VEGF基因可变剪接在根尖周囊肿骨破坏过程中的作用

Effects of alternative spliced variants of VEGF on the bone destruction of radicular cyst

目的本研究以根尖周囊肿为疾病模型,根据其临床特点确定骨破坏速率,并利用免疫组织化学方法观察间质中微血管密度;同时原代培养囊壁成纤维细胞,检测其中各种VEGF异构体对微血管增生及破骨细胞诱导效率的影响,探讨VEGF可变剪接在牙源性囊肿骨破坏过程中的作用。方法选取19例根尖周囊肿,以CD34抗体免疫组化染色凸显血管内皮细胞,并计算血管腔面积,曲面断层影像进行病变面积的测量,并选取其中13例新鲜手术标本进行根尖周囊肿囊壁成纤维细胞培养,制备条件培养基和诱导破骨细胞,并进行RNA的提取及realtime PCR检测VEGF异构体的相对表达量。χ~2检验用于评估CD34阳性的血管腔面积和临床特点。血管腔面积、各种异构体相对表达量、囊肿进展速率和破骨细胞诱导效率的关系由线性回归及Spearman’s等级相关评估。结果 VEGF-121a/总VEGF和VEGF-165a/总VEGF正相关于微血管管腔面积,后者则与囊肿骨破坏速率正相关;而VEGF和VEGF-189a的相对表达量正相关于破骨细胞诱导效率,VEGF-165b/总VEGF和VEGF-189b/总VEGF则与破骨细胞诱导效率成负相关。结论提示VEGF-xxxa型可能主要促进囊肿的骨坏过程,而VEGF-xxxb型则可能有抑制效果。

Objective This study took the radicular cyst as the model,determined the rate of bone destruction on the basis of the clinical characteristics,measured the microvessel density in interstitium by immunohistochemistry; The fibroblasts were isolated from capsule,through which the association of various VEGF isoforms with microangiogenesis and osteoclastogenesis were analyzed to investigate the role of the alternative splicing of VEGF in the process of bone destruction of odontogenic cysts. Methods We selected 19 cases of periapical cysts. The immunohistochemical staining with CD34 antibody was used to stain vascular endothelial cells,and the area of the vascular cavity was calculated. The lesion area was measured by the dental panoramic tomography. Thirteen cases of fresh specimens were prepared to culture the apical cystic fibroblasts. We prepared conditioned medium and induced osteoclasts,and extracted RNA to detect the relative expression of VEGF isoforms by real time PCR. The Chi-square test was used to assess CD34-positive vascular lumen area and clinical features. Logistic regression and Spearman's rank correlation were used to analyze the relations between vascular lumen area,the relative expression of various isomers,the rate of cyst progression and the osteoporosis induction efficiency.Results The ratio of VEGF-121 a/total VEGF and VEGF-165 a/total VEGF were positively associated the area of microvessels,and the area of microvessels was positively associated with bone destruction of odontogenic cysts; On the other hand,the relative quantitative expression of VEGF and VEGF-189 a were positively associated with osteoclastogenesis,however,VEGF-165 b/total VEGF and VEGF-189 b/total VEGF were negatively associated with osteoclastogenesis. Conclusion VEGF-xxxa isoforms may promote the bone destruction,while VEGF-xxxb variants may play an inhibitory effects.