Experimental Study Of ND-1 Combined With Temozolomide On MGMT And Apoptosis Affecting Glioma In Situ Model

Objective:(1)To explore the establishment of an intracranial mice glioma model.By culturing human glioma U87MG cells and inoculation into the brain of nude mice,the effective conditions and methods for establishing an intracranial mice glioma model were mastered.(2)To study the sensitivity and reliability of intracranial mice glioma model by different methods.MRI scan and in vivo imaging were performed on the intracranial mice glioma model to explore the best detection method in this study.(3)To study the efficacy of ND-1 combined with temozolomide(TMZ)in the treatment of mice with intracranial gliomas,and to explore the relationship between mechanism of action and O~6-methylguanine-DNA methyltransferase(MGMT),vascular endothelial growth factor(VEGF),death receptor(Fas),pro-apoptotic factor Bax and anti-apoptotic factor Bcl-2.Method:(1)Human glioma cell line U87MG was cultured,and a cell suspension of1×10~7/ml was prepared and injected into the striatum of BALB/c nude mice at 10ul/injection.The number of cells per nude mouse was 1×10~5.A total of 10 rats were used to establish an intracranial mice glioma model.After 2 week,nude mice were dissected and observed.(2)Fluorescein mcherry was transfected into human glioma cell line U87MG,cultured to stably express luciferase-expressing cell line(U87MG-Luc),and 5 nude mice with intracranial gliomas was established.After 1 week,tumor volume was measured by in vivo imaging system and MRI scan twice a week.The pros and cons of the two detection methods were investigated and the most suitable detection method was determined.(3)Thirty-six mice with intracranial gliomas were established,and nude mice(fluorescence?1×10~5)were randomly divided into model group,ND-1 group,low TMZ group(25 mg/kg)and high TMZ group(50 mg/kg),low TMZ combined with ND-1group(low combined group)and high TMZ combined with ND-1 group(high combined group),a total of 6 groups,6 in each group.They were continuously given drug by oral administration for 12 days.The body weight and tumor volume were measured every 3days during the study period.After the end of administration(day 13),HE staining was used to detect the histopathology of tumor mass,Tunel method was used to detect tissue apoptosis,and Western Blot was used to detect the expression of MGMT,VEGF,Fas,Bax and Bcl-2.Result:(1)Human glioma cell line U87MG was inoculated into the striatum of BALB/c nude mice.The number of cells was about 1×10~5/only,and the inoculation volume was10ul,a total of 10 mice.After 2 week,one nude mouse died and the other nude mice were dissected.The modeling is successful with the naked eye.(2)At the time of primary tumor formation(modeling for 1 week),the fluorescence imaging of the nude mice was 10~5 and 10~6 class.At this time,the tumor was not detected by MRI scan.When the fluorescence of the nude mouse was 10~7,the MRI scan tumor volume was 14.625 mm~3.When the fluorescence imaging of the nude mice was 10~8,the tumor volume of the MRI scan was 19 mm~3.When the fluorescence imaging of the nude mice was 10~9,the tumor volume of the MRI scan was 35.525 mm~3.(3)Compared with the vehicle control group,the tumor volume of the ND-1,low TMZ group,high TMZ group,low combination group and high combination group was significantly decreased(P<0.05).The expression of MGMT in tumor tissues of low TMZ group and high TMZ group was significantly increased(P<0.01).The expression of Fas in the ND-1 group,low TMZ group,high TMZ group,low combination group and high combination group was significantly increased(P<0.01).The expression of proapoptotic factor Bax was significantly increased in the high TMZ group,low combined group and the high combined group(P<0.05),and the expression level of anti-apoptotic factor Bcl-2 was significantly decreased in the low combined group and the high combined group(P<0.05).The increase of Bax/Bcl-2 ratio in the low combined group and the high combined group was statistically significant(P<0.05).And the body weight of the high TMZ group was significantly decreased(P<0.05).Conclusion:The optimal number of cells inoculated to establish the intracranial mice glioma model was 1×10~5/only,the survival rate was 90%,and the tumor formation rate was 100%.The best detection method for tumor volume is the living imaging system.The combination of ND-1 and TMZ in the treatment of glioma can improve the efficacy,reduce the side effects,and alleviate the resistance of glioma cells to TMZ.The mechanism is related to the down-regulation of MGMT expression,up-regulation of Fas expression and up-regulation of Bax/Bcl-2 expression.