Expression and Significance of Bcl-2,Bax,Fas and Caspase-3 in Different Phases of Human Hemangioma

Summary： The relationship between Bcl-2, Bax, Fas, caspase-3 and development of hemangioma and the molecular mechanism was investigated. By using immunohistochemical S-P method, proliferating cell nuclear antigen was detected. According to the classification of Mulliken in combination with PCNA expression, 27 cases were identified as proliferating hemangioma and 22 cases as involutive hemangioma. Five normal skin tissues around the tumor tissue served as controls. By using immunohistochemical technique, the expression of Bcl-2, Bax, Fax and Caspase-3 was detected. The cells expressing Bcl-2, Bax, Fax and cappase-3 were identified as hemangioma endothelia by immunohistochemical staining of Ⅷ factor. The average absorbance （A） and average positive area rate of Bcl-2, Bax, Fas and caspase-3 expression were measured by using HPIAS-2000 imaging analysis system. The results showed that the expression of Bcl-2 in the endothelia of proliferating hemangioma was significantly higher that in involutive degenerative hemangioma endothelia and vascular endothelia of normal skin tissue （P〈0.01）. The expression of Bax, Fas and Caspase-3 in the endothelia of involutive hemangioma was obviously higher than in the endothelia of proliferating hemangioma and normal skin tissue （P〈0.01）. The expression of BAx and Fas in endothelia of proliferating hemangioma was higher than in those of normal skin tissue （P〈0.05）. It was suggested that Bcl-2, Bax, Fas and caspase-3 might be involved in the development and involution of hemangioma. Bcl-2 could promote the growth of hemangioma by inhibiting apoptosis of endothelia. Bax, Fas and caspase-3 promote the switch of hemangioma from proliferation to involution by inducing the apoptosis of hemangioma endothelia.

Expression of HLA-G in Hemangioma and Its Clinical Significance

This study examined the roles of HLA-G in the pathogenesis,development and immune tolerance of hemangioma.From 2000 to 2007,52 paraffin-embedded specimens (26 from males and 26 from females) of skin capillary hemangioma and 7 samples of adjacent normal skin tissues were collected.Four fresh specimens of hemangioma were also harvested.All samples were HE-stained and proliferative cell nuclear antigen (PCNA) was immunohistochemically detected by using SP method.The samples were classified into proliferative group and degenerative group according to the Mulliken criteria and the expression pattern of PCNA.SP method and quantum dots double staining were applied to detect the expression of HLA-G and PCNA in hemangioma and normal tissue samples.The expression of HLA-G was detected by RT-PCR.The results showed that among the 52 samples of hemangioma,29 were of proliferative type and 23 degenerative type,and of the four fresh samples of hemangioma,2 were of proliferative type and 2 degenerative type.SP method results showed that HLA-G was expressed in both proliferative and degenerative hemangioma,but not in normal tissues.The quantum dots double staining exhibited that HLA-G expression was significantly higher in proliferative group than in degenerative (P<0.05) and normal groups (P<0.05),but there was no statistically significant difference between the latter two groups (P>0.05).RT-PCR revealed that HLA-G was transcribed in both the proliferative and degenerative hemangioma tissues,but not in normal tissues.We are led to conclude that the elevated expression of HLA-G in proliferative hemangioma cells may lead to immune tolerance,which allows cells to escape immune surveillance and proliferate.On the other hand,the lower expres-sion of HLA-G in degenerative hemangioma may result in immune cells-induced degeneration of hemangioma.

Treatment of Hemangioma by Transfection of Antisense VEGF Gene

The effect of transfection of antisense vascular endothelial growth factor （VEGF） gene on the growth of hemangioma was studied. A total of 49 cases of capillary hemangiomas of the skin were collected. Immunohistochemical method was used to detect the expression of PCNA in hemangioma tissues. According to the finding, 49 cases of hemangiomas fell into proliferating phase （27 cases） and involuting phase （22 cases） respectively. Another 5 cases of normal skin tissues adjacent to the tumor tissues served as control. Immunohistochemical staining was performed to detect the expression of VEGF in the tumor tissues and the normal tissues. The average absorbance （A） values and the average positive area rate of VEGF were measured by image analysis system （HPIAS-2000）. Endothelial cells from the tumor tissues in proliferating phase were cultured. Eukaryotic expression vector was constructed by sub-cloning, and transfected into human hemangioma endothelial cells by using cation liposome as vector. The expression of VEGF mRNA and protein was detected by RT-PCR and indirect immunofluorescence assay （IFA）, respectively, and the biological characteristics of the transfected endothelial cells were examined by MTT assay and flow cytometry （FCM） after transfection. Immunohistochemical results showed that the expression of VEGF in proliferating endothelial cells was remarkably higher than those in involuting endothelial cells and normal endothelial cells （P〈0.01）, but there was no significant difference in the expression of VEGF between involuting endothelial cells and normal ones （P〉0.01）. Electrophoresis and sequencing indicated that the eukaryotic expression vector containing antisense VEGF gene, i.e. pcDNA3.1-VEGF, was success- fully constructed. After VEGF antisense RNA recombinant was transfected into hemangioma endothelial cells, RT-PCR revealed that the expression of VEGF mRNA in pcDNA-VEGF （V） group and blank group was obviously higher than that in pcDNA-VEGF （A） group, and that the expression of endogenous VEGF mRNA in pcDNA-VEGF （A） group was significantly inhibited. Immunohistochemical result demonstrated that, compared with blank group, there was statistically significant difference between pcDNA-VEGF （A） and pcDNA-VEGF （V） groups （P〈0.01）, but there was no significant difference between pcDNA-VEGF （V） group and blank group （P〉0.05）. The activity of endothelial cell proliferation was reduced significantly after transfection, and obvious apoptosis occurred in hemangioma endothelial cells after transfection of antisense VEGF. It was suggested that VEGF plays an important role in the pathological change of hemangiomas by promoting endothelial cell proliferation and angiogenesis. Antisense VEGF gene transfection could effectively inhibit the growth of hemanioma endothelial cells.