Study On The PTEN Expression And Mutation In Human Gliomas And The Inhibitory Effect Of PTEN Constructs On The Proliferation And Invasion Of Gliomas

Gliomas are the most common primary brain tumors. Although currently available combined therapy including operation , radiotherapy , chemotherapy are used ,the prognosis of patients with malignant gliomas is still very poor . Cell invasion and metastasis are the most important malignant phenotype of gliomas. As the knowledge of tumor biology and molecular genetics increased, people keep exploring various kinds of new treatment methods to suppress the glioma cell proliferation for improving the result of treatment. It has been verified that alteration of multiple genes is involved in the molecular pathogenesis of tumor development. So the gliomas is the same as the tumor in other sites of the body, it's a disease involving mutiplegenetic changes.In a number of our previous studies , it had been demonstrated that EGFR gene amplification or overexpression is the early initiating molecular event in the development of malignant gliomas. EGFR overexpression will activates one of the most important downstream target AKT and its signaling pathways via PI3K. As a recently detected tumor suppressor gene , PTEN has the regulatory effect on the EGFR-PI3K-AKT signaling pathway. However, as to our knowledge, the expression and PTEN and its'role in glioma progression has not been fully investigated, so the present study aims at better understanding of the role of PTEN and the inactivation of EGFR-Pi3K-AKT pathway in the treatment of malignant gliomas.Protocol of the present studyThe first part of this study focused on the expression and gene mutation of PTEN in 50 samples of gliomas. Iirimunohistochemical study was used for examing PTEN expression and the expression of its relevant genes including FAK,Ki67, MMP2, MMP9 . PTEN expression was also studied by Western blotting. The relationship among the expression level of FAK,Ki67, MMP2, MMP9 and their correlation with degree of malignancy of gliomas was analyzed . PTEN gene mutation was detected by PCR-SSCP and DNA sequencing.In the second part, wild PTEN was transfected to human glioblastoma cell line TJ905 and rat gliobiastoma cell line C6 which both have lower expression of PTEN , and empty vector was also transfected as control. The positive transfected clones were randomly selected and identified by Western blot and in situ hybridization. MTT and TUNEL methods were used to evaluate cell proliferation and apoptosis. Cell invasion was examined by Transwell method, and cell migration by spheroid/wound-healing method. Flow cytometry was used for cell cycle analysis, and GFAP expression was also detected for evaluation of cell differentiation by Western blot.In the third part, in vivo study was carried out. Parental C6 cells and C6 cells trasfected with PTEN were implanted stereotactically into the right caudate nucleus of SD rats as control and transfected group. Rats with well-established cerebral gliomas were treated with PTEN and empty vector as treated and control treated group. Each group consisted often rats. The general behavior and survival of the rats were observed, the dynamic MRI and histopathological changes of the rumors and the expression of PTEN in glioma cells were examined.In the final part, we combined PTEN with antisense-AKT2 genes to transfect into C6 cells for observation of the antiglioma effect of combined gene therapy. Expression of PTEN and AKT2 was studied by Western blotting. MTT and TUNEL methods were used to evaluate cell proliferation and apoptosis. Tumor invasion was examined by Transwell method. The cells transfected with PTEN and antisense AKT2 were also implanted into the SD rats for in vivo study.Results Expression and Mutation analysis of PTENPTEN expression decreased correspondingly with the asscending of tumor grade, the protein expression level in grade III and IV was lower than that in low-grade gliomas. The mutation rate of exon 5/8 of PTEN is 18.75% in Primary GBM and no mutation was detected in low-grade gliomas. In immunohistochemical study, the positive stained-cell rate of FAK, MMP2/9 and Ki67 were increased with the degr...