| Glioblastoma multiforme is one of the most frequent human brain tumors. It cannot be cured by excision because gliomas possess extraordinary migratory ability and infiltrate into adjacent normal tissue and carries a poor prognosis because of refractoriness to conventional radiation therapy or chemotherapy. These migratory tumor cells are responsible for recurrent tumor growth.Recent evidence suggests that stem cells have the homing ability and can be used as delivery vehicles for brain tumor therapy. Bone marrow-derived mesenchymal stem cells are well suited for clinical application because they are easily isolated, can be expanded in culture, and can be genetically manipulate using currently available molecular techniques.Firstly, we constructed glioma model, observed the tropism of MSCs to tumor and then implanted MSCs by various routes into glioma-bearing rats to observe the tumor outgrowth with bioluminescence imaging in vivo and in vitro, and also investigated the activity of MSCs on glioma.Part one: Constructing 9Lluc and MSCRL cells expressing firefly or renilla luciferase.The PGL3-Basic plasmid and phRL plasmid (Promega) was digested and the cDNA fragment encoding luciferase(F-luc or R-luc) was isolated; the gene for F-luc or R-luc was cloned into the Lentivirus expression plasmid pBPLV and construct pBPLV-PGL3 and pBPLV-RL. The plasmids were transfected into 9L or MSCs, and obtained 9Lluc and MSCRL cells using flowcytometry. 9Lluc or MSCRL cells were diluted into appropriate cell culture media in 96-well plates to image. The results showed bioluniescence was correlated to the number of tumor cell and F-luc or R-luc was stable over time in vitro, which indicated 9Lluc and MSCRL was constructed successfully.Part two: To establish 9L/Wistar glioma models and compare the responses of F344 and Wistar rats to intracerebral implantation of 9L glioma cells.Adult male Wistar rats were anesthetized and placed in a stereotaxic apparatus. A burr hole was made at an appropriate location (1 mm posterior to bregma and 3mm right to midline) through the skull. A needle was inserted 5 mm ventral to the dura, and the 9Lluc cells (1×10~5 9Lluc cells) marked with DAPI were implanted stereotactically. The needle was removed slowly and the hole immediately was sealed with sterile bone-wax. The control animals were transplanted with PBS. The tumor growth was detected with BLI at 0, 7, 14, 21 days after implantation. Animals were injected intraperitoneally with 150mg/kg D-luciferin, placed into the light-tight chamber of the CCD camera system and a grayscale body surface reference image was taken under weak illumination. After the examination, the animals were sacrificed and obtain the brain. The tissue was processed for HE or immunohistochemistry for GFAP and Vemintin.All animals developed solid tumors and the size of tumor augmented from 7 to 21 days. Histologically tumors were hypercellular with neovascularization and hemorrhage. The tumors exhibited malignant characteristics by light microscopy. It was found no or faint GFAP or Vemintin immunoreactivity in tumor cells but strong staining in fibre-net. The results was coincidence with the malignancy gliomas and 9L/Wistar glioma models were established.Then we compared the characterization of 9L glioma tumors in Wistar rats and Fischer344 rats. A total of 1×10~~5 9Lluc cells per animal was injected stereotactically into the right caudate nucleus in Wistar or F344 rats. The characterization of tumor was detected by BLI in vivo at 7, 14, 21days after implantation and expression pattern of GFAP, Vemintin, CD4, CD8 and morphology were investigated using immunohistochemistry and histochemistry. The tumor exhibited malignant characteristics as well as extensive infiltration of the brain parenchyma in two models. Tumors showed progressive growth from the 7th to the 21st day in 9Lluc/F344 glioma models, while tumor was detected only at the 7th day in 9Lluc/Wistar; and few CD4 and CD8 positive cells were found in syngeneic 9Lluc/F344 models yet lots of CD4 and CD8 positive cells around and in the tumors of 9Lluc/Wistar.Part three: Bioluminesence imaging tracking mesenchymal stem cell homing to glioblastomas in vivo and in vitroThe cell migration assay was performed using double-chamber cluture dishes, Transwell. The medium of 9L was replaced with fresh DMEM the next day after initial culture. Then the supernatant was collected and added to the multiple well plates, adding the Transwell insert. MSCs (2.0×10~5 cells) with medium free of serum were placed in the upper chamber. The Transwell was placed at 37℃, 5% CO2 for 24h, then cells on the membrane with 8μm pores in the upper chamber was fixed with 4% paraformaldehyde and stained with crystal violet. Results of cell migration assays are expressed.9L cells marked with PKH26 were implanted into the nude mice. One week after 9L glioma cells implantation, animals were placed in a stereotaxic frame, and 1×10~6 MSCRL cells was injected into the contralateral, left frontal lobe at the following coordinates: 3.0 mm lateral and 1.0 mm caudal to bregma; depth, 4.5 mm from dura. The control animals were implanted MSCRL without 9L glioma cells. Using serial bioluminescence imaging, migration of parenchymally injected cells was observed.MSCs have the tropism in vivo and in vitro. The tropism ability of MSCs was correlated with the number of tumor cells in vitro. MSCs collected at the injection site at the first day, migrated to the midline at the 7th days and could be seen extensively in the range of glioma tissue, but seldomly in the normal cerebral tissue around at the 14th days after implantation. This feature was evident in the junction area between tumor and normal tissue.Part four: Mesenchymal stem cells can inhibit rat glioma growth in vivo and in vitroThe 9Lluc rat glioma cells were cultured alone or with conditional medium of MSCs or human embryonic kidney cells in 6-well plates for 3 days. Cells were trypsinized and counted under microscopy. The number of 9Lluc cells was determined with a flowcytometer. Mesenchymal stem cells were isolated and injected stereotactically into the contralateral brain parenchyma (3.0 mm lateral and 1.0 mm caudal to bregma; depth, 5.0 mm from dura), ventricles (1.0 mm lateral and 3.0 mm caudal to bregma; depth, 2.0 mm from dura), tumor site (1.0 mm posterior to bregma and 3.0mm right to midline) and femoral vein of rats at the 7th day after implanted 9Lluc gliomas. The characterization of tumor was detected in vivo using BLI at 0, 7, 14days after MSCs implantation and expression pattern of CD4 and CD8 positive cells were investigated using immunohistochemistry.We cocultured 9Lluc with MSCs or HEK cells and enumerated the total number and the number of 9Lluc cells 3 days later by flowcytometry. The proliferation of 9Lluc cells was inhibited to a significantly greater extent by cocultivation with MSCs (0.14±0.016×10~~6) than 9Lluc alone (1.0925±0.068×10) or with HEK cells (1.97±0.150×10~6) (P<0.01). The same results were obtained in animals. Animals injected with mesenchymal stem cells showed a transient tumor outgrowth at the 7th days after MSCs implantation, and by 14th day tumors regressed obviously, and some of them disappeared in tumor site team. Mesenchymal stem cells also evoked an immune response when injected into Fischer344 rats, and obvious infiltration of both CD4 and CD8 positive lymphocytes was detected in the tumor site, while few CD4 and CD8 cells were infiltrated in the specimens of tumor without implanted with MSCs.Conclusion: We established the glioma model of rats, observed the tropism of MSCs to tumors in vivo and in vitro, and detected the antitumor effect of MSCs, which was correlated with immune responses or soluble factors secreted by MSCs.
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