Experiment Of Glioma Treated With PTEN Overexpression

As the most common primary intracranial primary tumor, glioma has high morbidity and mortality. It accounts for 30%-50% of the adult intracranial, which is increasing in recent years. The conventional treatments of malignant tumor, such as surgery, radiation therapy, chemotherapy, biological therapy, all are not effective for gliomas. Therefore, the patients'average survival period is still short. So far this deadly cancer cannot be cured fundamentally. Thus the new therapy becomes a big task. As a fresh treatment for a disease, gene therapy is concerned by more and more people. Common gene therapy includes gene replacement and faulty gene supplement. Transfer tumor cells by tumor-suppressor gene are the most effective gene therapy to kill or inhibit tumor cells.PTEN (phosphates and tensing homologue deleted on chromosome 10) the tumor-suppressor genes, which are lack of chromosome 10 and homologous with tension protein genes, are one of the tumor suppressor genes with double specificity. It is discovered recently and lacked in the expression of gliomas stem cells. PTEN genes are located in chromosome 10 q23.3, including 9 explicit the son, and 8 introns. The 5th explicit son are coded the 122 and 123 amino acids. This coding sequence has the same sequence with protein serine/threonine phosphates and protein tyrosine phosphates catalytic center, so this region has double specificity phosphates'function. This function plays an important role in inhibiting tumor, which not only can dephosphorylated in phosphoric acid serine/threonine and tyrosine as protein phosphatase, but also can inhibit 1-3 instill acylating phosphate-the product of PI3K) kinase (3,4,5 inositol acylating phosphate 3 phosphoric acid (PIP3)-the phosphorylation of phosphate ester and plays a negative regulatory role in the function of PI3K enzyme signaling pathways, as fat phosphatase. The dephosphorylation of D3 PIP3 is an important mechanism of inhibiting cell growth and promoting apoptosis. PTEN protein can restrain the activities of PI3K kinase signaling pathways, such as PKB/Akt cell survival proliferation signal activation which can promote the G1 cell cycle P70S6-kinase activation. PTEN gene has become a market prospect as one kind of tumor suppressor genes.To further explore the effect of PTEN genes and proteins in gliomas, we deeply analyzed the PTEN functions in gliomas through clinical research and basic research. Through the research we examined PTEN gene level and protein levels in gliomas and the relationship with cell apoptosis. Through the basic research we transferred the exogenous wild-type PTEN into glioma cells transfected, observed the therapeutic effect of wild-type PTEN to gliomas cells in vitro and in vivo, and detected the possibility for glioma, for providing the theory basis for gene therapy. The research of PTEN-PI3K-Akt-NF-κB and the downstream target genes signaling pathways have not been reported at home and abroad.1:The research of relationship between PTEN expression in the normal brain tissue and gliomas and cell apoptosisObjective:Explore the relationship between PTEN (phosphatase and tensin homology deleted on chromosome ten) expression in the normal brain tissue and gliomas and cell apoptosis.Methods:Test people normal brain tissue and the human.brain PTEN protein levels in gliomas organization expression using immunohistochemistry SP and Western blot; detect normal brain tissue and human brain mRNA level in gliomas PTEN expression using semi-quantitative RT-PCR; and detect normal brain tissue and cell apoptosis in human glioma using TUNEL.Result:1. PTEN is over-expressed in normal brain tissue samples, exists in gliomas tissue samples. The positive expression increased following the levels (Fig1),100%,85.7%,63.6%,50% for normal to higher-level (table 1). There is statistically significant between low-level gliomas (Ⅰ-Ⅱ), and high-level gliomas (Ⅲ,Ⅳ) (P< 0.05).2. PTEN protein expression level in normal brain is higher than in the human brain with gliomas. The higher level of gliomas, the lower PTEN protein expression obviously (Fig2), which showed 0.667±0.203, 0.475±0.169,0.237±0.087,0.153±0.046 relatively (table2). Furthermore glioma express has significant differences (P< 0.05).3. PTEN mRNA level in normal brain is higher than in the human brain with gliomas. The higher level of gliomas, the lower PTENmRNA expression obviously (Fig2), which showed 3.767±0.865.2.530±0.619,1.853±0.436,0.688±0.140 relatively (table2). Furthermore glioma express has significant differences (P< 0.05).4. Cell apoptosis rate in normal brain obviously lower than in human gliomas, the two groups express significant difference (P< 0.05) (Fig4). Moreover apoptosis rate decreased with the increase of the expression level, 3.2±0.5%,46.4±6.7%,37.2±5.2%,21.2±5.1% from normal to higher-level (table 4). There is close relationship between apoptosis rate, the gliomas level and mRNA and protein expression level PTEN (P< 0.05), because when PTEN mRNA and protein expression level express rise, cell apoptosis rate decrease.Conclusion:PTEN mRNA and protein expression level of in the brain with elevated levels of gliomas, express drop, closely related with PTEN cell apoptosis, may become a new instructions targets diagnosis..2:The influence of PTEN gene recombinant adenovirus for cell proliferation of glioma, apoptosis and the cell