| Hemangioma is the most common benign tumor of infancy,characterized by rapid growth in early life and subsequent spontaneous regression in the following years.The pathogenic mechanism of infantile hemangioma has not been explicated.There are lots of therapies on hemangioma,while the curative effect is not defined.Exploring the pathogenesis ofhemangioma and the mechanism of drugs are helpful to prevent and treat hemangioma.Presently,knowledge about the therapeutic effects of Pingyangmycin and Dexamethason on hemangiomas is obtained from clinical observation and from in vitro study on endothelial cells. So far,the studies on endostatin are mostly related with tumor and there is no investigation on hemangioma.In this study hemangioma endothelial cells(HEc)and human umbilical vein endothelial cell(HUVEC)are primary cultured,and treated with PYM,DX and YH-16,the effect and mechanism of these drugs are observed,three dimensional(3D)angiogenesis model and nude mice hemangioma model were reproduced in order to provide feasible models for more study on hemangioma.The effect of Pingyangmycin on.hemangioma blood vessels is investigated with the 3D angiogenesis model.The hemangiomas are mainly caused by the hyperplastic of the vascular endothelial cells. Successfully isolating and culturing the HEC will provide experimental material for researching the biological behavior of hemangioma.The observations of the hemangioma histology might be used to study its histological development.This investigation may help better understanding the mechanism of hemangioma pathogenesis and provide evidences for future therapy.The total thesis includes three parts:Part One:Culture endothelial cells in vitro,reproduce three dimensional(3D)angiogenesis model and nude mice hemangioma modelExperiment One Primary culture and identify HEC and HUVEC in vitroObjective:To primary culture and identify HEC ancl HUVEC.Methods:1.Gathering 15 cases(aged from 2 months to 18 months,female 9 cases and male 6 cases)of infant skin proliferating hemangiomas.To primary culture the endothelial cells of. infant skin proliferating hemangioma by explants outgrowth culture.2.Gathering 6 cases postpartum novelty umbilical cords,primary culture the endothelial cells of umbilical cords with digestion treatment.We use the sterile hemangioma and umbilical cords as material to isolate and culture the HEC and HUVEC with M199 medium added 10%fetus bovine serum,and in the culture box of 5%CO2,37℃and saturated humidity.Change the culture liquid every two days. We make them to survive from generation to generation after the ECs have grown up to the single layer cells.We observed ECs by phonics microscope,identified ECs by observing expression of the factorⅧrelated antigen which only in endothelial cells by SP immunohistochemistry and transmission electron microscope.The tissue slice was observed by HE staining.Result:All two kinds of ECs were cultured successfully,they spreaded as cobblestone on the bottom of cell culture dishes.High-speed growth was found after three weeks.The cells were observed under a phase contrast microscope for cell morphology analysis and appeared as epithelium.The expression of the factorⅧassociated antigen was positive in the isolated cells and umbilical vein endothelial cells analyzed by SP immunohistochemistry and transmission electron microcopy.The results showed that the cells isolated were the vascular endothelial cells of the hemangioma and the umbilical cord.The tissue slice was observed by HE staining,the result showed that the cell nucleus express blue,the cell cytoplasm express light red and the red blood cell in the vascular are reddish-orange.The nucleus of the local tissue were denseness,this showed that vascular in the hemangioma were hyperplastic obviously.Conclusion:The vascular endothelial cells of the infantile skin hemangioma and umbilical cord can be cultured from generation to generation by explants outgrowth culture and explants outgrowth culture respectively in vitro with highly purity.Experiment Two Establish a three dimension(3D)model for angiogenesis of hemangioma and preliminary study of the modelObjective:To establish a three dimension(3D)in vitro model for angiogenesis ofhemangioma and preliminary study of the model.Methods:A small fragment ofhemangioma biopsy is embedded in fibrin gel in a well of culture plates and incubated in a serum-free,buffered-salt,minimal medium to set up the three-dimension(3D)in vitro model for angiogenesis of hemangioma.The model is cultured in the culture box of 5%CO2,37℃and saturated humidity.The culture medium including M199 medium added 10%fetus bovine serum is changed every three days.And pingyangmycin as an interference factor is used to observe its effect of blood vessels.We observed the model by photics microscope and fluorescence microscope and HE staining.Result:In the model,a complex network of microvessels grows out from the 3 tissue fragments from the day 4th to 7th day after culture,and on the 8th to 10th day after culture a compact network of microvessels come into being and microvessels furcating and crossoverring,then tending to be stationary.Microvessels grow slowly to stagnate on the 11th to 14th day after culture.The compact network around the tissue fragment was confirmed to be blood vessels by immunohistochemistry and electron microscopy.Microvessels grow fastly to stagnate after 1 day to 2 day putting pingyangmycin into the model.The expression of the factorⅧassociated antigen was positive in the microvessels analyzed by SP