Establishment Of Brain Metastasis Model In Nude Mice By Modified Carotid Artery Injection And Study Of Cryptotanshinone In The Treatment Of Brain Tumor

PART 1 Establishment of Brain Metastasis Model in Nude Mice by Modified Carotid Artery InjectionObjective: To establish cop GFP and Nluc labeled MHCC97-H human hepatoma cells.Use these cells to construct a model of brain metastasis in nude mice by modified carotid artery injection method,in which can be observed by in vivo imaging system.Methods: The lentiviral vector plasmid carrying the target gene was used to transfect 293 FT cells by the calcium phosphate method,and the lentivirus was packaged to infect MHCC97-H human hepatoma cells.The positive cells were selected by puromycin,and the monoclonal cell strain stably expressing cop GFP and Nluc were purified by limiting dilution assay.The phenotypes of tumor cells such as proliferation,migration and invasion of stable strain and original strain were compared in vitro.The brain metastasis model of nude mice was established by modified carotid artery injection,and the growth of intracranial metastasis was monitored by in vivo imaging system.Results: Successfully established a stable strain of MHCC97-H human hepatoma cells which was labeled by cop GFP and Nluc.The stable strain and the original strain were used to establish the model by modified carotid artery injection method,and there was no statistically significant difference in body mass change and survival curve between the two groups of tumor-bearing nude mice(P>0.05).The formation of brain metastasis in nude mice with stable strain can be monitored in real time by in vivo imaging system,and the intensity of bioluminescence signal is gradually enhanced with the growth of tumor.Conclusion: MHCC97-H human hepatoma cells labeled by cop GFP and Nluc were successfully established and triumphally constructed a model of brain metastasis in nude mice by modified carotid artery injection method.This experimental model can be non-invasively and continuously visually monitored by in vivo imaging system,which provides an ideal murine model for the study of the brain metastasis mechanism of liver cancer and the development of therapeutic drugs.PART 2 Study of Cryptotanshinone in the Treatment of Brain TumorObjective: To study the effect of cryptotanshinone combined with tumor necrosis factor-related apoptosis inducing ligand(TRAIL)on apoptosis of glioma U87 cells and its related mechanism.Methods: U87 glioma cells were cultured in vitro,and cryptotanshinone was treated separately at different time and concentration,western blot method was used to detect the expression changes of DR4,DR5,STAT3 and p-STAT3(Tyr705).Control group,cryptotanshinone group,TRAIL group and cryptotanshinone combined with TRAIL group were set up.The effect of each group on the survival rate of U87 cells was detected by MTT method.DAPI staining and Annexin V/PI double staining flow cytometry were used to detect the effect of each group on apoptosis of U87 cells.Western blot method was used to detect the expression of caspase family proteins and the expression of STAT3 and p-STAT3(Tyr705)in signal pathway in each group.Results: Cryptotanshinone increased the expression of DR4 and DR5 and decreased the expression of p-STAT3(Tyr705)in U87 cells in a time-dependent and dose-dependent manner.Cryptotanshinone combined with TRAIL significantly inhibited the viability of U87 cells(P<0.01)and promoted the rate of apoptosis(P<0.01).The combination group significantly promoted the expression of caspase family proteins and decreased the expression of p-STAT3(Tyr705)(all P<0.05),and the effect of the combination of the two was better than that of cryptotanshinone or TRAIL alone(P<0.05).Conclusion: Cryptotanshinone combined with TRAIL can synergistically inhibit the proliferation and promote apoptosis of glioma U87 cells.The mechanism may be related to the up-regulation of DR4 and DR5 expression and inhibition of STAT3 pathway by cryptotanshinone.