cycleObjective:Analyze the PTEN tumor-suppressor genes'influence to U251, U87, A172 gliomas, apoptosis and cell proliferation, anddiscuss the cycle of its possible mechanism.Methods:Transfer restructuring adenovirus (Ad-PTEN-GFP) carrying PTEN and green fluorescent protein or empty carrier adenovirus (Ad-GFP) to U251, U87, A172 glioma cell lines. Detect cell growth curve by MTT; detect apoptosis by TUNEL; detect cell cycle distribution and apoptosis rate after carrying Ad-PTEN-GFP by flow cytometric analysis ion; test PTEN,CIAP1, CIAP2 XIAP, Survivin mRNA level by half quantitative polymerase chain reaction (PCR) (RT-PCR); test PTEN,AKT,p-AKT,IκB,P65,CIAP1,CIAP2,XIAP,Survivin,CyclinD1,P27,Bcl-2,Bax protein levels by Western Blot.Result:1. Test results of Carrying PTEN adenovirusThe highest rate reached in the third day after Ad-PTEN infection.when infections (M.O.I) was 100 plural, the fluorescent expression rate were all more than 80%(Fig1,2,3), which accorded with the requirement of the carrier of gene therapy.2. The MTT detection for cell growth curveWhen the MOI=100, the proliferation activity degree reduced compared to spectrophotometry Ad-PTEN-GFP carrying group and Ad-GFP carrying group each time (P< 0.01 Fig 7), with a statistically significant difference. Increased PTEN significantly reduced glioma cell proliferation activity.3. TUNEL method to detect apoptosisWhen MOI=100, adenovirus carrying U251, U87, A172 cell lines, cell apoptosis proportion in which respectively taking 1d,3d,5d carrying Ad-PTEN-GFP group were significantly higher than not carrying group. In the fifth day, apoptosis rate was highest (Fig 6,9 P< 0.01).4. Detect cell apoptosis and cell cycle changes by streaming instrument Compared with Ad-GFP cell, Ad PTEN-GFP-carrying U251, U87, A172 cells, apoptosis rate increase gradually; compared with no righteous sequence, blank control group apoptosis rate significantly increased. It noticed that PTEN genes over-active obvious lead to glioma cell apoptosis (Fig 1). The cell cycle analysis show that 5 days after the Ad-PTEN-GFP carrying U251, U87, A172 cell, G1 phase cells increased, S period cells decreased, the cell cycle block proportion in G1 phase (Fig 8).5. mRNA level of PTEN and IAP family by RT-PCR detection when M.O.I= 100 in carrying U251, U87, A172 cell line 1,3,5 days, compared with Ad-GFP group, Ad-PTEN-GFP group's CIAP2, CIAP1, XIAP, Survivin mRNA expression level was significantly lower, and IAP family mRNA expression level is reduced to the minimum level on the third day. MRNA of IAP Expression level family and mRNA of PTEN Expression level were negatively correlated (Fig 4,5; table 3,4 P< 0.01).6. Western blot test PTEN and related target genes protein level When M.O.I= 100 in carrying U251, U87, A172 cell line 1,3,5 days, Ad-PTEN-GFP group protein expression level changed obviously compared to Ad-GFP group AKT,p-Akt,IκB,P65,IAps,CyclinD1,P27,Bcl-2 family, and the protein expression level was closely related to PTEN protein expression level (Fig 5, table 4).Conclusion:High expression PTEN can significantly inhibite gliomas U251, U87, A172 proliferation and apoptosis-oriented lines, which may be relevant to PTEN-PI3K/AKT-NF-κB signaling pathways activated. Through this channel regulation various factors related to family, such as apoptosis IAPs and Bcl-2 family, and through regulating the cell cycle related factors cause the cell cycle block.3:Experimental study of restructuring nude glioma growth by PTEN genesObjective:Study the therapeutic effect of nude tumor-burdened using the Ad-PTEN gliomas, and discuss its possible mechanism.Methods:Build nude glioma model, inject tumor carrying Ad-PTEN-GFP or empty Ad-GFP, observe tumor growth and nude survival, detect apoptosis using TUNEL method; Immunohistochemical check PTEN protein levels and P65 change.Results:1. Situation of tumor growth and survival time of nude U251 cells to 7×105 nude about 20 days after inoculation tumor nodules are visible form, inject inside 5 times, tumors Ad-PTEN group of tumor size obviously, a slow growth control volume small, the treatment for 6 weeks after tumor size inhibition rate, as compared with control 82.50% was significant difference (p <0.05 Fig5). U251 and Ad-GFP treatment group nude tumor growth and survival condition worse quickly after transplantation, two groups died off the average survival time in 28～80 days. Nude respectively to 54±7 days and 58±8days, Ad-PTEN nude treatment group was significantly better than living condition of the other two groups, tumor growth slow until 120 days after transplantation end still has 2 only observe nude alive, the mean survival time plus or minus 10 days,103 generate time significantly extend (Fig6).2. Each target protein and apoptosis in the expression Immunohistochemical display P65 protein mainly express PTEN and in the nucleus, with occasional cytoplasm after expression, shaded into tan, brown particles. Ad-PTEN treatment group significantly higher in P65 PTEN expression expression, reduced significantly, and apoptosis increase (Fig 1-3). Its expression differences with a statistical significance.Conclusion:Each cell nude are visible transplantation after inoculated the growth of tumors, Ad-PTEN after treatment can significantly inhibit the growth of tumors in nude, extend survival time. Immunohistochemical results suggest that PTEN possibly by PTEN-P65 pathways promote tumor cell apoptosis body.