| BackgroundInfantile hemangiomas (IH) is a common benign tumor in infants, occurring with high rate8.7%-12.7%from1to12months after born[1-3]. Most IH will regress spontaneously without any scar after earlier proliferative phase. However, some serious IH may affect appearance or even threaten life. Current treatment options include surgery, drug, laser and combination of them. The clinical efficacy is not perfect, because of unclear understanding on pathphysiology and pathogenesis.There are five clinical characteristics of IH:①Specific natural process:rapid growth of tumor from born and spontaneous regression after one year old.②More female patients:the rate between male and female is1:3-5[5-6]。③Increasing rate on head and neck:the rate of IH on head and neck is60%[7].④More white patients:the white people has greater chance than black and Asian people[8].⑤This disease responds to corticosteroid treatment[9-10].To investigate the pathphysiology and pathogenesis of IH, it is necessary to establish an animal model. There are five animal models of IH reported as follows:the chicken comb and wattle model[11]; malignant endotheliomas and angiosarcomas nude mice model induced by injections of1,2-DMH[12]; transgenic mice carrying MT gene linked to Py early region regulatory sequences[13]; human hemangioma nude mice model by hemangioma endothelial cell (HemEC) suspension inoculation[14]; human hemangioma nude mice model by tissue block transplantion[15].Targeting a stable and reliable IH animal model, we compared the feasibility of establishment of human hemangioma animal model by HemEC suspension inoculation and tissue block transplantion.Objective1. To isolate and culture hemangioma endothelial cells (HemEC) from fresh infantile hemanioma tissue.2. To compare the feasibility of establishing human hemangioma animal models by HemEC suspension injection and tissue transplantation.Methods1. Specimens of proliferative infantile hemangioma (<3-month-old; untreated) were obtained from Department of Stomotology in Qilu hospital Shandong University and Department of Hemangioma in Linyi tumor hospital. The clinical diagnosis were confirmed in Department of Pathology in Qilu hospital. This study has already got approval from their parents.2. The tissue was washed by PBS and cut to pieces. Half of the tissue was used for culturing HemEC, the other half was for establishing IH tissue trasplantation mice model. And a few pieces were kept to test the expression of GLUT1by immunohistochemistry (IHC). 3. HemEC was isolated from adherent proliferative IH tissue and tested the expression of vWF and CD34by immunocytochemistry (ICC).4. BALB/c nude mice (n=12/group) were28±3.2-day-old and20.5±2.1-g-weight. The IH tissue transplantation group:fresh proliferative IH tissue was transplanted into both sides of back or oxter of nude mice; HemEC was injected into both sides of back or oxter of nude mice. The experimental mice were monitored for tumor change and sacrificed to harvest tumor at7,30and60days. The harvested tumor were fixed for HE staining.Results1. GLUT1is strongly expressed in obtained tissue, which proves that the tissue is from proliferative IH.2. The morphology of cultured HemEC is polygon or fusiform. The specific endothelial cells marker vWF and CD34are both positive expressed in HemEC.3. In the establishment of HemEC injection mice model, all of both sides of12nude mice were successfully tumor-formed. At day7, the tumor was about1mm×1mm×1mm with few new formative vessels; at day30, the tumor was5mm×4mm×3mm with a large number of vasculars surrounded by HemEC; at day60, the tumor was2mm×3mm×2mm with some HemEC apoptosis.4. In the establishment of IH tissue transplantation mice model, one nude mice was dead by infection, two mice lost their transplanted tissue by lost lines, the other9nude mice were successfully tumor-formed. At day7, the tumor was merged together with body and showed a lot of vasculars; at day30, the tumor was2mm x2mm x2mm with large area of cells apoptosis; at day60, the tumor has already been absorbed by body.Conclusion1. It is available to culture HemEC from IH tissue.2. It is not appropriate to establish IH animal model by IH tissue transplantation.3. Injection of HemEC in nude mice is a stable and specific IH animal model. Infantile hemangiomas are the most common benign tumors in infancy. Although natural history and progression of these diseases are well described, their origin remains unclear. Remarkable progress has been gained in the past two decades towards understanding the pathology of these lesions. New studies have created sophisticated hypotheses on the origin of this disease. These include theories of placental origin, somatic endothelial mutation, and extrinsic factors creating an environment for growth. While none of current hypothesis explains all the characteristics of IH, continued research targeting pathology will be helpful for new treatment.IH is composed of various cell types including hemangioma endothelial cells (HemEC). Recent studies have found that HemEC express P2adrenergic receptors (β2-AR) and β3-AR [1-2]. Propranolol (PRO) is thought to exert its effects on IH progress by three different molecular mechanisms: vasoconstriction, inhibition of angiogenesis and induction of apoptosis.