The Study Of MIFNγ-secreting Bone Mesenchymal Stem Cells For The Treatment Of Rat Intracranial Glioma

Malignant glioma is the most common primary intracranial neoplasm.Despite dramatic advances in imaging, surgical technique, and adjuvantradiation therapy and chemotherapy strategies, the prognosis for patients withmalignant glial tumors remains dismal. Because glioma exertimmunosuppressive effects and immue amnesty, people hope immunegenetherapy can cure glioma. The failure of immune genetherapy is due to lowefficiancy of viral vector and the inability to address the highly invasive natureof this disease. Malignant glial cells often disseminate throughout the brain,making it exceedingly difficult to target and treat all intracranial neoplasticfoci. The cure of glioma need not only kill tumor cells, but also recoveryinjured nerve cells. It plays a important role of curative effects on glioma tofind a vector that can aim directly at micrometastasis and prepare brain.The use of adult BMSCs has recently been explored. In vitro,BMSCs canproliferate, differentiate, migrate. The use of BMSCs as delivery vehicles fortumor-toxic molecules represents the first experimental strategy aimedspecifically at targeting disseminated tumor pockets. Investigators havedemonstrated that BMSCs possess robust tropism for infiltrating tumor cells,and that they can be used to deliver therapeutic agents directly to tumorsatellites, with significant therapeutic benefit. These technologies representimportant progress in the development of a treatment strategy that canspecifically target disseminated neoplastic pockets within the brain. BMSCscan differantiate into neuron and improve neurological disorders. The use ofBMSCs therapies for brain tumors holds significant promise and may emergeas an important therapeutic modality for patients with malignant glioma.IFNγ can attack tumor directly,upregulate class Ⅰ MHC and class ⅡMHC antigen expression,activate macrophage,T lymphocytes and NK cells,present antigen,inhibit tumor angiogenesis,promote tumor apoptosis.The major goal of this study is to treat rat glioma by BMSCs transfectedmIFNγ in vitro and in vivo.1.The separation and verification of BMSCsWe separated and purified the BMSCs from rat bone marrow by densitygradient centrifugation on a density gradient solution with Percoll. Positiveexpression of CD29 , CD44 and Musashi were detected withimmunohistochemistry test. In order to indicate multipotent differentiation,weinduced BMSCs to differentiate into osteoblast and adipocyte by incubationwith different culture media. The results demonstrated that these cellsexpressed CD29(46%),CD44(70%) and Musashi(12.85%),have the capacityto differentiate into osteoblast and adipocyte.2.Migration and differentiation of BMSCs in vitro and in vivoIn vitro,we observed the migration of NIH3T3 cells and BMSCs to C6glioma cells using double-chamber culture dishes , Transwell(Costar) ,Migration number of BMSCs was significantly much more than NIH3T3 cells.The nestin positive BMSCs was 3.17%,the demonstrated a few of BMSCshad NSCs characteristic. we observated differentiation of BMSCs into neuroncultured in DMEM/F12(containing bFGF,EGF and B27) for 7d after 24hpreinduced by 1mM BME. The identity of differentiated BMSCs wasconfirmd by immunohistochemistry staining for MAP-2 and GFAP,positivecells were 57.04% and 41.53% respectively. The results demonstrated BMSCscould differentiated to neurons and astrocytes.In vivo,Hoechst33342 labeled BMSCs were injected into the contralateralstriatum of rat glioma model. We examined BMSCs migration 14d and 21dafter transplantation. Some of BMSCs were found in the corpus callosum andblood vessel wall 14d after transplantation. Large numbers of BMSCs werefound abroad in the corpus callosum,cerebral cortex and brain vascular wall21d after transplantation. The results of immunocytochemical stainingsuggested that implanted BMSCs expressed MAP-2 and GFAP.3 . Construction and transfecting BMSCs of recombinant plasmidpLEGFP-mIFNγTo construct a recombinant plasmid pLEGFP-mIFNγ for developing thegenetical therapy of tumor by BMSCs. mIFNγ gene obtained from plasmidpcDNA3.1-mIFNγ were inserted into pLEGFP-C1 to form a new plasmid,pLEGFP-mIFNγ. pLEGFP-C1 and pLEGFP-mIFNγ were transduced intoPA317 packaging cells with lipofectamine 2000 respectively. Transducedefficacity was monitored by GFP. Transformants were selected in mediumcontaining G418. The titer of PA317/PLEGFP-C1 andPA317/pLEGFP-mIFNγ culture supernant were 5.5×105CFU/ml and1.7×105CFU/ml. BMSCs were transduced by PA317/PLEGFP-C1 andPA317/pLEGFP-mIFNγ culture supernant. The protein expression of mIFNγgene in BMSCs/pLEGFP-mIFNγ was 5.73ng/106/48h, detected with enzymeof linked immunoadsorden assay(ELISA).4 . The effect on C6 glioma cells of BMSCs transfected withpLEGFP-mIFNγIn vitro,growth and migration of C6 glioma cells were examined indifferent conditioned culture medium. BMSCs/pLEGFP-mIFNγ culturesupernant inhibited C6 glioma cells growth by MTT assay and inhibitedmigration using wound healing and matrigel drip meathods.In vivo,we observed the survival time of rats within 60 days in eachgroup. The mean survival time of rats in the control and BMSCs/pLEGFP-C1groups was (19±1.15) days and (22.30±1.19) days respectively(P>0.05),whilethat in treatment group was (43±5.52)days and there were five rats outlivedbeyond 60 days. The tumor volume in the control and BMSCs/pLEGFP-C1groups was of no difference but the tumor volume(40.67±9.29mm3) intreatment was significantly smaller than that in the control group(40.67±9.29mm3)(P<0.05). The necrosis area ratio in treatment was largerthan that in the control. The CD4+ and CD8+ lymphocytes infiltration intreatment group was more than that in the control group throughimmunohistochemical examination(P<0.05).Our results confirm that BMSCs/pLEGFP-mIFNγ can induce anti-tumorimmunal reaction,inhibit tumor growth in rats and prolong rat survival.BMSCs can act as vector of treating glioma by genetherapy.