Notch-1 Adeno-associated Viral Vectors. Targeting Silence Build Its Human Glioma Cancer Stem Cells Into Nude Mice

Objective To construct the recombinant adeno-associated virus vector expressing Notch-1 gene short RNA interference(RNAi) and to use it block the Notch-1 gene expression, investigate its effection on tumorigenesis of human glioma stem cells.Method The Notch-1 template DNA sequence was designed, and the recombinant vector of plasmid pAAV-MCS2/siRNA-Notch-1 were constructed. The recombinant adenovirus vector AAV-siRNA-Notch-1 was obtained by homologous recombination with pAAV-MCS2/siRNA-Notch-1, pAAV-RC and pHelper in bacteria QBI-293A. Glioma stem cells were isolated from U251 human glioma cell line with serum-free medium. The human glioma stem cells were transfected in optimum moi multipe of infection, then we divided these cells into three group:rAAV-EGFP-Notch-1 group, rAAV-EGFP group and control group. Each group's cells would be subcutaneously injected into 5 nude mice.20 days after, mice were sacrificed.Immunohistochemistry was used to determine the expression of Notch-1 gene.Result The pAAV-MCS2/siRNA-Notch-1 was successfully constructed by the results of PCR, and the rAAV with a titer of 1.2×1012 (VP/L) was obtained. The glioma stem cells from human glioma cell line U251 have been successfully isolated and they can expressed CD 133 and Nestin positive. Compared to the control group, the volume of rAAV-EGFP-Notch-1 group decreased obviously and immunohistochemistry testified the expression of Notch-1 protein in this group decreased significantly too.Conclusion The viral vector was successfully prepared, and it may be used in further investigation of Notch-1 function in tumor stem cell(TSC). Blocking Notch-1 gene expression can effectively suppress the growth rate of human gliomas stem cells in subcutaneous tumor, which provides a new way to treat human glioma.