liver endothelial cells than normal vascular endothelial cells have enhanced angiogenesis and the role of drug resistance

Title: liver endothelial cells than normal vascular endothelial cells have enhanced angiogenesis and the role of drug resistanceAuthor: Xiong YuquanDegree-granting units: Fudan UniversityKeywords: liver cancer;; tumor vascular endothelial cells;; normal vascular endothelial cells;; angiogenesis;; sorafenib;; drug resistanceSummary:Primary liver cancer is a common malignant tumor, surgical resection remains the most effective treatment, but 5-year recurrence and metastasis after radical resection rate of 60-70%, which is the main reason affecting long-term effect of liver cancer. Inhibition of tumor angiogenesis, survival of the tumor can be reduced nutrient supply, thereby inhibiting tumor Neodymium Magnets growth. For unresectable liver cancer and liver cancer metastasis and recurrence prevention, anti-angiogenesis is a promising approach. Angiogenesis inhibitors to treat cancer is based on the theory of new and remodeling of tumor blood vessels is different from the rest of the normal blood vessels. Vascular heterogeneity of tumor angiogenesis in recent years, research focus, from the molecular level the heterogeneity of tumor blood vessels, helping to monitor the evolution of the tumor, estimated the degree of malignancy and may provide new targets for cancer therapy. Evidence that the tumor vascular endothelial cell growth factor receptors, signal transduction molecules and adhesion molecules such as molecular and cellular activity associated with the expression of vascular endothelial cells differ from normal, abnormal expression of these molecules may contribute to tumor vascular endothelial cells with normal cell function in vascular endothelial cells vary. So far, reports from the breast, colon, and glioma and other tumors carve election cultured endothelial cells than normal endothelial cells have a stronger angiogenesis and chemotherapy http://www.everbeenmagnet.com/en/products/110-sintered-neodymium-magnets resistance, but has not been detected against the tumor vascular endothelial cells Drug sensitivity studies to generate reports, and no isolated and cultured liver endothelial cells and to study their angiogenic ability and other characteristics of the study reported. Currently, sorafenib and other anti-angiogenic drugs in the treatment of liver cancer progress, prolong survival, but its effect is not lasting, typically several months after the emergence of drug resistance, tumor growth again, and its resistance mechanism is not clear. The issue to be isolated from human liver and cancer through the tissues of the vascular endothelial cells subcultured, and detection of tumor vascular endothelial cells and normal endothelial cell function differences, simultaneous detection of tumor vascular endothelial cells and vascular endothelial cells against normal angiogenesis differences in drug response and to explore differences in the molecular mechanism of its occurrence, with a view to improve the efficacy of anti-angiogenic drugs to find new ways.1 research platform for tumor vascular endothelial construction of a human liver and cancer tissues of vascular endothelial cell isolation, identification and subculturedThe number of endothelial cells is extremely limited, the separation and purification affected by many factors, how to mix a variety of tumor cells into endothelial cells isolated and purified the key. We used CD105 antibody cross-linked magnetic activated cell sorting method, and to optimize the human liver and cancer tissues for vascular endothelial cell sorting, and the subculture. Liver cancer tissues from isolated CD105-positive cells was a typical "cobblestone" morphology of endothelial cells; isolated from liver tissue CD105-positive cells does not have this feature, and the shuttle long fibrous phenotype. AFP and RT-PCR, flow cytometry showed that CD68-positive sorting of cells up to 99% of CD105 expression, but did not express AFP and CD68, thus excluding the tumor cells and macrophages pollution; Immunocytochemistry analysis showed positive points Ⅷ factor expression in selected cells as a positive, more than 95% positive sorted cells were Ⅷ factor and acetylated low density lipoprotein uptake test positive; sorting cells in Matrigel gel can form capillary structure; flow showed CD105, CD31, CD34, CD144, VEGFR1 and VEGFR2 markers such as vascular endothelial cells derived from CD105-positive cell sorting expressed on both. While sorting the positive cells were subcultured to 20 generations or more. These results indicate that by optimizing the immune magnetic separation method, we successfully obtained high-purity sorting of CD105-positive liver cancer CD105 positive endothelial cells and normal endothelial cells, and can be successfully subcultured, isolated and cultured liver endothelial cells can be used cancer angiogenesis research.