缺氧条件对成骨细胞VEGF和TGF-β1表达影响的实验研究  

EXPERIMENTAL STUDY ON THE EFFECT OF HYPOXIA ON THE EXPRESSION OF VEGF AND TGF-β_1 OF RAT MANDIBULAR OSTEOBLASTS

作　　者：关键[1] 王健平[1] 车笑非[2] 李德超[1] 

机构地区：[1]佳木斯大学口腔医学院颌面外科,黑龙江佳木斯154004 [2]佳木斯市妇幼保健院口腔科

出　　处：《中国修复重建外科杂志》2008年第8期984-988,共5页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：黑龙江省青年科学技术专项资金资助项目(QC05C47);黑龙江省卫生厅重点资助项目(2004-001)~~

摘　　要：目的研究CoCl2对体外培养的鼠下颌骨成骨细胞的作用,观察缺氧条件对成骨细胞VEGF及TGF-β1表达的影响,为临床牵张成骨技术的分子生物学机制研究提供理论依据。方法取新生24h内Wistar大鼠下颌骨,改良酶消化法培养和纯化成骨细胞,采用HE染色、ALP组织化学染色、Ⅰ型胶原免疫组织化学、钙结节染色行细胞形态学及组织学鉴定,并绘制生长曲线。取第3代最佳生长状态的鼠下颌骨成骨细胞,用150μmol/LCoCl2处理(实验组),设正常状态下培养为对照组,分别于培养后0、3、6、9、12、24h,采用免疫荧光技术检测VEGF及TGF-β1表达。结果HE染色示成骨细胞形态多样,呈三角形、多角形、圆形及鳞片形等不规则形态,突起长短不一向外伸展,胞核清晰可见;胞浆丰富,呈粉红色,胞核蓝紫色,有时可见2个细胞核,密集处细胞形态不明显,分界模糊,仅见大圆核细胞。ALP组织化学染色示细胞胞质呈黑色颗粒,细胞核轮廓不清,细胞密集区可见大量阳性细胞。Ⅰ型胶原免疫组织化学染色示实验组阳性细胞均匀可见,胞浆呈棕黄色,胞核周围尤为明显,空白对照组细胞未见棕黄色颗粒。钙结节染色示成骨细胞于盖玻片上培养15d后,细胞聚集成不透明区域,呈结节样;茜素红染色后,有橙红色着色。培养各时间点对照组细胞激光共聚焦显微镜下可见较弱的VEGF表达荧光;实验组细胞随缺氧时间延长,VEGF表达荧光强度值增加,于术后9h达高峰,随后降至正常水平。培养各时间点激光共聚焦显微镜下可见两组细胞均有TGF-β1表达荧光,对照组各时间点荧光强度值均略高于VEGF对照组;实验组TGF-β1表达在缺氧3h短期成倍增加,随缺氧时间延长表达逐渐降低。结论经新生大鼠下颌骨培养的成骨细胞性状稳定;适当缺氧可诱导成骨细胞VEGF和TGF-β1表达增强。Objective To observe the effects of cobalt chloride （CoC12）-simulated hypoxia on VEGF and TGF-β1 expression and to provide theoretical basis for deciphering the molecular mechanism of clinical distraction osteogenesis. Methods The mandibular osteoblasts were obtained from newborn Wistar rats within 24 hours and cultured and purified through modified enzymatic digestion. The morphological and histological changes of cells were evaluated by the HE staining, the histochemical staining for ALP, the collagen I immunohistochemistry staining and the calcified nodules staining, and the growth curves were drawn. The best cells of the 3rd-passage rats were treated with CoC12, and then immunofluorescence was used to detect the expressions of VEGF and TGF-β3, at 0, 3, 6, 9, 12 and 24 hours after culture. Results The HE staining demonstrated that the cellular forms were diverse, triangular, polygonal, circular and scaly and so on. The prominence varied in length and extended outwards. The nucleus was clearly discernible. The cytoplasma was rich and pink, with the nucleus royal purple. Sometimes 2 cell nuclei were seen. At the crowded place, cellular form was not clear, the dividing line was indistinct, and just the great-circle nuclear cells could be seen. The ALP immunohistochemistry staining demonstrated that the cell butcher nature appeared black pellets, the cell nucleus outline was unclear, and at the cell compact district, massive masculine cells could be seen clearly. The collagen I immunohistochemistry staining demonstrated that masculine cells were seen evenly, cytoplasma appeared yellowish brown especially around the nucleus. However, yellowish brown pellets were not seen in negative cells. The osteoblast calcium tubercle staining demonstrated that the cells gathered in the opaque region with the shape of tubercle after 15 days of culture. After alizarin red staining, the reddish orange pigmentation appeared. At various time points, weak VEGF fluorescence was seen in the cells in the control group under the la

关 键 词：成骨细胞TGF-β1 VEGF 缺氧 CoCl2 

分 类 号：R363[医药卫生—病理学]