Study Of Gene-modified Bone Marrow Mesenchymal Stem Cells For The Treatment Of Gliomas

Objective(1)To investigate the culture method and biological character of adult rat bone marrow derived mesenchemal stem cells (MSCs).(2)To investigate the distribution of enhanced green fluorescent protein (EGFP) transfected MSCs after implanted into rat brain glioma.(3)to investigate the treatment effect of IL-4 transfected MSCs on rat brain glioma.Methods(1)MSCs were separated from adult rat bone marrow by gradient centrifugation and expanded in vitro. 2～3 passage cells were observed under microscope. The cells surface marks were detected by immunocytochemistry. Cells were induced in vitro under different conditions and the ALP, oil red O and NSE were detected respectively after induction.(2)Enhanced green fluorescent protein (EGFP) was transfected into MSCs in vitro by liposome and established the rat C6 glioam model. The MSCs modified by EGFP were implanted into rat brain tumor and the distribution of MSCs was observed under fluorescent microscope.(3)MSCs were transfected by mIL-4 and selected by G418. The mIL-4 expression was detected by ELISA and RT-PCR. BrdU labeled MSCs -mIL-4 were implanted into the rat brain tumor. After treatment the survival time was observed and the tumor volume was calculated by MR. The histological changes were observed by H.E. staining and the expression of CD4, CD8, mIL-4 and BrdU were detected by immuohistochemystry.Results(1)Most of mesenchemal stem cells were long or flat fusiform shape under microscope and positive for CD44, CD105 while negative for CD45. Cells can differentiate into osteoblasts, adipocytes and neural cells.(2)MSCs could be stably transfected by EGFP and green fluorescent was detected under fluorescent microscope. The rat brain tumor was identified as glioma by H.E. staining and GFAP positive. EGFP labeled MSCs mainly distributed into brain tumor and the edge.(3)After transduction MSCs could stably express mIL-4 mRNA and the production of mIL-4 in cultured supernatants (1.0×10~6cells/24h) was 65.78±2.28np/ml. The BrdU labeled MSCs mainly distributed with brain tumor and the edge. Survival time was obviously prolonged after treatment by MSCs-mIL-4 and tumor volume (mm~3) was much smaller than the control groups (55.08±5.14 vs.227.72±10.23, 279.54±18.84, P<0.01). Necrosis was observed in tumor tissue companied with the infiltration of CD4 or CD8 positive T lymphocyte. The expression of mIL-4 protein could be observed in MSCs-mIL-4 group.Conclusions(1)Adult rat bone marrow derived mesenchemal stem cells could be esily cultured and expanded in vitro and have the property of self-renew and multilineage potential.(2)MSCs could be trasfected by exogenous gene.(3)MSCs possess the extensive tropism for rat brain glioma.(4)Gene therapy using mIL-4 transfected MSCs could inhibit the growth of glioma.(5)Gene modified MSCs may be a new tool for gene therapy of malignant brain neoplasms.