| Background and Objection:Glioma is the most common intracranial tumor. High grade astrocytoma, as the most common kind of glioma, usually recurrents in six months after operation. In recent years, it was reported that over80%of patients with high grade astrocytoma had only an average survival time of less than2years despite of the comprehensive therapy including operation, radiotherapy and chemotherapy. Patients with glioblastoma multiforme, especially, had only a median survival time of less than one year. Moreover, secondary damage of brain tissue would usually accompany with the comprehensive therapy. Thus, it seems that little progress had been made in therapy of malignant glioma in the past decades.Glioma as a kind of solid tumor with characteristics of invasion and infiltration, relies on abundant blood supply for its rapid growth. It was reported that tumor size would be kept in a diameter of2-3mm without blood supply. The blockage of tumor blood supply inhibits the growth and metastasis of tumor, or at least, shrinks the tumor to minimal size. Thus, anti-angiogenesis treatment had been considered to be the most potential therapeutic strategy.Endothelial vessels have always been considered as the only blood supply system in tumors not until the discovery of vasculogenic mimicry (VM) in1999by Maniotis et al. VM is a formation of fluid-conducting channels by highly invasive and genetically dysregulated tumor cells. More and more studies have proved the existence of VM in various tomors. Patients suffered from tumors with VM had a five-year survival rate of nearly0. Researchers suggested that the existence of VM might be responsible for the poor clinical prognosis as they provided additional blood supplies at hypoxic environment caused by anti-angiogenesis, which leaded to a higher tumor invasion and survival capability. Hence, the combination of anti-endothelial vessel treatment and anti-VM treatment might had a better outcome in anti-angiogenesis strategy. However, studies regarding anti-VM therapy were far from sufficient.Transforming growth factor-β (TGF-β) has found to be overexpressed in high grade gliomas but in a low level in low ones and normal tissue. Recently a TGF-β antagonist, isoxanthohumol, has been reported to inhibit VM formation in breast cancer cell line MDA-MB-231. Thus, we wondered if the inhibition of TGF-β could also suppress VM formation in glioma. Furthermore, TGF-P had been proved to be correlated with endothelial-angiogenesis in tumors. All these indicated us that TGF-β might be a potential target of both anti-endothelial vessel and anti-VM, although the detailed mechanisms were far from clear.Matrix metalloproteinase (MMP) family plays an important role in tumor invasion, metastasis and angiogenesis. It had been proved that MMPs, especially MMP-2and MT1-MMP, are essential in the process of VM formation in melanoma. TGF-P signaling pathways were reported to regulate MMP-2and MT1-MMP according to some other reports. Thus, we hypothesized that TGF-β might regulate VM formation by modulating the expression of MMPs.Chapter one:Construction of U251MG cell line stably transfected with TGF-β shRNA plasmidObjective To construct a U251MG cell strain stably transfected with TGF-β shRNA plasmid, a model for studying the influence of TGF-P on VM formation.Methods TGF-P shRNA was acquired from chemical synthesis and PCR. The shRNA was cloned into pYrl.1eukaryotic expression plasmid and tested by restriction enzyme cutting and sequencing. The plasmid was transfected into U251MG cells using Lipofectamine2000TM. Then the transfected cells were selected by G418. The stably transfected cell strain was enlarged for further experiments thereafter.Results The TGF-P shRNA plasmid was successfully constructed. U251MG cell strains with low expression of TGF-β were successfully established. RT-PCR showed that there were significant differences (F=778.513, P<0.001) between groups of TGF-β inhibited by plasmids transfection. The pYr1.1A group and the pYr1.1B group had significantly higher TGF-P expressions than Mock group (P=0.011and P<0.001, respectively). ELISA showed that there were significant differences (F=4170.561, P<0.001) between groups. The pYr1.1A group and the pYr1.1B group had significantly higher TGF-β concentrations than Mock group (P<0.001and P<0.001, respectively).Conclusion U251MG cell strains with low expression of TGF-β were successfully established. These would help to study the influence of TGF-β on VM formation in glioma. Chapter two:The influence of TGF-β gene silence on VM formed by U251MG in vitro and expression of VM-related genesObjective To study the influence of TGF-β gene silence on VM formed by U251MG in vitro and expression of VM-related genes.Methods The capability of VM formation in U251MG with different treatments was evaluated by three-dimentional culture and tube formation assay. RT-PCR was employed to study the expression of VM-related genes including EphA2, VE-cadherin, MMP-2, MMP-9, MT1-MMP and LAMC2. Gelatin zymography was used to detect the activities of MMP-2and MMP-9.Results VM formation assay showed that there were significant differences (F=22.509, P<0.001) between groups. The pYr1.1A group and the pYr1.1B group had significantly shorter total length of tubes per field than U251MG group (P=0.041and P<0.001, respectively). RT-PCR showed that there were significant differences (F=74.519, P<0.001) only between groups of MTl-MMP expression. The pYr1.1A group and the pYr1.1B group had significantly lower MT1-MMP expressions than U251MG group (P=0.015and P<0.001, respectively). While the pYr1.1B+rhTGF-β group had significantly higher MT1-MMP expressions than U251MG group (P=0.038). Zymography showed that there were significant differences between groups of MMP-2-pro and MMP-2-active expression (F=79.015, P<0.001and F=30.524, P<0.001, respectively). The pYr1.1A group and the pYr1.1B group had significantly lower MMP-2-active expressions than U251MG group (P=0.015and P<0.001, respectively). While the pYr1.1B+rhTGF-β group had significantly higher MMP-2-active expression than U251MG group (P=0.029). The pYr1.1A group and the pYr1.1B group had significantly higher MMP-2-pro expressions than U251MG group (P=0.028and P<0.001, respectively).Conclusion TGF-β is required for VM formation in glioma cell line U251MG. MT1-MMP participates in TGF-β-induced VM formation.Chapter three:The influence of TGF-β gene silence on VM formation in U251MG xenograft derived from BALB/c nude mouseObjective To study the influence of TGF-β gene silence on VM formation in U251MG xenograft derived from BALB/c nude mouse.Methods To establish the model of BALB/c nude mouse loaded with U251MG mass. The expression of TGF-β was confirmed by RT-PCR and ELISA. The number of days that tumor grows from cells injection to a size of lcm3was recorded and compared between groups. VM in xenograft was identified by CD34-PAS dual staining. The number of VM in each group was compared using tube formation assay. Immunohistochemistry was employed to detected the expression of MT1-MMP.Results The model of BALB/c nude mouse loaded with U251MG mass was successfully established. The time that tumor grows from cells injection to a size of1cm3in shRNA group had significant differences between groups (F=91.467, P<0.001). The pYr1.1B group took significantly longer time than the U251MG group (P<0.001). TGF-β mRNA had significant differences between groups (F=970.049, P<0.001). shRNA group was significantly lower than U251MG group (P<0.001). TGF-β and MT1-MMP expression had significant differences between groups (F=157.290, P<0.001and F=73.063, P<0.001, respectively). TGF-β expression in shRNA group was significantly lower than that in U251MG group (P<0.001). MT1-MMP expression in shRNA group was significantly lower than that in U251MG group (P<0.001). The number of VM tubes had significant differences between groups (F=15.030, P<0.001). shRNA group was significantly less than U251MG group (P<0.001).Conclusion TGF-β down-regulation could result in a significantly impaired capability of VM formation in U251MG It was meaningful for inhibiting tumor blood supply. |
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