| Background and objective:Infantile hemangioma, the most common tumor of infancy, is a benign vascular neoplasm resulting from abnormal proliferation of endothelial cells and angiogenesis. Infantile hemangioma has a female dominance of2to5:1and mostly afflicts the cranio-faciocervical region following by the limbs and body. Based on the histological characteristics of hemangioma, Mulliken divided the course of hemangioma into three phases:proliferative phase, involuting phase and involuted phase. In proliferative phase, endothelial cells divide, proliferate and accumulate rapidly, basilar membrane thickens accompanying by the accumulation of mastocytes, macrophagocytes and perithelial cells. In involuting phase, the updating of endothelial cells slows down and parenchymal cells are being replaced by fibrofatty tissue. In involuted phase, fibrofatty tissue keeps depositing in the capillary.The greatest difference between hemangioma and other benign tumors is that85to90percent of infantile hemangiomas can involute by themselves, despite that the involuting process could last for ten years or so. Differences of location, size and growth rate of hemangioma during this process tend to influence children’s appearance, body function and mental health, and many of them may cause different kinds of deformities. For instance, tumors in airway may compress trachea causing dyspnea, few oversized hemangiomas may lead to abnormity in hemodynamics causing cardiac insufficiency, fast growing tumors may bring about skin ulceration, haemorrhage and severe pain causing troubles while children are growing and developing. Therefore, positive measures should be adopted to treat infantile hemangioma. Current therapeutic methods include the use of glucocorticoid, laser treatment, isotope applying, cold therapy, the administration of interferon, surgery, etc. All of these methods contain both curative effects and complications, so that their clinical applications are restricted.In2008, Leaute-Labreze from Bordeaux Children’s Hospital of France, accidentally discovered hemangioma withering while treating two hemangioma children, one with HOCM(hypertrophic obstructive cardiomyopathy) and the other with increasing cardiac output, by oally taking propranolol. In these two case, the tumors became smaller and softer, and the skin tension and color weakened. Thereafter, they treated other9hemangioma children by oally taking propranolol with the consent of their parents. In the first24hours after propranolol administration, all children’s tumors faded and softened, following by the shrink of their size. This method created a new path on propranolol treating infantile hemangioma, and the research was published in the New England Journal of Medicine, causing attentions around the world. Since then, many researchers reported optimistic results after treating infantile hemangioma with propranolol. And propranolol is more frequently used in infantile hemangioma based on its precise effects, less pain causing, and fewer complications.Propranolol is a widely used non-cardioselective beta-adrenergic antagonist, it can competitively antagonize the activation of neurotransmitter and catecholamine to beta-receptor. Propranolol is normally used to treat cardiac diseases, such as hypertension, SVT (supraventricular tachycardia), IHD(ischemic heart disease) and arrhythmia; it is also widely adopted in treating children with cardiac disease or infantile hyperthyroidism. Its approximately40years’ clinical use has proved propranolol’s fine security and survivability, but the mechanism of propranolol treating hemangioma is far from elucidated. Researchers and doctors have been trying to explain the specific mechanism on infantile patients, but they all failed due to children’s lack of compliance with follow-up and the lack of appropriate animal models on which experimental results can be recorded. We establish an animal model and explore the probable mechanism of propranolol’s effects on infantile hemangioma. By furthering our knowledge on this mechanism, we shall make some improvement on treating hemangioma and redusing complications.MethodPart One:The tissue of infantile hemangioma was harvested from a2month-old boy with cutaneous and subcutaneous hemangioma on his right neck. The parents described that the tumor growed fast recently with the area of2.3cm×2.1cm and hadn’t been treated. This hemangioma sample was resected under aseptic condition, and some part of this sample was investigated by pathologic examination and immunohistochemisty dye, and the rest was preserved under0-4℃after the fibrofatty tissue was removed. It was then sent to the Animal Experimental Center. Cut the tissue into3×4×5mm on superpurgative working table. Chose ten BALB/nu/nu nude mice for experiment, each of which was about5-6weeks old, weighing20g and sex unlimited. Injected2.5%of Pentobarbital Sodium (45mg/kg) into the abdomen of the mice for anesthesia, aned planted the hemangioma tissue subcutaneously (4implantations each mouse) behind the neck. The surgery took less thanl hour. Measured the maximum diameter "a" and transverse diameter "b" via vernier respectively on day14,21,28,35,42,49, and56, and