Neuroblastoma Cells In Boyden Chamber In Vitro Invasion Model And Cxcl12 And Its Receptor Antagonist Amd3100 On The Migration And Invasion Of Neuroblastoma

Objective:Neuroblastoma is one of the most common malignant tumors of children,it owns the high invasion. In order to study the invasion feature of the NB, we build the new cell lines of NB ourselves to offer a good model for the study biological characteristics of the NB. Boyden chamber owned the ability to determine the invasion and metastasis of the tumors. The chamber owns the shape of the barrel,the bottom owns the polycarbonate microporous which is coated the reconstituted basement membrane.The tested cells were layed the upper chamber,the lower chamber owned the chemotactic liquid which attracted the cells migration. The invasive cells began to adhere to the surface of the artificial basement membrane,and then secreted the matrix metalloproteinase to digest the artificial basement membrane.The cells moved the porous membrane,and finally attached to the other side of the membrane. The quality of the cells reflect the invasive which attached to the other side of the membrane.To investigate the specific condition of the Boyden chamber adapt to the neuroblastoma cells,we tested four different cell lines under the different concentrations and times to gain a stable cell density and time as a standardized condition of the neuroblastoma.Methods:Use Boyden chamber in vitro assay to investigate the invasion ability of the four cell lines.100μl four cell suspension of various concentrations(1.5×105,2.0×105,2.5×105,3.0×105/ml) was added into the upper chamber of the Boyden chamber, and the cells were allowed to penetrate the membrane for 24,36,48 and 60 hours respectively. The invasive rate was gotten as the quantity of cells on the under surface of the membrane.Results:Bovden chamber invasion models showed that the invasion of four different cell lines was not exactly same.The invasive quantity of tumor cells was still low at 24 hours after the incubation, and increased dramatically at 48 hours, and after 48 hours tended to be smooth. Besides, the invasive quantity went high as the cells concentration added increaed, while not dramatically when above 2.5×105/ml. Under the same conditions,the invasion ability of cells with the capacity of transfer the bone marrow was larger than the cells without capacity of transfer the bone marrow.Conclusion:The experiment successfully established the invasion model in vitro of Boyden chamber that was adopted to the neuroblastoma cells.It was significant to the measurement of the invasive ability in vitro of the neuroblastoma cells and the transfer mechanism and influence factors of the cell. Objective:The malignant tumors owned the invasion and the metastasis characteristics.The ability of the metastasis and the trend were the main deciding factors for the tumor cells matastasis.Recent studies showed the some malignant tumors transferred the specific organs. The chemokines played an important role during the organ-specific metastasis of the tumors.The CXCL12-CXCR4 biological axis played an important role in the organ-specific metastasis of the variety tumors.The neuroblastoma metastasized to the bone marrow.The unique metastatic pattern and the high CXCL12 levels in the micro-environment of the bone marrow, the general expression of the CXCR4 of the neuroblastoma cells, we speculated that neuroblastoma cells used CXCR4-mediated response to activate the tumor cells through blood vessels and the interstitial,and formed the bone marrow metastasis of the NB.We used the Boyden chamber to test different concentrations of the CXCL12 induce the NB cells migration and invasion,and then used the CXCL12 specific receptor antagonist AMD3100 blocked the biological axis to study the role of the CXCL12/CXCR4 during the invasion and metastasis of the NB cells.Methods: Use Boyden chamber in vitro assay to investigate the migration and invasion ability of the human neuroblastoma cell lines. The cell lines were incubated with or without 100ng/ml AMD3100, then induced by differfert concentration CXCL12(30,100,300ng/ml) or without.Results:Boyden chamber assay in vitro indicated that the ability of Chinese neuroblastoma cells migration and invasion were enhanced significantly by CXCL12 in in vitro culture system. The quantity of the trans-membrane cells under 100ng/ml CXCL12 was the most. AMD3100could inhibit the cells migration and invasion.Conclusion:CXCL12 played an important role of the neuroblastoma cells migration and invasion,,and AMD3100 has the potential therapeutic significance.