| Glioma is the most common primary malignant tumor in the central nervous system. Owing to the feature of its invasive growth, the exsistance of BBB(Blood-brain barrier), glioma is hard to be removed completely by the currently therapy. As other tumours,gliomas results from the activation of proto-oncogenes and inactivati-on of tumor suppressor genes. Recently, PI3K/AKT (phosphorinositide 3-kinase/ Serine threonine kinase) pathway was found to be related with the initiation and development of glioma. PTEN is a important gene inhibitting PI3K/AKT pathway and it can also participate cell growth,migration thorough MAPA,FAK pathway.we used Ad-PTEN and PI3K inhibitor LY294002 to investigate the role of PI3K/AKT pathway on growth and invasion effect in human glioma cells LN229.The present study was divided into three parts.In the first part of this study, Ad-PTEN was amplified with HEK293 cells and detected its titer.Then Ad-PTEN was identified by western-blot test. MTT method was used to evaluate IC50 of LY294002 in human glioma cell line LN229 with or without Ad-PTEN infected. The result showed that the IC50 of LY294002 in the LY294002 group was 8.32μmol/L,and the IC50 of LY294002 in LY294002+Ad-PTEN group was 16.33μmol/L that were both in the safety range of clinical use.The second part was focused on the study in vitro. MTT method was used to evaluate cell proliferation. Flow cytometry was used for cell cycle analysis. Annexin V-FITC was used for apoptosis analysis. The invasion ability was detected by Transwell analysis and migration ability was studied by scratch assay. Western blot was used to evaluate the expression of proteins. The results showed that compared with control groups, significant suppression of growth after 48 hours were found in LY294002 group, Ad-PTEN group and combined group by MTT assay. The analysis of Flow cytometry indicated G0/G1 arrest and the percent of S phase cell were decreased, which were treated by LY294002 group, Ad-PTEN group and combined group. The detection of cell apoptosis showed the apoposis rate of three therapy groups was significant decreased.The synergy effect of inducing tumor cell apoptosis was better than either used alone. The results also showed that when given the LY294002 and Ad-PTEN, the invasion and migration ability of LN229 cell line significantly decreased. western blot showed that three treatment groups,the express-ion of p-AKT,PCNA,Bcl-2,MMP-2,MMP-9 and Cyclin D1 were down-regulated.But the expression of Caspase-3 were up-regulated.And the expression of PTEN was significantly up-regulated in Ad-PTEN group and combined group.Vivo study was carried out in the third part. Nude mice bearing xenografted subcutaneous LN229 gliomas were divided into six groups:①control group;②D M SO group;③Ad-nonsense group;④Ad-PTEN group;⑤LY294002 group;⑥Ad-PTEN+LY294002 group(combined group).Two weeks after establishing subcutane-ous explanted LN229 glioma model in nude mice, we started to measure the tumor volume and give the therapy. The results showed that during the observation period of 4 weeks, the tumor growth rate in three treatment groups significantly slowed down and the tumor volumes were much smaller than those in control groups. The results of related protein expression were the same as those of the in vitro study.Conclusions:1.IC50 of LY294002 in LY294002 group is significantly decreased than that in LY294002+Ad-PTEN group. They both in the range of clinical limite use.2.LY294002 and Ad-PTEN could induce cell cycle G0/G1 arrest, induce cell apoptosis,inhibit the cell invasion and migration, suppress the growth of the LN229 cells in vitro and vivo. The suppressive effect of LY294002 combined with Ad-PTEN is better than either using alone, which indicate the synergy effect.3.The study in vitro and in vivo confirmed:LY294002 combined with Ad-PTEN can up-regulate the expression level of PTEN gene, repress the PI3K/AKT pathway downstream protein like PCNA,Bcl-2,MMP-2,MMP-9,cyclin D1 and FAK, while up-regulat the expression of anti-oncogene Caspase-3. So LY294002 combined with Ad-PTEN can induce the alteration of biological behavior of the tumor cell, produce an anti-tumor effect. and it may become a new way for gene therapy of malignant gliomas by using small molecule inhibitor and adenovirus carrying anti-oncogene.
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