The culture of temporary tumor-like bone marrow mesenchymal stem cells (TT-BMSC) and the detection of cell biology property
Yi Chen, Lin Cong, Ximeng Yin, Baotie Dong, Yaxin Han, Guanjun Tu
Ann Transplant 2011; 16(3): 49-58
Background: Bone marrow mesenchymal stem cells (BMSC) are common seed cells for transplantation. However, there are some limitations in their use that have not yet been resolved. Our research modified tumor-related genes temporarily on BMSC which simulated tumorigenesis temporarily in vitro. The cells were named temporarily tumor-like bone marrow mesenchymal stem cells (TT-BMSC).
Material/Methods: Cultivation of TT-BMSC: BMSC were cultured and identified, then the BMSC were transfected MMP-2 gene expressive vector and screened for 4 weeks by G418. Finally, the anti-oncogene PTEN of BMSC was knockdown by ribonucleic acid interference (RNAi) using PTEN gene special small interfering ribonucleic acid (SiRNA). The detection cell biology property of TT-BMSC in vitro: Methyl thiazolyl tetrazolium (MTT) assay and Cell cycle analysis for cell proliferation, Matrigel Invasion Assay for invasion and migration, and the cell model of ischemia and anoxia in vitro for survival.
Results: RT-PCR and Western blot results indicated MMP-2 expression increase significantly after transfection of the MMP-2 expressive vector in the BMSC, while PTEN mRNA and protein expression decrease significantly after PTEN RNAi, and the longest duration of the PTEN RNAi is 15 days. MTT assay and Cell cycle analysis indicated TT-BMSC cell growth vigor is reinforced significantly (P<0.001). Matrigel Invasion Assay showed that TT-BMSC can go through the matrigel successfully (P<0.001). The ability of TT-BMSC to tolerate ischemia and anoxia increased significantly in the model of ischemia and anoxia in vitro (P<0.001).
Conclusions: We cultivated TT-BMSC successfully and TT-BMSC possessed a powerful ability to survive, proliferate, invade and migrate in vitro.
Keywords: bone marrow mesenchymal stem cells, Matrix metalloproteinase-2, phosphatase and tensin homolog deleted on chromosome ten, ribonucleic acid interference