| BackgroundHemangiomas are the most common tumors in infants and young children.Infantile hemangiomas are the majority of them.The use of drug injection and oral propranolol has achieved good results and formed an ideal and standardized treatment guideline.Kaposi-form hemangioendothelioma(KHE)is a minority in hemangiomas,but it is a serious one in all kind of hemangiomas.KHE was first reported by Zerberg et al.in 1993.It is an aggressive tumor with tissue morphology similar to Kaposi's sarcoma,which develope rapidly and often accompany with thrombocytopenia and consumptive coagulopathy.In the latest classification of hemangiomas and vascular malformations published by the International Society of Research on Hemangiomas and Vascular Malformations,it is defined as "vascular tumors with locally aggressive behavior or borderline" and it is also cleared that Kasabach-Merritt phenomenon(KMP)is a typical characteristic of KHE.Kaposi-form hemangioendothelioma is mainly manifested as a giant invasive localized hemangioma without self-healing tendency in clinic.It is often accompanied by thrombocytopenia,hypofibrinogenemia,microvascular,consumptive anemia,and severe coagulation.All of these symptom are defined as KMP.Due to its severe symptoms,acute onset,progressive exacerbation of symptoms in a short period of time,KHE that occurs in deep tissues such as the mediastinum and retroperitoneum has difficulty in timely diagnosis,and most of the affected individuals are infants and young children.All these leads to a high mortality rate of 12%-30%.The pathogenesis of Kaposi-like hemangioendothelioma and the mechanism leading to thrombocytopenia and coagulation dysfunction are still unclear.There is no unified opinion on the treatment methods and treatment specifications of KHE at home and abroad.So KHE is the most prominent and difficult issues among all hemangiomas.Animal models are important tools for exploring the etiology,pathogenesis,control technology and drugs of disease.However,no successful animal model of Kaposi-like hemangioendothelioma has been reported.At present,the main research line of hemangiomas is the mechanism of initiation and regulation of angiogenesis.Therefore,we envision a new way to construct an in vitro animal model that reflects the angiogenesis process of Kaposi-like hemangioendothelioma.Embed KHE tissue block sections in fibrin gel and cultured in plates.This create a three-dimensional model of angiogenesis of Kaposi-like hemangioendothelioma.This construction method can solve the key problem of KHE animal model construction.The completed model can provide a good model for the study of angiogenesis of hemangiomas.It can be used for drug effectiveness and effects,the development and transformation of KHE,the principle and development of KMP,and provide new possibilities and new ideas for related research on KHE and KMP.Method1.Tissue preparation:Immediately immersed tissue of surgically resected proliferative hemangiomas into Hanks' solution at 4 ?,wash the blood salamander,and then immerse into serum-free EGM-2 medium.Remove normal skin and excess fat at room temperature.Cut into 1mm3 tissue pieces for later use Sent the remaining surgical specimens to routine pathological and immunohistochemical experiments2.Preparation of culture system(double-layer sandwich method):Add penicillin,streptomycin,aminocaproic acid,and fetal bovine serum to EGM-2 medium containing endothelial cell growth factor kit to make a complete medium.Add proteinogen(final concentration 2.5mg/ml)into medium and mix with thrombin(0.5U/ml).Then quickly add them to the culture wells of a 24-well culture plate.After 10 minutes,a lower gel was formed(amount of about 0.8ml).Implant a tissue block of 1 mm3,and then add the mixture to the upper layer to form an upper gel(amount of about 0.5 ml).Finally add 1ml of the complete medium.Each case can be repeated to prepare several wells in a 24-well culture plate according to experimental requirements,which is convenient for group comparison3.Incubation:Place the culture plate in a humid incubator at 37? containing 5%CO2,and change the culture medium once every two days.Observe the angiogenesis while changing the medium4.Identification of cultured blood vessels(observation of HE staining and immunohistochemical experiment):take out the whole gel after culture and make into frozen sections for HE staining.And compared the immunohistochemical results with the one of specimen source.Result2-4 days after the Kaposi's hemangioendothelioma tissue block was implanted into the fibrin gel culture system,microscopic observation showed that small "dendritic" blood vessels extended outward from the edge of the tissue block,and the new blood vessels stagger into a network.structure.The growth is fast and the number of new blood vessels gradually increases.After 12-16 days,the growth situation is stable.The new blood vessel network can cover the entire field of vision under a 50x microscope,and a clear lumen-like structure can be seen under a 200x high power microscope.In the case of continuous nutrient supply,lysis and necrosis of the lumen-like structure began to occur after 26-30 daysTake the cultured tissue pieces into frozen sections and then subject to HE staining.After the staining was completed,the original tissue blocks conformed to the characteristics of Kaposi-like hemangioendothelioma.The cross-section of the "dendritic" structure extending around the tissue was circular or oval.The hollow lumen and tube wall consisted of a single layer of flat epithelial cells with deep-stained spindle nucleiTake another section for immunohistochemical experiments.The results are negative for GLUT-1,while the results of D2-40,CD31 and CD34 are all positive,which is consistent with the immunohistochemical results of tissue donors after surgery.Also consistent with currently recognized immunohistochemical characteristics of hemangioendothelioma.Confirming that the newly formed vascular network is composed of Kaposi-like hemangioendothelioma vascular epithelial cells.ConclusionThe fibrin gel culture system can complete the cultivation of a three-dimensional angiogenesis model of Kaposi-like hemangioendothelioma.The new born blood vessels form a "dendritic" network structure,which extends outward from the original tissue block.The new born blood vessels can be seen in 2-4 days The structure is stable from 12-16 days.Under the condition of continuous nutrition supply,the system can survive for more than 26 days.This model provides an excellent and accurate animal model of Kaposi-form hemangioendothelioma.The culture time is short,the survival time is long,the operation is simple,and a considerable time window for experimental research can be provided.The fibrin gel culture system truly reflects the tumor activity and angiogenesis process of Kaposi-form hemangioendothelioma in human.This model can be used for observational research on the effectiveness and effect of drugs,the occurrence and development of Kaposi-like hemangioendothelioma,the principle and development process of the Carmel phenomenon,and provides new possibilities and new research approach for KHE and KMP. |
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