①Both of the autonomic nervous system and epinephrine play a key role in the control of vascular tone. Epinephrine causes either vasoconstriction or vasodilatation by activating α1or β2-AR, respectively.β-AR inhibitors without α-AR antagonistic effects like PRO inhibit epinephrine regulated vasodilatation and this leads to vasoconstriction of HemEC.②During the earlier growth phase of IH, there is increased expression of two major proangogenic factors:basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). Recent publications have reported that the expression of VEGF is reduced by β-AR inhibitors which leads to inhibition of angiogenesis[3].③poptosis is mediated by a lot of pathways, with the β2-AR being the probable receptor involved. It has been hypothesized that by blocking the β2-AR, PRO induces apoptosis at an increased rate[4].Purpose1. To detect the expression of β1, β2, β3-AR on infantile hemangioma tissue and HemEC.2. To observe the function change of HemEC after various specific β1,β2, β3-AR inhibitors treatment.3. To study the relationship between β2-AR and pathogenesis of IH by lentivirus transfection skill.Method1. Fresh tissue of proliferative IH, VM and LM were obtained by surgery. Fix the tissue and test the expression of β1, β32, β3-AR on these tissue by IHC.2. Isolate RNA and protein from proliferative and involution IH, then detect the expression of β1, β2, β3-AR on RNA and protein level.3. Isolate RNA and protein from proliferative IH and adjacent tissue, then detect the expression of β1, β2, β3-AR on RNA and protein level. 4. Isolate RNA and protein from HemEC and HUVEC, then detect the expression of β1,β2,(33-AR on RNA and protein level by qPCR and western blotting.5. HemEC was treated with gradient doses of PRO. The ability of proliferation and tube-formation was detected by MTT and tube-formation assay respectively.6. HemEC was treated with optimized dose of specific β1,β2,(33-AR inhibitors. The ability of proliferation and tube-formation was detected by MTT and tube-formation assay respectively.7. Lentivirus combined with β2-AR gene interfere were designed and synthesized. The transfection rate on HemEC was detected by qPCR and western blotting.8. In vitro, HemEC was treated with PRO or ADRB2-si to knock down expression of β2-AR, and then returned expression of β2-AR by ADRB2-hi transfection. The ability of proliferation and tube-formation was detected by MTT and tube-formation assay respectively.9. In vivo, the tumor in HemEC injection mice model was treated with PRO or ADRB2-si to knock down expression of P2-AR, and then returned expression of β2-AR by ADRB2-hi transfection. The change of tumor was observed.Result1. The expression of β2-AR is positive in proliferative IH, but negative in VM and LM. The expression of (31and β3-AR are negative in proliferative IH, VM and LM.2. The expression of β2-AR is higher in proliferative IH than that in involution IH. There are not differences between expression of β1and β3-AR in proliferative and involution IH.3. The expression of β2-AR is higher in proliferative IH than that in adjacent tissue. There are not differences between expression of β1and β3-AR in proliferative IH and adjacent tissue.4. The expression of β2-AR is higher in HemEC than that in HUVEC. There are not differences between expression of β1and β3-AR in HemEC and HUVEC.5. The optimized dose of PRO on affecting proliferation and tube-formation abilities of HemEC is10μM.6. PRO or ICI (β2-AR specific inhibitor) inhibits the proliferation and tube-formation abilities of HemEC completely. There is partly inhibition from CGP (β1-AR specific inhibitor) and no effect from SR (β3-AR specific inhibitor).7. By qPCR and WB, ADRB2-si knocks down the expression of β2-AR on HemEC; ADRB2-hi increases the expression of β2-AR on HemEC.8. PRO and ADRB2-si inhibit the proliferation and tube-formation abilities of HemEC and slow down the tumor growth on IH tumor model, which could be returned by ADRB2-hi. Conclusion1. The expression of β2-AR is positive in proliferative IH, but negative in VM and LM. The expression of β1and β3-AR are negative in proliferative IH, VM and LM.2. The expression of β2-AR is higher in proliferative IH than that in involution IH. There are not differences between expression of β1and β3-AR in proliferative and involution IH.3. The expression of β2-AR is higher in HemEC than that in HUVEC. There are not differences between expression of β1and β3-AR in HemEC and HUVEC.4. Blocking β2-AR inhibits the proliferation and tube-formation abilities of HemEC completely. There is partly inhibition from blocking (31-AR and no effect from blocking β3-AR.5. Blocking β2-AR reduces the tumor-forming on HemEC injection mice model, and recover the expression of (32-AR can reverse it. |
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