| BackgroundThe objective of this study is to probe into the in vitro and in vivo photodynamic effects of a new third generation photosensitizer (ZnPcS4-BSA) that compounds zinc phthalocyanine tetra-sulfonate (ZnPcS4) and bovine serum albumin (BSA) on U251 human glioma, in order to provide early fundamental researches for the clinical applications of ZnPcS4-BSA.Glioma is the most common malignancy in neurosurgery with rather high incidence rate and mortality rate and very bad prognosis. The current treatment of gliomas is mainly based on surgery, chemotherapy and radiotherapy, whose efficacy is not so desirable. To seek more effective treatment methods for gliomas is what medical and health-care workers and scientific researchers around the world have been struggling for. Photodynamic therapy (PDT) is a new medical technology under research and development. Since it was put into clinical research in the 1970s, PDT has been applied in the treatment of many benign and malignant diseases, and the therapeutic effects are quite satisfactory. In addition, PDT is also used in the treatment of gliomas, whereas its effects need further observation, large samples and long-term follow-up studies. Photosensitizer plays a significant role in PDT of gliomas, for different photosensitizers may lead to quite different therapeutic effects and adverse reactions. The first generation photosensitizers are all complex porphyrin mixtures with uncertain compositions, and their structures have not been completely defined yet; their action spectra are not ideal, for the maximum excitation wavelength is 630 nm, at which wavelength the tissue penetration is relatively poor and the depth of interaction is less than 0.5 cm, so it is not ideal for treating large and deep-seated tumors; the time interval between drug administration and irradiation is long; the in vivo residence time is long; light is avoided for over four weeks, and the photosensitive side effect on skin is significant. The second generation photosensitizers have made great improvement on the basis of the first generation, but their selectivity and enrichment capacity in tumor tissues are still limited. This study adopts ZnPcS4-BSA, a third generation photosensitizer whose excitation wavelength is closer to red emission, action time shorter, metabolism faster and targeting effect better. As for the PDT effect of this photosensitizer on gliomas, no research has been done up to now. Under such background, ZnPcS4-BSA is used to conduct a study on the in vitro and in vivo photodynamic effects of ZnPcS4-BSA on U251 human glioma and its mechanism of action is probed into.This study can be divided into two parts:the first part mainly focuses on the in vitro photodynamic killing effect of ZnPcS4-BSA on U251 human glioma cells, probes into the optimum drug concentration, the optimum time for drug use and the optimum laser dosage, and then discusses its killing mechanism; the second part, based on the experimental results of the first part, makes a further study of the in vivo photodynamic killing effect of ZnPcS4-BSA on tumor-bearing nude mice, and then analyzes its killing mechanism.Part I:Study on the in Vitro Photodynamic Killing Effect of ZnPcS4-BSA on U251 Human Glioma Cells and Its MechanismObjective:To probe into the optimum drug concentration, the optimum time for drug use and the optimum laser dosage through discussing the photosensitization of ZnPcS4-BSA-mediated photodynamic therapy (PDT) on U251 human glioma cells cultured in vitro; to provide experimental data and theoretical basis for further researches on the in vivo applications of ZnPcS4-BSA to animals as well as for future clinical treatment of gliomas through discussing its killing mechanism. Methods:1. Employ conventional methods for thawing and subculture of U251 human glioma cells.2. Uptake pattern of ZnPcS4-BSA Use UV-Visible spectrophotometer to scan the absorption peaks of ZnPcS4-BSA and draw the concentration standard curve according to those peaks; determine the content of ZnPcS4-BSA in U251 cells; work out the optimum incubation time of U251 cells under the action of ZnPcS4-BSA.3. Toxic effect of ZnPcS4-BSA on U251 human glioma cells Grouping: Negative control group:only add normal saline, no ZnPcS4-BSA. 8 groups with photosensitizer of different concentrations:the concentrations were 0.78125,1.5625,3.125,6.25,12.5,25,50, 100μmol/L, respectively. Treatment:No irradiation for all groups. Use CCK-8 to determine the cell viability.4. Toxic effect of simple laser irradiation on cells Irradiate 5 groups of U251 cells with laser at the dosage of 25,50,100,200 and 400 J/cm2, respectively; add no photosensitizer to any of the 5 groups and determine the cell viability by using CCK-8.5. The influence of laser dosage on the cell killing capacity of ZnPcS4-BSA-mediated PDT Add ZnPcS4-BSA (30μmol/L) into 5 groups of U251 cells. After 4 hours of incubation, irradiate them with laser at the dosage of 25,50,100,150 and 200J/cm2, respectively. Determine the cell viability by using CCK-8, and then arrive at the optimum laser energy in ZnPcS4-BSA-mediated PDT.6. The influence of concentration of ZnPcS4-BSA on ZnPcS4-BSA-mediated PDT Grouping: Negative control group:U251 cells cultured without ZnPcS4-BSA, nor laser irradiation. 