The Effect Of Percutaneous Puncture Introduction Of 630nm Laser On Hematoporphyrin Derivative-mediated Transplantation Of Lung Adenocarcinoma A549 Cells In Nude Mice

Objective To observe the effect of percutaneous puncture introduction of 630 nm laser on hematoporphyrin derivative-mediated transplantation of lung adenocarcinoma A549 cells in nude mice and provide the theoretical basis for the clinical application of interstitial photodynamic therapy.Methods Twenty-four lung adenocarcinoma A549 cell subcutaneous transplantation tumor nude mice model was constructed and divided into four groups randomly.It will be treated accordingly according to the following requirements when the average tumor volume reached 250-300mm~3:the blank control group(without any treatment),the photosensitizer group(intratumoral injection of photosensitizer but no 630 nm laser irradiation),the laser irradiation group(percutaneous puncture 630 nm laser irradiation but no photosensitizer injection)and the HPD-PDT combination group photodynamic(intratumoral injection of photosensitizer followed by percutaneous puncture 630 nm laser irradiation).The photosensitizer injection method was chosen to be injected into the tumor at a dose of 10 mg/kg from three sites of the tumor then avoid light for 4 hours;Fiber introduction is performed by using a 30 ml syringe needle inserted to the front end of the fiber,then adjust the distance between the front of the fiber and the tip of the needle when percutaneous puncture to ensure that the tumor is completely covered by the light spot.The laser power was maintained at 100 m W/cm~25minutes per irradiation and 1minute interval,15 minutes intermittent irradiation and the irradiation was performed once every 2 days for 3 times.Nude rats with tumors were executed after 2 weeks of percutaneous transluminal photodynamic therapy,and the tumor tissue was obtained for subsequent experiments.(1)Record the long and short diameter of the tumor and calculate the volume of the transplanted tumor,then plot the tumor growth curve and compare the change of the volume of the transplanted tumor in each group.(2)Calculate the tumor inhibition rate,then plot the tumor inhibition rate curve and compare the tumor growth inhibition of each group.(3)HE staining was performed to observe the pathological morphological changes of tumor cells and intercellular matrix in each group.The degree of tumor tissue injury was evaluated by observing the necrosis of tumor cells.(4)TUNEL staining of transplanted tumor tissue sections was be used to observe the apoptosis in transplanted tumors and calculate the apoptosis index of each group.(5)RT-q PCR(real-time fluorescence quantitative PCR)was employed to detect the expression levels of VEGF,HIF-1αand BAX mRNA in the transplanted tumor tissues.(6)The protein expression levels of VEGF,HIF-1αand Bax were detected by Western blot.Results(1)The growth rate of transplanted tumor in the blank control group,photosensitizer group and light group was no significant change than before and the tumors grew significantly while the growth rate of the HPD-PDT group was significantly slower and the tumor volume was reduced.(2)The tumor volume inhibition rates were 0,3.4%,8.7%,and 40.6%respectively in the blank control group,photosensitizer group and light group.The tumor suppression rate was significantly higher in HPD-PDT group compared with the other groups,the differences were statistically significant(P<0.05).(3)After the transplantation of tumor tissue with HE staining,the number of tumor cells in the blank control group,the photosensitizer group and the light group were relatively large which were closely arranged and large and deep-stained nucleus under microscope.In the HPD-PDT group,different degrees of necrotic tumor cells were observed under microscope and the number of tumor cells was significantly reduced and the cell arrangement was disordered.(4)Positive apoptotic cells were significantly increased in the HPD-PDT group after TUNEL staining and the apoptotic phenomenon was most obvious after TUNEL staining.The apoptosis indices of each group were 0%,4%,6%,8.7%and 35%respectively and the differences treated with HPD-PDT combination was statistically significant compared with those of the other three groups(P<0.05).There was no statistical difference in apoptotic index between the three groups of the blank control group,the photosensitizer-only group and the light-only group(P>0.05).(5)The results of RT-PCR showed that the mRNA expression of BAX was relatively increased in the HPD-PDT combination treatment group and the mRNA expression of VEGF and HIF-1αwas significantly decreased when compared with the blank control group,photosensitizer group and light group,the difference was statistically significant(P<0.05).There was no statistical difference between the three groups of blank control group,the photosensitizer group and the light group(P>0.05)(6)The results of Western blots method showed that the BAX protein expression level was increased and the protein expression levels of VEGF and HIF-1αwere decreased in the HPD-PDT combination treatment group compared with the blank control group,the photosensitizer group and the light group,the differences were statistically significan(P<0.05).There was no statistical difference between the three groups of blank control group,the photosensitizer group and the light group(P>0.05).Conclusion The 630 nm laser introduced by percutaneous puncture had an inhibitory effect on the growth of hematoporphyrin derivative-mediated transplanted tumors in lung adenocarcinoma A549 cells in nude mice,that suggesting the effectiveness of mesenchymal photodynamic therapy for lung cancer;it could induce apoptosis of tumor cells and the mechanism was related to upregulation of BAX gene expression;it could reduce the expression levels of HIF-αand VEGF genes that indicating photodynamic therapy could inhibit the formation of neovascularization factors.