The Treatment Of The C6 Glioma By Transplantation Of The Stem Cells Transfected The TRAIL Gene

Objective1. To culture and propagate Bone Marrow Mesenchymal Stem Cells(BMSCs) and Neural Stem Cells in vitro, transplanted into rat with glioma.we try to find applicable cell line as transplant donor by detecting the two kinds of cells biological characteristics.2. In order to support proofs for virus package,to construct and identify PDC315-TRAIL vector,the TRAIL gene were extracted form spleen of SD rat.3. To explo(?)e the possibility of the BMSCs as gene therapy vectors, the TRAIL were transfected into BMSCs by lipofection to observe the gene expression and cell activity.4. For futher study on BMSCs as the gene therapy vector in the treatment of intracranial tumors, the TRAIL-BMSCs were co-cultured with Rat C6 glioma cells,then to detect its effect on tumor cell growth and apoptosis.Method1. The rat hippocampus tissue were separated in sterile state and expanded in vitro systems.The different generations of neural stem cells were cryopreservated and revival,and the biological characteristics were dentified by immunocytochemistry.2. To establish C6 rat gliomas model and treat them by transplanting bone marrow stromal cells and neural stem cells,4 rats were randomly drawed from each group to perform MRI detection when the tumors were grown in 3ws and 4ws. Perfused and drown at different time points, the brains were cut paraffin sections to do HE staining,GFAP and BrdU immunohistochemistry.3. The TRAIL gene were extracted from the rat spleen, and eukaryotic gene plasmid PDC315-TRAIL were constructed.4. The TRAIL was transfect into GFP-BMSCs when GFP-BMSCs were passaged into P4 and P5 by liposome. The TRAIL expression value was detect after 24 hours and 48 hours by immunofluorescence, mRNA expression by RT-PCR, TRAIL expression by Western blot,and the effect of GFP-BMSCs proliferation after transfection by MTT.5. GFP-BMSCs transfected the TRAIL after 24 hours were co-cultured with Rat C6 glioma cells. Dectect the bystander effect by MTT colorimetry and its apoptosis effect on tumor cells by Hochest stain.Result1. The neurospheres were nestin-positive cells and the differentiated cells were (3-tubulin,glial fibrillary acidic protein and 04 positive cells. The morphous,the growth velocity and the other characters of the neural stem cells have no change after cryopreservation and resuscitation.2. The transplantation of BMSCs and NSCs was safety,the cells could survival around tumor tissues and differentiate.It was hardly to examine BrdU positive cells in tumor tissue.And the number of BrdU positive cells was obviously degressive frorm the external tumor tissue to internal.3. The TRAIL gene was successfully extracted from the SD rat spleen. It was 873bp fragments after PCR recycling. Dissected with EcoR I and BamH I enzyme, the cloning products were sequenced and identified by, Shanghai ShengGong Co.LTD. This series has been shared in the three gene and proteins banks of United States, Japan, and Europe.It was awarded the nucleic acid database Number EF030546 and protein data Number:ABK32522.1.4. The TRAIL protein was detected expressed in cell membrane and kytoplasm both at 24 hours and 48 hours after transfected by immunofluorence,but both in control cells and in empty vector cells were negative.Meanwhile,the fluorescence of 24 hours' was more stronger than in 48 hours'.Also the gene were at a high expressed state in experimental group in RT-PCR and Western blot,but negative in control cells and empty vector cells.The MTT showed there was no significant effect on cells survial.5. The GFP-BMSCs with TRAIL can significantly inhibit C6 cells proliferation at the ratio of 62.7% and promote C6 cells apoptosis,the group with GFP-BMSCs had a great apoptosis and inhibitory rate,higher than the control group and empty vector group.Conclusion1. The glioma could be treated by NSCs and BMSCs as the bio-carrier transfected the aim gene,for they could target glioma cells and pursuit the tumor cells infiltrated into the nomor tissues.2. The TRAIL gene could be extracted form SD rat's spleen and liver,but it's more in spleen.3. The eukaryotic plasmid PDC315-TRAIL was successfully imported into the BMSCs by lipofection in vitro.The BMSC-TRAIL vector could express target gene stablely,and have no obvious effect on cells activity.4. TRAIL could induce C6 glioma cells apoptosis significantly.5. BMSCs, more convenient than NSCs as the bio-carrier,can significantly inhibit C6 cells proliferating, hasten C6 cells apoptosis and have significant bystander effect.