The effects of heat on the biological activity of recombinant human bone morphogenetic protein-2
The effects of heat on the biological activity of recombinant human bone morphogenetic protein-2
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DOI：
10.1007/s00774-005-0623-6
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发表时间：
2005-11-01
影响因子：
3.3
通讯作者：
Takaoka, K
中科院分区：
文献类型：
--
作者：
Ohta, H;Wakitani, S;Takaoka, K
本研究旨在研究热对重组人骨形态发生蛋白（rhBMP）-2 骨诱导活性的影响。 rhBMP-2 样品在 50、70、90 或 100 摄氏度下加热 15 分钟，或 1、2、4 或 8 小时，或在 120 摄氏度下高压灭菌 15 分钟。在体内和体外系统中测定了加热前后rhBMP-2的骨诱导活性。对于体内测定，将5μg rhBMP-2样品浸渍到多孔胶原盘（直径6mm，厚度1mm）中，冷冻干燥，并植入ddY小鼠的背部肌肉中。三周后，从宿主体内取出植入物，并通过射线照相检查是否有异位新骨组织。通过单能 X 射线吸收测定法对新骨量进行量化。通过将浓度为 100 ng/ml 的 BMP 样品添加到 MC3T3-E1 细胞培养物中来测定 rhBMP-2 的体外活性。 48小时后，测量碱性磷酸酶活性。根据体外测定数据，在 50 或 70 摄氏度加热后，体内或体外测定系统均未发现骨诱导活性显着降低，除非蛋白质在 70 摄氏度下持续加热 8 小时。然而，加热至 90 摄氏度以上并持续较长时间会导致 rhBMP-2 的生物活性以时间和温度依赖性方式降低。 rhBMP-2 在暴露于 120 摄氏度或超过 120 摄氏度的温度时会失活。
This study was designed to investigate effects of heat on the bone-inducing activity of recombinant human bone morphogenetic protein (rhBMP)-2. rhBMP-2 samples were heated at 50, 70, 90, or 100 degrees C for 15 min, or 1, 2, 4, or 8 h, or autoclaved at 120 degrees C for 15 min. The bone-inducing activity of the rhBMP-2 before and after heating was assayed in in vivo and in vitro systems. For the in vivo assay, 5 mu g rhBMP-2 samples were impregnated into porous collagen disks (6 mm in diameter, 1 mm thickness), freeze dried, and implanted into the back muscles of ddY mice. Three weeks later, the implant was harvested from the host and examined for ectopic new bone tissue by radiography. The new bone mass was quantified by single-energy X-ray absorptiometry. The in vitro activity of the rhBMP-2 was assayed by adding the BMP sample at a concentration of 100 ng/ml to cultures of MC3T3-E1 cells. After 48 h, the alkaline phosphatase activity was measured. After heating at 50 or 70 degrees C, no significant reduction in bone-inducing activity was noted in either in vivo or in vitro assay systems unless the protein was exposed to sustained heat at 70 degrees C for 8 h, based on in vitro assay data. However, heating above 90 degrees C and for longer periods led to a decrease in the biological activity of the rhBMP-2 in a time-and temperature-dependent manner. rhBMP-2 was rendered inactive when exposed to temperatures at or in excess of 120 degrees C.