Research Of Monocyte Chemoattractant Protein-1/s Effect In The Tropism Of Bone Marrow-derived Mesenchymal Stem Cells For Glioma Cells

Objective: To detect the cases of C6glioma cells secreting monocytechemoattractant protein-1and bone mesenchymal stem cells expressing CCR2（thereceptor of monocyte chemoattractant protein-1）, explore the role of monocytechemoattractant protein-1in bone mesenchymal stem cells migration in response toglioma cells.Methods:1.Bought SD male rats, took thighbones and tibias, separated bonemesenchymal stem cells, characterized by immunocytochemistry using known celltype–specific surface markers;2.Bought and subcultured C6glioma cells;3.CollectedC6glioma cell-conditioned medium, preserved at-20°C for reserve;4.Qualitativelyresearched monocyte chemoattractant protein-1DNA in C6glioma cells using PCR,detected the case of C6glioma cells secreting monocyte chemoattractant protein-1usingELISA;5. Qualitatively researched CCR2DNA in bone mesenchymal stem cells usingPCR, detected the case of bone mesenchymal stem cells expressing CCR2usingimmunocytochemistry;6. Transwell assay was used to observe bone mesenchymal stemcells migration toward C6glioma cells in vitro, there were three groups: GroupA(C6glioma cell-conditioned medium), GroupB(C6glioma cell-conditioned mediumsupplemented with monocyte chemoattractant protein-1neutralizing antibody),GroupC(serum-free LG-DMEM medium). Counted the number of bone mesenchymalstem cells migrated into the low-chambers in different groups and statistic analysed thedifferences among them.Results:1. Detected bone mesenchymal stem cells type–specific surface markersusing immunocytochemistry, the results showed that CD45expression was negative,caryon were purple, the cytoplasm and cell membrane did not dye; CD44/CD90/CD105 expressions were positive, the cytoplasm and cell membrane dyed yellow.2.Qualitatively researched monocyte chemoattractant protein-1DNA in C6glioma cellsusing PCR, the result showed that a band appeared at658bp, and the band was accordedwith the length of monocyte chemoattractant protein-1gene in the length; detected thecase of C6glioma cells secreting monocyte chemoattractant protein-1using ELISA, theresult showed that the differences between the monocyte chemoattractantprotein-1concentrations of different groups had statistical significance, P<0.05.3.Qualitatively researched CCR2DNA in bone mesenchymal stem cells using PCR, theresult showed that a band appeared at409bp, and the band was accorded with the lengthof CCR2gene in the length; detected the case of bone mesenchymal stem cellsexpressing CCR2using immunocytochemistry, the result showed that CCR2A andCCR2B expressions were positive, the cytoplasm and cell membrane dyed yellow.4.The results of Transwell assay showed that the difference between GroupA andGroupB had statistical significance, P<0.05; the difference between GroupA andGroupC had statistical significance, P<0.05.Conclusion:1. The primary cultured cells were bone mesenchymal stem cells.2.C6glioma cells could secrete monocyte chemoattractant protein-1.3.Bonemesenchymal stem cells could express CCR2.4. Monocyte chemoattractant protein-1played an important role in the tropism of bone marrow-derived mesenchymal stemcells for glioma cells.