estimated tumors’ volume change based on V=π/6ab2. On day30and60, harvested4samples respectively for HE, CD31and GLUT1dye, then observed their indexes.Part Two:1. From October2010to December2012, Laser Cosmetic Center of Guangzhou General Hospital of Guangzhou Military Region collected the information of10out-patient and in-patient children suffering hemangiomas who had received oral propranolol treatment, among whom there were4boys and6girls with ages of52days-10months, average age of4.5months and weight of5.1-8.6kg, average weight of7.9kg. Among the cases, there were6on the face,2on the neck,1on the limbs and1on the back. Tumor area was5cm×4cm maximum and1.0cm×0.6cm minimum. All patients had not received any other kinds of treatments and they were all in good health without medical history of genetic and communicable diseases, etc. Three kinds of tissue specimens of hemangioma tumors were collected by2mm-diameter skin ring respectively from four temporal points of before, one month after, three months after and half year after patients’ taking the oral medicine. After fixed by formaldehyde, the four specimens were immediately kept in paraffins, forming the corresponding four groups of before, one month after, three months after and half year after group.2. HE staining and paraffin section immune histochemistry method (Streptomyces avidin-peroxidase link method, SP method) staining were applied after routine sectioning of the organizations block. We noted slitting organization slice block to ensure that the layers of tissues from the epidermis to the subcutaneous hemangioma could be observed under the microscope. After HE staining of tissue sections the basic structure of the tissue was observed. Each specimen detected the expression of CD31, vascular endothelial growth factor (VEGF) and its receptor (VEGFR). CD31was commonly used in the vascular endothelial cell marker. CD31was detected in order to observe the density of blood vessels in the tumor tissue of the hemangioma. Each slice wass observed at high magnification (20×10,40×10).3. We observed the result through the microscope (BX-51upright fluorescence microscope by Olympus Company) after tissue staining, then used computer digital software (Image-pro plus. Version6.3.1.542) to capture tissue sections images marked VEGF, VEGFR and measured the mean optical density.ResultPart One:The pathological examination of the specimen of human body hemangioma was in accordance with the pathological changes of hemangioma. The dyeing results of CD31and GLUT1both demonstrated Strong Positive. The wound begun to heal seven to ten days after the specimen of hemangioma was transplanted into the skin of the nude rat. However, as the wound had not been sewed up, some tissue blocks were displaced, making them exposed by the wound. There had not been obvious changes for ten to twenty days, but from the21st day, the tissue blocks begun to shrink. On the60th day, the skin of back of the nude rat tended to be flat, with no tissue blocks observed but being touched. The HE dyeing was undertaken on the30th day and the60th day, demonstrating the characteristics of fibrosis pathology of translated hemangioma. The hemangioma did not grow and the results of the dyeing of CD31and GLUT1demonstrated Negative. The establishment of the animal model failed.Part Two:Clinical observation of Propranolol treatment of infantile hemangioma-after taking propranolol orally for six months, the volume and color of hemangioma reduced and faded. The effect of the treatment was obvious. During the treatment, one child suffered from mild diarrhea. It got better on its own two days later without being given any intervention. The full structure of the tumor tissue from the epidermis to subcutaneous hemangioma and the tumor changing process was observed after HE staining. The result showed that the tumor gradually shrinked. Immunohistochemistry detection showed that CD31marker expresses in vascular endothelial cells on the cell membrane from which the density of blood vessels can be observed after taking the medication for one month, three months and six months. Compared with the case without taking the medicine, it confirmed that vascular density gradually decreases, and the difference is obvious. That is, the microvascular density within the blood vessels in tumor tissue gradually decreased. Compared with the case before taking the medicine, the VEGF and VEGFR expression intensity of the tumor tissue gradually narrowed after taking the medicine after a month, three months to six months. The distinction was sharp.Conclusion1. In order to establish an animal model for infantile hemangioma, it is necessary to possess appropriate donor and receptor and select multi-part planting; and it is also a necessity to build a suitable environment for growing such as estrogen according to the knowledge of hemangioma gained over the years.2. The main mechanism of Propranolol treatment of infantile hemangioma is to achieve the purpose of curing hemangioma by reducing the expression of VEGF, VEGFR to inhibit blood vessel growth, reduce angiogenesis, and promote vascular endothelial cell apoptosis. |
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