dispersion and no obvious fusion and adhesion between particles can be synthesized through the synthesis method of gold nanoparticles in this experiment.2.Gold nanoparticles did not show significant cytotoxicity to normal glial cells,gallic acid inhibited normal glial cells and brain glioma U251 cells to different degrees,and the inhibition degree of glioma cells was more obvious.Au@GA attenuates the cytotoxicity of gallic acid alone.Gold nanoparticles can improve the radiotherapy sensitivity of glioma,and Au@GA can significantly improve the radiotherapy sensitization of tumor. |
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