5 groups with photosensitizer of different concentrations:the concentrations were 20,40,60,80 and 100μmol/L, respectively; continue to culture for 4 hours and then irradiate them with laser at the dosage of 150 J/cm2. Treatment:Use CCK-8 to determine the cell viability and calculate inhibition rate; work out the optimum concentration of ZnPcS4-BSA in ZnPcS4-BSA-mediated PDT.7. Use flow cytometry to detect apoptosis Take ZnPcS4-BSA with the concentration of 20μmol/L and cells from the irradiation group irradiated with laser at the dosage of 100 J/cm2 to perform the apoptosis detection experiment. Take cells cultured under the same conditions but with no PDT as the control group. Detect apoptosis on the flow cytometry and analyze it by WinMDI29 software.8. Use Real-time PCR to detect VEGF gene expression Take ZnPcS4-BSA with the concentration of 20μmol/L and cells from the irradiation group irradiated with laser at the dosage of 100 J/cm2 to detect VEGF gene expression. Take cells cultured under the same conditions but with no PDT as the control group. Detect VEGF gene expression by fluorescence quantitative PCR instrument, cDNA Synthesis Kit and All-in-OneTM qPCR Mix.9. Use SPSS17.0 software package to carry out statistical analysis of the data. Use average±standard deviation to express the results. When P<0.05, the difference was statistically significant.Results:1. U251 human glioma cells could grow well after thawing and repeated passages, showing pathological features of malignant glioma;2. It was found out from UV scanning that ZnPcS4-BSA reached its absorption peak at the wavelength of 684nm, according to which a standard curve was drawn; the correlation coefficient r=0.999, indicating good curve fitting. In addition, the uptake volume of ZnPcS4-BSA reached the peak after U251 human glioma cells were incubated with ZnPcS4-BSA for 4 hours.3. Treat cells simply with ZnPcS4-BSA of different concentrations without laser irradiation. The difference of cell viability between groups was of no significance. 4. Add no ZnPcS4-BSA when culturing U251 human glioma cells; simply give laser irradiation at different dosages. LSD analysis showed that the cell viability of the group with the largest dosage (400 J/cm2) was lower than that of any other group (P<0.001), and other groups showed no statistical difference after comparison.5. Fix the concentration of photosensitizer to be 30μmol/L; give laser irradiation at different dosages; as the laser dosage increased, the cell viability decreased.6. Fixed the laser dosage to be 200 J/cm2; carry out photodynamic reactions with administration of ZnPcS4-BSA of different concentrations. The cell inhibition rate increased as the concentration of photosensitizer increased; the half maximal inhibitory concentration (IC50) was 0.16mmol/L.7. Adopt flow cytometry to detect apoptosis. It was found out that compared with the untreated groups, the apoptosis ratio of PDT groups increased, marking significant difference (P<0.01). Under the optical microscope, it could be seen that the quantity of tumor cells reduced significantly; the residual tumor cells lost their normal shapes with soma shrinkage, color of cytoplasm becoming lighter, pyknosis of cell nuclei, and increase of impurities in the Petri dish.8. The VEGF gene expression of U251 human glioma cells in the PDT group was 5.29 times compared with the untreated groups, showing significant difference (P≤0.05).Conclusion:1. The optimum time of ZnPcS4-BSA-mediated PDT for U251 human glioma cells should be 4 fours after drug administration.2. ZnPcS4-BSA itself has no obvious toxic effect on U251 human glioma cells.3. When the laser dosage is lower than 200 J/cm2, simple laser irradiation will not damage U251 human glioma cells, while over high dosage will kill U251 human glioma cells.4. In the ZnPcS4-BSA-mediated PDT for U251 human glioma cells, the optimum laser dosage is 200 J/cm2.5. In the ZnPcS4-BSA-mediated PDT for U251 human glioma cells, the inhibition rate of U251 human glioma cells increases as the ZnPcS4-BSA concentration increases. The half maximal inhibitory concentration (IC50) is 0.16mmol/L.6. ZnPcS4-BSA-mediated PDT can achieve the antineoplastic therapeutic effect by inducing apoptosis.7. ZnPcS4-BSA-mediated PDT can induce an increase of VEGF gene expression.PartⅡ:Study on the in Vivo Photodynamic Killing Effect of ZnPcS4-BSA on Tumor-bearing Nude Mice and Its MechanismObjective:To build nude mouse models of U251 human glioma, probe into the in vivo photodynamic killing effect of ZnPcS4-BSA-mediated PDT on gliomas, and analyze its killing mechanism, so as to lay a theoretical foundation for future clinical applications.Methods:1. Adopt conventional methods to subculture U251 human glioma cell lines. Inoculate the nude mice each with 0.2ml of suspension containing 1×108U251 human glioma cells injected into their left armpits, and then continue to raise them under SPF conditions. Start the experiment when the tumors grow to 0.6-0.8cm in diameter.2. Averagely divide the 30 nude mice into five 