Expression Of Bcl-2 In Dermal Hemangioma And Construction Expression Of Bcl-2 Gene Vector

Objective To investigate the relationship between Bcl-2 and the growth of hemangimas and effect on biological therapy.At first we detected the expression of Bcl-2 in hemangiomas,include proliferating hemangiomas, involuting hemangiomas and nomal skin,by means of immunohistochemistry and in situ hybridization;Then we constructed the eukazyotic expression vector with sense and antisense inserted bcl-2 cDNA fregment; at the same time we cultured endothelial cell of proliferating hemangiomas: it is the basement of study the effect of bcl-2 in the growth of hemangimas and its therapy.Methods Specimens from 49 cases of dermal hemangiomas from the Department of Pathology in Renmin Hospital of Wuhan University from 1996 to 2002 were collected. The specimens were generally fixed by formalin, and embedded in paraffin. Sections of 5 μm thickness were made and attached to poly-L-lysine-coated glass slides. Hematoxylin and eosin (HE) staining were carried out routinely to identify the histological characters of hemangiomas. The phases of hemangiomas were identified by detecting the expression of proliferating cell nuclear antigen (PCNA) by immunohistochemistry. Based on Mulliken's standard, the hemangioma specimens were divided into two groups: proliferating hemangiomas (27 cases) and involuting hemangiomas (22 cases). Normal skin tissues around hemangiomas from 5 cases were also chosen for control. Immunohistochemical stainings and in situ hybridization were performed to detect the expression of bcl-2 in those three groups. Endothelial cells were identified by expressing Factor Ⅷ-related antigen. The average optical density and the rate of positive area of expression of Bcl-2 were analyzed using image analysis Image HPIAS-2000 Analysis System. Bcl-2 gene was subcloned into pEGFP-Cl vector from pBluscript-bcl-2 by means of gene cloning to obtain sense and antisense pEGFP-C1-bcl-2 plasmid.Confirmed by restriction enzyme analysis and nucleotidesequence determination.The vector asked PEGFP-Cl-bcl-2 and PEGFP-Cl-AS-bcl-2. Fresh speciments of proliferating hemangioma were collected to culture endothelial by the means of tissue pieces.The cells were identified by expressing Factor Ⅷ-related antigen and observation of cell modality.Results 1. The expression of Bcl-2 in proliferating hemangiomas was significantly higher than that in involuting hemangiomas and normal skin tissues(P≤ 0.05).No significant difference was found between the expression of Bcl-2 in involuting hemangiomas and that in normal skin tissues(P>0.05).2. Restriction enzyme digestion and agarose gelelectrophoresis which have fragment long of 0.63 Kb was antisense vector and which long of 1.3Kb was sense vector.Nucleotide sequence determination the sense and antisense received same sequence.3. The endothelial cell formed endothelial cell clones and contact-inhibited "cobbles tone". Factor Ⅷl-related antigen was positive.Conclusion 1. Bcl-2 participated in the development and involution of hemangiomas.2. A eukaryotic expression plasmid containing bcl-2 and GFP gene issuccessfully constructed in the study.3.The culture technique of tissue pieces for endothelial cell of hemangioma has been established .