Study On The Origin Of Endothelial Progenitor Cells In Infantile Hemangioma

RESEARCH BACKGROUNDInfantile hemangioma is a vascular benign tumor characterized by abnormal hyperproliferation of vascular endothelial cells.The incidence of infantile hemangiomas reaches 4%-5%,and the incidence of preterm infants can be as high as23%.It can affect all parts of the body,such as skin,muscle,bone,heart,liver,etc.About 60% occurs in the head and neck,25% occurs in the trunk,and 15% occurs in the limbs.Hemangiomas proliferate rapidly in the early stage,forming an endothelial cell mass with no clear vascular structure.In the later stage,some of the skin lesions slowly fade away,leaving fibrous fatty tissue behind.About the pathogenesis of IH,there are several theories,such as genetics and gene mutations,placenta chorionic ectopicism,stem cell theory,hypoxia theory and so on.However,no perfect theory can fully explain the etiology,pathogenesis and clinical features of IH.Therefore,exploring the pathogenesis of infantile hemangiomas plays an important role in the prevention and treatment of IH.Pathological studies show that proliferative hemangiomas have a large number of endothelial cells.So where does a large number of proliferating endothelial cells come from?At present,most viewpoints suggest that proliferative endothelial cells are derived from hemangioblast stem cells and endothelial progenitor cells.Endothelial progenitor cells(EPCs)are a group of embryonic angiogenic cells.It can proliferate and differentiate into precursor cells of mature endothelial cells.it is considered that cells labeled with CD34 and CD133 are EPCs.Recentstudies have shown that there are three major sources of EPCs: bone marrow,peripheral blood,and cord blood.In the early stage of hemangiomas without luminal structures,peripheral blood EPCs can not enter the fetal tissue successfully.The number of bone marrow-derived EPCs is extensive and widely distributed,which is not consistent with the incidence and clinical features of hemangiomas.So whether the endothelial progenitor cells of infantile hemangiomas proliferate from placental chorionic tissue,Which factors are involved in their differentiation.This is the significance of the research group for this study.A previous study of the research group found that The vascular endothelial growth factor(VEGF)and the Delta sample ligand 4(DLL4)were highly expressed in the rapid growth stage of infant hemangioma,And the two were related.Some studies have found that 548 cases of pregnant women who had chorionic venepuncture materials,the incidence of infantile hemangiomas in their children as high as 21.1%.The majority of hemangiomas are located in the head,neck and chest,and are mostly multiple.Chorionic villus stripping is widely used in prenatal diagnosis.During this process it may cause the fetus to expose the villi and cause related complications such as limb defects,hemangiomas,and other vascular defects,especially in the early pregnancy(8-9 weeks).The corresponding risk will be greater.Therefore,this project conjectured that during pregnancy,due to factors such as chorionic puncture,ruptured placenta,or partial infraction of placenta tissue,placental tissue EPCs enter the fetus with blood and colonize a certain site.Because during perinatal period,especially during fetal delivery,umbilical cord injury around the neck and the birth canal causes fetal and neonatal hypoxia,and promotes the production of hypoxia-inducible factor(HIF-1?)in large quantities,thereby enhancing the VEGF gene expression.The overexpression of these factors further recruits and stimulates the proliferation of vascular endothelial progenitor cells and differentiates into endothelial cells,forming a large number of newly formed capillaries,which constitute proliferative hemangioma tissues.In the regression phase,the number of EPCs was greatly reduced,and at the same time,the pericytes proliferated under peripheral vascular stimulation and further differentiated intoadipocytes,constituting the major cellular components of the hemangiomas during the subsided period.OBJECTIVE1.Immunohistochemical staining is used to detect the expression of VEGF/DLL4 in infantile hemangiomas and normal skin tissues during the proliferative phase and extinction phase.The correlation between the two was analyzed,To confirme that VEGF/DLL4 regulates the blood vessels of proliferative hemangiomas.Generated to regulate the occurrence of hemangiomas.2.Immunohistochemical SP method and immunofluorescence method were used to detect the expression of CD133,CD34 and CD31 in infantile hemangiomas.In order to confirm the presence of EPCs in proliferative infantile hemangiomas and EPCs are the cytological basis for hemangiomas.3.Immunohistochemistry and immunofluorescence were used to detect CD133 and CD34 in maternal placenta of infants with hemangiomas in infants,confirming the presence of EPCs in placenta.4.Placental chorionic tissue cells were isolated and cultured by enzyme digestion and density gradient centrifugation.Endothelial progenitor cells were identified in placental tissues of infants with infantile hemangiomas by immunofluorescence staining and flow cytometry.It is the cytological basis of the occurrence of hemangiomas in infants and young children;and the morphological observation of them to explore the optimal isolation and culture methods of endothelial progenitor cells.METHODSPart 1.50 infant hemangiomas specimens were collected,including 5 fresh specimens.After reviewing the data,all cases were divided into proliferative phase(n=26),regressive phase(n=24),and another 5 normal paraneoplastic tissues were taken as Control,All wax blocks were stained with HE to observe the pathological features ofhemangiomas in each stage.Immunohistochemical staining was performed using a HPIAS-10 high-resolution color pathology report management system to measure the mean optical density and positive area ratio of VEGF/DLL4 protein expression and perform quantitative analysis.Part 2.