Isolation Of MSCs And Explore The Mechanism Underlying The Tropism Of MSCs For Local Tumor

Background and Objective Bone marrow mesenchymal stem cells are a class of multi-potential cells can differentiate to adipogenic, chondrogenic, osteogenic or muscle . MSCs are known to have a tendency to accumulate at the site of tumors, and therefore can be utilized as a platform for targeted delivery of anti-cancer agents. This study is to isolation of human bone marrow-derived mesenchymal stem cells(Mesenchymal stem cells,MSCs)in vitro ,and to explore the mechanism underlying the tropism of hMSCs for local tumor in vivo.Methods (1) The bone marrow was obtained by aspiration from a healthy male volunteer, MSCs cells were isolated by HISTOPAQUE?-1077 densitygradient and cultured in expansion medium.(2)Stromal cells were analyzed by flow cytometry and confocol microscope using phycoerythrin(PE)–conjugated monoclonal antibody (mAb) to CD44,CD105,CD90,CD34,CD45,CD19. Isotype-matched mouse mAbs were used as negative control. Cells were analyzed using a FACSCalibur flow cytometer and CellQuest Pro Software. To investigate the differentiation potential of the mesenchymal cells, we culture the cells under conditions that were favorable for adipogenic, chondrogenic, or osteogenic differentiation. (3) Mice with established xenograft model for in situ breast cancer were injected with the human mesenchymal stem cells labeled with SPIO in vitro through the tail vein and then sacrificed at time point. The distribution of MSC-derived cells in tumor was then examined by immunohistochemistry. (4) Collect the fresh breast tumor tissue in aseptic conditions, tissues are mechanically minced and subsequently digested enzymatically with collagenase. Tumor-associated vascular endothelial cells in the resulting single-cell suspension are labeled with Abs against CD31 antigens and separated by capture with magnetic beads, and then endothelial cells were cultured in vitro. (5) Tumor-associated vascular endothelial cells were analyzed by flow cytometry and confocol microscope using phycoerythrin(PE)–conjugated monoclonal antibody (mAb) to CD34,CD31,vWF. Isotype-matched mouse mAbs were used as negative control. (6) A Modified Boyden chamber assay was used to investigate the in vitro migration of MSCs to tumor-associated vascular endothelial cells. MSCs were cultured in a 24-well tissue culture insert with an 8μm pore size membrane (BD Biosciences). Migrating cells were stained with crystal violet and counted using bright-field microscopy under 200×magnification.Result (1) Cells were cultured from marrow after density fractionation were spindle, highly homogeneous in shape and had a strong ability of proliferation. (2)Flow cytometry shown the expression of surface molecular markers CD44, CD105, CD90 in mesenchymal stem cells were 99.79±3.35%, 99.62±3.78%, 98.93±3.62% and were negative for reactivity to antigens CD19, CD34, or CD45; It was observed that surface molecular markers CD44, CD105, CD90 were highly expressed, especially in cytoplasm through confocol microscope; Bone marrow mesenchymal stem cells were induced to differentiate to bone, fat and cartilage successful, the result shown that MSCs have the potential to differentiate to lineages of mesenchymal tissue.(3)Mice with established xenograft model for in situ breast cancer were injected with the human mesenchymal stem cells labeled with SPIO in vitro through the tail vein, SPIO labeled cells were mainly in the local tumor.(4) Tumor-associated vascular endothelial cells isolated in vitro were adherent growth at 6-12-hour, cells were small polygonal, spherical at begin, gradually growing into spindle, after 3 ~ 4 days ,the cells were cobblestone-like arrangement.(5) Crystal violet staining showed that: groups of tumor-associated vascular endothelial cells, migration of MSCs across the membrane were obviously more than control group (p <0.05).Conclusion Bone marrow mesenchymal stem cells were easily to obtain and culture in vitro. The tropism of hMSCs for local tumor in vivo was related to local tumor vascular endothelial cells. OBJECTIVE:It is aimed to study the relationship between Snail and invasion and metastasis of cervical cancer, and to explore its relative molecule mechanism.METHODS: Snail over-expression plasmid was transfected into cervical cancer cell lines of Hela and Siha. Western blot was applied to analyze the corresponding molecules including Snail, E-cadherin and Vimentin. The invasion and metastatic ability was examined by in vitro cell wound healing experiment and Boyden chamber invasion assay.RESULTS :(1) Transfected with PEGFPC1/Snail in cervical cancer cell lines of Hela and Siha, the expression of E-cadherin was significantly lower than its counterpart, however, the expression of Snail and Vimentin was significantly higher than its control group .(2)The capability of wound closure in Siha and Hela cells transfected with PEGFPC1/Snail at 12h was significantly enhanced(p<0.05), the invading/migrating cells were obviously increased at 12h after transfecting with PEGFPC1/Snail in Siha and Hela cells(p<0.05). Up-regulation of Snail could enhance the capability of metastasis and invasion.CONCLUSION: Transcription factor of Snail could enhance the potential of invasion and metastasis of cervical cancer cell lines through induction of EMT( Epithelial-mesenchymal transition).