A Study On The Apoptosis And Biological Properties Of Human Hemangioma In Nude Mice Xenograft Model

Background and objectivesInfantile hemangioma is the most common benign tumor of infancy characterized by rapid growth in early life and subsequent spontaneous regression in the following years. Recently more and more researchers demonstrated the development of hemangiomas may be the results of imbalance of cell proliferation and apoptosis and increased apoptosis may be involved in the regression of hemangioma. But specimens in most of researches were obtained from clinical different hemangiomas . In this study, an animal model of human hemangioma on nude mice was established by xenografting human hemangioma tissue into nude mice subcutaneously. Thus the apoptosis and biological properties of human hemangioma in vivo can be observed to explore initiatively the natural regularity and cause of hemangioma regression. MethodsEstablish xenograft model: The original hemangioma specimen was obtained from a male infant aged 2 months by surgery. It was cut into small pieces 5mm × 4mm × 3mm in size thus followed by grafting subcutaneously into sixteen juvenile nude mice, 6 pieces each.The grafts were observed on volume, color, and texture in the life cycleGrafts were harvested at 2, 5, 7, 15, 30, 45, 60, 90, 120, 180days after grafting, respectively. Formalin-fixed and paraffin-embedded specimens were cut into 6 μ m sections and stained with HE for microscopic observation.CD31 monoclonal antigen was employed in immunohistochemistry on specimens harvested at different time points.TUNEL assay was employed to detect apoptotic cells and the Ki-67 antigen to detect dividing cells in the grafts> Immunohistochemistry and image analysis were employed to detect the expression of GR and VEGF at different time courses.Results> The grafts development typically exhibits an ischemia phase followed by a proliferative phase that continues into the involuting phase. During the ischemia stage, the most of grafts were observed decreasing in volume and turning pallor, and subsequently grew to achieve the apex of volume curvature at the end of 2nd month. At the end of the 6th month, most of the grafts were in regressing and turned pallor or slightly yellow, with fibrofatty appearance in dissection.> Under light microscopy observation, the original human specimen of hemangioma was consisted of plump cells crowded in lobes. Small and irregular lumens containing blood cells were recognizable among the cells.> In the ischemia phase(about2-15days) large scale of hyaline degeneration with scattered cell groups and coagulation necrosis was definite in sections cell groups grew in number and then grew in cellular density as evidenced on specimens harvested in the proliferation phase(about30-60days). Karyokinesis was frequent and the grafts resembled typical proliferative hemangioma in morphology. In the invloluting phase the grafts were mostly substituted by fibrofatty tissue with residual distended microvascular lumens.> Immunohistochemistry of mouse anti-human CD31: High level positive staining during the three phases identified that the epithelial cells in the living grafts were of human origin.> Endothelial proliferation was present throughout all the phases, but there were no demonstrable differences between the different phases. In contrast , a clear distinction was evident in apoptosis: TUNEL-positive endothelial cells were highly observed throughout the involuting phase but were sparse in the proliferating phase.> Positive staining of VEGF was recognized in grafts and located in cytoplasm of endothelial cells in proliferating phase, and was hardly recognizable in grafts in involuting and involuted phases.> Positive staining of GR was recognized in grafts and located in cytoplasm of endothelial cells in proliferating phase,and was hardlyrecognizable in grafts in involuting and involuted phases. Conclusion> Planting of human subcutaneous hemangiomas onto nude mice subcutaneously is a feasible way to establish a model of human hemangioma. In this model, the histological and biological properties of human hemangioma were retained with stable growth which resembles that of natural pattern.> The development of hemangiomas may be the results of imbalance of endothelial cell proliferation and apoptosis. And during the development phases of hemangiomas, apoptosis of the endothelial cells play the crucial role. The increasing apoptosis seems to be associated with spontaneous regression of hemangiomas.>^ VEGF not only effectively induce endothelial cell proliferation, but also its untimely discontinuation might lead to subsequent regression of the new endothelium.