(1)Collect the IH tissue archived paraffin blocks,organize the patient's medical records,and confirm that all the children did not receive any adjuvant treatment before surgery.A total of 50 patients were included,including 22 males and 28 females,aged from January to 2 years old.1.3 years old.All hemangiomas were histologically confirmed by three pathologists.Immunohistochemical staining and immunofluorescence staining,using the HPIAS-10 high-resolution color pathology report management system to detect the average optical density and positive area ratio of CD133,CD34,CD31 protein expression and quantitative analysis.(2)A total of 30 pregnant women delivered from the Department of Obstetrics of the Fifth Affiliated Hospital of Guangzhou Medical University from September2015 to September 2017 were selected.Among them,25 infants with neonatal susceptibility to infantile hemangiomas and 5 newborn infants who did not have hemangioma.After that,the placental tissue part of chorionic chorionic villi was harvested under sterile conditions.The qualitative analysis of placental chorionic tissue CD133 and CD34 were qualitative detection by immunohistochemistry and immunofluorescence.(3)Using the aseptic placenta tissue collected in the previous step,placental tissue cells were isolated and cultured by enzymatic hydrolysis combined with density gradient centrifugation.Qualitative analysis of CD133 and CD34 proteins was performed by immunofluorescence and flow cytometry to confirm placental tissue.There are endothelial progenitor cells.Cells stained with CD133(+)CD34(+)were identified as endothelial progenitor cells.(4)SPSS 17 was used to analyze and process the data.Each group of data was expressed as the mean standard deviation(X±s).The mean optical density and positive area ratio of each group were analyzed by one-way ANOVA and SNK(q)test.The positive area ratio was Make a bivariate correlation analysis.The provision P ? 0.05 considered the difference statistically significant.RESULTPart 1VEGF and DLL4 protein were highly expressed in the proliferative phase of infantile hemangiomas but not in the regression phase group and the normal skin group(P<0.05).There was no significant difference between the regression group and the normal skin group.(P>0.05).The correlation analysis of VEGF and DLL4 positive area ratio showed that the expression of VEGF and DLL4 was positively correlated in proliferative and regressive stages,VEGF: r=0.821,P=0.003;DLL4:r=0.830,P=0.003.Part 2(1)The IOD and positive area ratios of CD31,CD34,and CD133 proteins in proliferative hemangiomas were significantly higher than those in IH tissues and normal skin groups at the time of regression(P<0.05).There was no significant difference in the IOD and positive area ratio of CD31,CD34 and CD133 protein in the group(P>0.05).Immunofluorescence staining showed high expression of CD34 and CD133 proteins in proliferative hemangiomas,and low expression in hemangiomas and normal skin tissues during the remission period.(2)Placental tissue immunohistochemical staining showed that the trophoblast cells surrounding brown-yellow particles,CD34 and CD133 protein has a more significant expression,immunofluorescence showed that CD34 and CD133 protein in the placenta tissue is highly expressed.(3)Under the inverted microscope,the newly isolated mononuclear cells are round,the cell morphology is small and unevenly suspended at the bottom of the petri dish;after 48 h,cells adhere to the wall;after 3 d,the adherent cells have tiny colonies formed around The cells were colonial-centered and germinated outward.On the 7th day,they were mainly round cells in the center and surrounded by typical colonies of spindle cells.On the 10 th day,the spindle cells had typical line-like arrangement.Adjacent cell clusters connect to each other to form a vascular network-like structure.After 14 days of culture,spindle-shaped cells gradually shortened and gradually became rhombic.The adherent cells gradually showed a typical paving stone-like change on the 28 th day of culture.(4)Cellular immunofluorescence showed that there were endothelial progenitor cells expressing both CD34 and CD133 in cells isolated by this method;Flow cytometry showed that CD34 and CD133 dual-labeled endothelial progenitor cells exist in placental tissues isolated and cultured by this method.CONCLUSIONS1.VEGF and DLL4 are highly expressed in proliferative hemangioma tissues,and they are lowly expressed or not expressed in the regressive stage and normal skin tissues,and there are significant differences.It was proved that VEGF-DLL4/Notch signaling pathway may participate in the pathological process of IH.2.EPCs markers CD34 and CD133 proteins are highly expressed in proliferative hemangiomas and placenta tissues,suggesting that EPCs of infantile hemangiomas may share homology with placental tissue EPCs and are precursor cells of endothelial cells.The pathological process of hemangiomas is based on cytology.3.EPCs can be successfully isolated from maternal placenta and normal placental tissues of infants with infantile hemangiomas.