缺氧状态下体外培养成骨细胞血管内皮生长因子和骨形态发生蛋白2的表达及意义

目的研究缺氧对体外培养成骨细胞血管内皮生长因子（VEGF）和骨形态发生蛋白2（BMP-2）表达的影响及意义。方法采用酶消化法获取人成骨细胞并进行原代培养，倒置显微镜下观察成骨细胞的生长形态；采用Gomori改良钙钴法和免疫组织化学SABC法分别检测原代培养成骨细胞碱性磷酸酶（ALP）和骨钙素（OCN）的表达，进行原代培养成骨细胞鉴定。细胞传至第2代时，按继续培养条件的不同将细胞分为缺氧组（氧分压〈4．8kPa、氧容积比〈5％）和常氧组，在传代培养24、48、72h时分别收集2组细胞，采用免疫组织化学SABC法检测VEGF和BMP-2的表达，应用图像分析系统进行半定量检测，结果以平均灰度值表示，平均灰度值低示表达水平高。结果原代培养成骨细胞可表达ALP和OCN，初步确定经体外培养获得了具有生物活性的成骨细胞。在培养24、48、72h时，缺氧组成骨细胞VEGF表达水平均明显高于常氧组（平均灰度值分别为123．53±7．38 vs 141．21±6．03、116．45±6．34 vs 138．37±5．04、108．11±5．37 vs 136．65±6．54，均P〈0．01），且缺氧组成骨细胞VEGF表达水平随缺氧时间延长而逐渐升高。缺氧组成骨细胞BMP-2表达水平在培养24h时明显高于常氧组（平均灰度值为143．28±2．82 vs 146．91±2．06，P〈0．01），而在培养48、72h时与常氧组比较，差异均无统计学意义。结论缺氧可诱导成骨细胞VEGF的表达上调；而缺氧延长则不利于成骨细胞BMP-2的表达。缺氧对VEGF和BMP的早期表达调控可能与骨修复的启动相关。

Expressions of VEGF and BMP-2 in the osteoblasts cultured under hypoxia and their significance

Objective To study the expressions of VEGF and BMP-2 in the osteoblasts cultured under hypoxia, and their significance. Methods Osteoblasts were obtained by enzyme digestion, primarily cultured in vitro, and then observed under an inverted phase contrast microscope. The expressions of ALP and OCN in primary passage were detected by modified Gomori staining and SABC staining respectively to identify the osteoblasts. The second-passage cells were divided into hypoxia group (PO2 < 4.8 kPa, and oxygen volume < 5%) and control group, and were harvested 24, 48, and 72 hours after culture. The the expressions of VEGF and BMP-2 were investigated by SABC straining. Mean gray value of the positive-stained cells was measured by adigital image processing software to evaluate the levels of the expressions. Resuts ALP and OCN were detected in the primary-passage cells, showing that the cells cultured in vitro were osteoblasts with bioactivity. At 24, 48, and 72 hours, the expression level of VEGF in the hypoxia group was significantly higher than that in the control (gray value: 123.53 ± 7.38 vs. 141.21 ± 6.03 at 24 hours, 116.45 ± 6.34 vs. 138.37 ± 5.04 at 48 hours, and 108.11 ± 5.37 vs. 136.65 ± 6.54 at 72 hours, all P < 0.01). In addition, the level of VEGF expression in the hypoxia group increased with the hypoxia time. In the hypoxia group, the expression level of BMP-2 was significantly higher than that in the control at 24 hours (gray value: 143.28 ± 2.82 vs. 146.91 ± 2.06, P < 0.01), and was similar with the latter at 48 and 72 hours. Conclusions Hypoxia may upregulate the expression of VEGF in osteoblasts, while downregualte the expression of BMP-2. These may be related to the initiation of bone reconstruction.