The Study Of Effect And Mechanism Of 630nm Laser On Human Lung Adenocarcinoma Cell Xenograft Model In Nude Mice Mediated By Hematoporphyrin Derivatives

Objective To investigate the effect and mechanism of 630 nm laser on human lung adenocarcinoma cell xenograft model in nude mice mediated by hematoporphyrin derivatives(HPD)and provide theoretical basis for clinical photodynamic therapy(PDT).Methods Twenty-four human lung adenocarcinoma A549 cell xenograft model in nude mice was established and randomly divided into four groups When the average tumor volume reached 100-150mm3: the blank control group(no photosensitizer and no laser irradiation),the photosensitizer group(with photosensitizer but without 630 nm laser irradiation),the laser irradiation group(only 630 nm laser irradiation but without photosensitizer)and the PDT group(630nm laser irradiation after the photosensitizer was given),make corresponding processing respectively.Low dose multiple exposures were used.In other words,photosensitizer was injected into the tumor at the dose of 10mg/kg and in order to make the photosensitizer as evenly distributed as possible,we injected it uniformly from three sites of the tumor.After avoiding light for 4 hours,illuminated by 630 nm laser with energy density 80 mw/cm2 at a distance of 5 cm for 10 mins.The long and short diameters of the tumor were measured by vernier caliper and the volume was calculated.Tumor samples of the four groups were taken 2 weeks later.(1)The tumor growth curve was plotted,the tumor volume growth changes in each group was compared and the tumor growth inhibition rate was calculated.(2)Hematoxylin-eosin(HE)staining was performed for routine pathological observation of tumor sections,the pathomorphologic changes of cells,stroma and blood vessels in several groups of tumors were compared.(3)Terminal Deoxynucleotidyl Transferase(Td T)mediated d UTP nick end labeling(TUNEL)staining was used to observe and compare the apoptosis induced by photodynamic therapy,and the apoptosis index was calculated.(4)Real-time reverse transcription-polymerase chain reaction(RT-q PCR)was used to detect the expression level of angiogenesis related factors VEGF,HIF-1? and apoptosis related factors Bax and Bcl-2 m RNA in the transplanted tumor tissues.(5)Western blot was employed to detect the expression of angiogenesis related proteins VEGF,HIF-1? and apoptosis related proteins Bax?Caspase-3 and Bcl-2.Results(1)The tumor inhibition rates of blank control group,photosensitizer group,laser group and PDT group were 0,5.6%,7.8% and 51.6% respectively.Compared with the other three groups,the tumor growth inhibition rate of the photodynamic treatment group was significantly increased and the difference was statistically significant(P < 0.05).(2)After HE staining of tumor tissues,the nuclei were blue,and the cytoplasm and stroma were red.The sections can be seen under the microscope that there were a large number of lung cancer cells in the four groups,which were closely arranged,with large hyperchromatic nuclei.The interstitium was loose,and the staining was pink,the results indicated that the animal model of lung adenocarcinoma A549 was successfully established.(3)Tunel staining revealed that more apoptotic cells were found in the PDT group,and the apoptosis index was calculated.Compared with the other three groups,the difference was statistically significant(P < 0.05).There was no significant difference between the other three groups(all P > 0.05).(4)RT-PCR results showed that compared with the other three groups,the m RNA expressions of VEGF,HIF-1? and Bcl-2 in the PDT group decreased,while the expression of Bax m RNA increased(P < 0.05),and the differences were statistically significant.But there was no statistically significant difference between the other three groups(all P > 0.05)?(5)Western blot results showed that protein expressions of VEGF,HIF-1? and Bcl-2 decreased in the PDT group,while protein expressions level of Bax and Caspase-3 increased(P < 0.05),indicating statistically significant differences.There was no statistical difference between the other three groups(all P > 0.05).Conclusion The 630 nm laser mediated by HPD can significantly inhibit the growth of human lung adenocarcinoma xenograft tumor in nude mice,the mechanism of which is related to the inhibition of tumor angiogenesis by down-regulating VEGF and HIF-1? gene expression,and the promotion of tumor apoptosis by up-regulating Bax?Caspase-3 and down-regulating Bcl-2 gene expression.