5 groups at random, control group, irradiation group, photosensitizer group, low-dose PDT group, and high-dose PDT group. The control group had not any treatment. The irradiation group was simply given laser irradiation (wavelength:670nm, dosage:200 J/cm2). As to the photosensitizer group, photosensitizer ZnPcS4-BSA was only injected into the tail veins of the mice at the dosage of 2 mg/kg, with no irradiation, and avoiding light for 24 hours. As to the low-dose PDT group, photosensitizer ZnPcS4-BSA was injected into the tail veins of the mice at the dosage of 1 mg/kg; after 4 hours of light avoiding, the group was given laser irradiation (wavelength:670nm, dosage: 200 J/cm2); then avoid light for another 20 hours. As to the high-dose PDT group, photosensitizer ZnPcS4-BSA was injected into the tail veins of the mice at the dosage of 2 mg/kg; after 4 hours of light avoiding, the group was given laser irradiation (wavelength:670nm, dosage:200 J/cm2); then avoid light for another 20 hours. These procedures were repeated once a week, and three times later, the nude mice were executed to remove their tumors, measure the volumes and weights of the tumors, and then calculate the tumor inhibition rate. Tumor samples of each group were stained by HE staining, and observed under the optical microscope.3. Employ immunohistochemistry to determine the VEGF gene expression of tumor tissues in different groups, observe and count the cells with positive expression of VEGF under the optical microscope.4. Detect and count the mean microvessel density (MVD) under the optical microscope.5. Adopt TUNEL to detect the apoptosis index of tumors, and observe under the optical microscope.6. Use SPSS17.0 software package to carry out statiscal analysis of the data. Use average±standard deviation to express the results, adopt one-way analysis of variance for comparison between groups and LSD for pairwise comparison. When P≤0.05, the difference was statistically significant.Results:1. Animal models were successfully built, and the tumor formation rate was 100%.2. The comparison of tumor volume between the control group and the irradiation group and between the control group and the photosensitizer group showed no statistical significance (P>0.05); the comparison of tumor volume between the control group and the high-dose PDT group and between the control group and the low-dose PDT group showed statistical difference (P<0.05). The comparison of tumor weight between the control group and the irradiation group and between the control group and the photosensitizer group showed no statistical significance (P>0.05); the comparison of tumor weight between the control group and the high-dose PDT group and between the control group and the low-dose PDT group showed statistically significant difference (P<0.05). The tumor inhibition rate of the low-dose PDT group and the high-dose PDT group was 57.488% and 67.150%, respectively, significantly higher than that of the control group and the photosensitizer group.3. When observing under the optical microscope after PDT, decrease of tumor cell density, soma shrinkage, pyknosis of cell nuclei and more necrosis could be seen.4. Compared with the control group, the expression of VEGF and MVD of the irradiation group and the photosensitizer group both had no statistical difference, while the low-dose PDT group and the high-dose PDT group could increase VEGF and MVD. VEGF had positive correlation with MVD, with the correlation coefficient r=0.999, P<0.001.5. The comparison of apoptosis index between the irradiation group and the control group and between the photosensitizer group and the control group showed no statistical difference (P>0.05); the comparison between the low-dose PDT group and the control group showed statistically significant difference (P<0.05); the comparison between the high-dose PDT group and the low-dose PDT group showed statistical difference (P<0.05). It could be seen under the optical microscope after PDT that more cells showed apoptosis.Conclusion:1. In the in vivo ZnPcS4-BSA-mediated PDT for U251 human glioma, nude mouse models of transplanted human glioma can be successfully built.2. Without energy radiation, photosensitizer ZnPcS4-BSA itself will not be activated to produce photodynamic reaction and then generate cytotoxicity; tumor growth cannot be inhibited simply by laser irradiation on tumor-bearing nude mice; no obvious toxic effect is produced on the tumor-bearing nude mice simply by administration of ZnPcS4-BSA. Within a certain range, the tumor inhibition effect of ZnPcS4-BSA-mediated PDT improves as the dosage of photosensitizer increases.3. After ZnPcS4-BSA-mediated PDT, the expression of VEGF in tumor tissues increases, and its mechanism may relate to hypoxia that is caused by PDT. The expression of VEGF has positive correlation with MVD, and during the neovascularization of U251 human glioma, MVD gradually increases as the expression of VEGF increases. 4. In the ZnPcS4-BSA-mediated PDT to the nude mice bearing U251 human glioma, there exists a mechanism in which tumor cells are induced to apoptosis. |
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