(2) tumor vascular endothelial cells and normal endothelial cell function moreTo compare CD105 ~ + TEC (tumor vascular endothelial cells) and CD105 ~ + NEC (normal vascular endothelial cells) and HUVEC (human umbilical vein endothelial cells) and functional differences, we are in complete serum-free medium in continuous culture cells, the result CD105 ~ + TEC proliferation was significantly higher than CD105 ~ + NEC and HUVEC; in serum-free culture medium of cultured cells display CD105 ~ + TEC anti-apoptotic ability was significantly higher than CD105 ~ + NEC and HUVEC; scratch experiments showed that CD105 ~ + TEC's migration is higher than CD105 ~ + NEC and HUVEC; 3D Magtrigel rubber serum-free culture medium to form capillary network structure and function showed that CD105 ~ + TEC angiogenic capacity than CD105 ~ + NEC and HUVEC; cell adhesion showed that CD105 ~ + TEC ability to interact with tumor cells than CD105 ~ + NEC and HUVEC; drug trials show CD105 ~ + TEC of doxorubicin and 5 - fluorouracil resistance was significantly higher than CD105 ~ + NEC and HUVEC. This illustrates the CD105 ~ + TEC with CD105 ~ + NEC and functional differences in HUVEC, showing stronger proliferation, anti-apoptosis, migration, angiogenesis, and tumor cell interaction capabilities and greater resistance to chemotherapeutic drugs, should be more applicable to the mechanism of tumor angiogenesis in vitro.3 tumor vascular endothelial cells and endothelial cells of normal vascular response to sorafenib treatment differences in its mechanismAlthough the election took part in the current culture of tumor vascular endothelial cells and to explore its function studies, but not on vascular endothelial cells against tumor angiogenesis drug reaction reports. We compared the tumor vascular endothelial cells and normal endothelial cells to sorafenib's response differences: cell proliferation experiments showed that sorafenib at low concentrations the effects of various cell lines was no significant difference, when the concentration is greater than 5μM sorafenib when, CD105 ~ + TEC than CD105 ~ + NEC and HUVEC showed higher resistance; 5μM sorafenib concentrations, the tubule formation assay and spheroid assay experiments showed that, HuVEC and CD105 ~ + NEC can not form a capillary network structure or the occurrence of budding structures, and CD105 ~ + TEC able to form a capillary network structure and the occurrence of budding structures, indicating that tumor endothelial cells more resistant to the role of sorafenib. We then examined the cell lines before and after treatment in the sorafenib its p-STAT3, STAT3, p-Akt, Akt, p-MAPK and MAPK signaling molecules changes, the results show relative HUVE and CD105 ~ + NEC, CD105 ~ + TEC in sorafenib for 24 hours before and after expressing higher levels of p-AKT and p-STAT3, the P-MAPK expression level of the basic lower and P-MAPK expression in the rebound after sorafenib treatment increased. Tip shorter dosing interval or in combination with other medications may help to improve the efficacy of sorafenib.Conclusion1 used to optimize the immune magnetic separation method can be effectively purified liver and cancer tissues in vascular endothelial cells, and can be subcultured.2.CD105 ~ + TEC and compared CD105 ~ + NEC HUVEC in endothelial cell-specific molecule expression were different.3.CD105 ~ + TEC and compared CD105 ~ + NEC HUVEC with functional differences, showed a stronger ability to survive and chemotherapy resistance.4.CD105 ~ + TEC and compared CD105 ~ + NEC HUVEC soraafenib more resistant to the effects, its mechanism and CD105 ~ + TEC express higher levels of p-AKT and p-STAT3, but express low levels of P- MAPK, and P-MAPK expression in the rebound after sorafenib treatment-related increase.Value of1 sorting to obtain purified liver cancer and peripheral vascular endothelial cells, and can be subcultured, the mechanism can be used for liver cancer angiogenesis and anti-angiogenic therapy research.(2) to explore the CD105 ~ + TEC for more resistance to sorafenib's mechanism for improving the efficacy of sorafenib for liver cancer provides new clues.Innovation1 The first successful subculture from the liver tissue sorting of CD105 ~ + vascular endothelial cells.2 for the first time validated against tumor angiogenesis, vascular endothelial cells more resistant to drugs sorafenib and explore its possible molecular mechanism.Degree Year: 2009