immunohistochemistry.The cells in the microvessels were the vascular endothelial cells observed by transmission electron.Conclusion:This model represented the in vivo status of hemangioma partly and could be an acceptable model for in vitro study.However,it has the disadvantages of unstable growth rate and regression time.Therefore,this model can not fully reflect the in vivo human hemangioma and further study needs to be carried out for establishing better model for investigation. Experiment Three Establish a human hemangioma in nude mice xenograft modelObjective:To establish a human hemangioma model in nude mice.Methods:A small flesh fragment of strawberry hemangioma specimen was cut into small pieces 5mm×4mm×3mm in size thus followed by grafting subcutaneously into eight juvenile nude mice (BALB/c),8 pieces each.The grafts were observed on volume,color,and texture in the life cycle. Grafts were harvested at 10,12,14 and 16weeks after grafting,respectively.Formalin-fixed and paraffin-embedded specimens were cut into 3～4μm sections and stained with HE for histopathological examination.Results:After 3～4 weeks,all of the tumor samples survived,had hardly change.Parts of the grafts were augmented a little in volume at the end of 6thweeks.Most of the grafts were held the line or decreasing in volume at the end of 8thweeks and turning pallor and rigidity at the end of 13thweeks.At the end of the 16thweeks most of the grafts were in regressing and turned pallor or slightly yellow,with fibrofatty appearance in dissection.The shape of cells in the grafts were multiplicity,there had more collagen fiber and fibroblast.The expression of the factorⅧassociated antigen was positive in the grafts analyzed by SP immunohistochemistry.Conclusions Simplex tissue graft into nude mice is operated easy,the volume of grafted hemangioma in nude mice did not change obviously.Most of the grafts were encapsulated by fibrous tissue and absorbed in the end.This model can not to be a good model for study of hemangioma for its great difference between grafts and clinical fact.It needs more work fish for hemangioma animal models.Part Two:Effect of YH-16,PYM and DX on HEC and HUVECObjective:To investigate the function of YH-16,PYM and DX on HEC and HUVEC.Methods:To primary culture HEC and HUVEC.①Different dosage of YH-16,PYM and DX were applied in the HEC and HUVEC,and the effective concentration of YH-16,PYM and DX were examined by MTT method.The test was divided into groups randomly as drugsexperiment group,PBS-blank control group and M199 medium-negative control group.In the following 12h,24h and 36h interventions,the morphous of EC was observed by photology inverted microscope,the cytoactive was detected by method of MTT and the cell cycle was investigated by flow cytometry(FCM).Results:1.ECs were necrosis under the role of YH-16,PYM and DX,many cells debris can be seen under the light microscope.2.The MTT absorptance decreased after treatment(P＜0.05).3. The analysis of cell cycle in HEC and HUVEC showed that the percentage of G1.phase cells increased(P＜0.05)and that of G2 cells phase decreased(P＜0.05)in all three groups,and the percentage of S phase decreased in the group YH-16 and group DX,and increased in the group PYM.While the percentage of G1 phase and G2 phase cells increased(P＜0.05),and that of S phase cells decreased(P＜0.05)in HUVEC The PI of all three groups decreased(P＜0.05), compared with the normal control.Conclusion:1.EC can be arrested before enters the DNA compose stage by all the three drugs.2. All the three drugs can induce EC apoptosis.3.All the three drugs can change the morphous of EC,and prevent the EC mixed tighter to form blood vessel.4.PYM and DX have the ability to injury the DNA directly,and that take part in the blockage of cell cycle,apoptosis DX arrests HEC and HUVEC enters the replication stage and inhibits cells proliferation.YH-16,PYM and DX inhibit ECs proliferation and growth by arresting DNA composing.Part Three:Effect of YH-16,PYM and DX on expression of VEGF, MMP-2 and Shh in HEC and HUVECObjective:To observe the effect ofYH-16,PYM and DX on expression ofVEGF,MMP-2 and Shh in HEC and HUVEC.Methods:To primary culture HEC and HUVEC.The test was divided into five groups randomly, the effective concentration of YH-16,PYM and DX as experiment group,and PBS as blank control group and M199 medium as negative control group.The expression of VEGF,MMP-2 and Shh were detected by RT-FQ-PCR(real-time fluorescence quantitative polymerase chain reaction).Results:1.The expression of VEGF,MMP-2 and Shh were obviously decreased under the role of YH-16,PYM and DX(P＜0.05).2.The expression of Shh in HEC was higher than in HUVEC and the expression of shh in HUVEC was extremely lower(P＜0.05).3.The expression of Shh was higher than that of VEGF and MMP-2 in HEC(P＜0.05).4.The correlation between VEGF and MMP2,VEGF and Shh,MMP-2 and Shh are significant at the 0.05 level(2-tailed).Conclusion:1.YH-16,PYM and DX can inhibit the expression of VEGF,MMP-2 and Shh in HEC and HUVEC.2.VEGF,MMP2 and Shh can influence each other.3.The expression of Shh in HEC was more higher than in HUVEC and the expression of shh in HUVEC was extremely lower.The expression of Shh was higher than that of VEGF and MMP-2 in HEC.It showed that the Shh maybe had a great role in the path0genesy of hemangiomas,the level of Shh can be used to as a molecular marker for the identification of HEC from HUVEC,and should to be a target of gene therapy to hemangioma in future.
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