Purification of a skeletal growth factor from human bone.
Purification of a skeletal growth factor from human bone.
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从人骨中纯化骨骼生长因子。
DOI：
10.1021/bi00257a037
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发表时间：
1982
期刊：
影响因子：
2.9
通讯作者：
Baylink,DJ
中科院分区：
文献类型：
--
作者：
Farley,JR;Baylink,DJ
John R.法利和大卫J. Baylink* 摘要：从脱矿人骨基质中分离纯化出一种骨骼生长因子。剂量为6 μ g/mL的纯化因子显著增加了无血清培养物中鸡胚骨细胞的增殖率（对照的292%，p< 0.0001），但对以相同初始密度接种的鸡胚皮肤细胞没有影响。该因子对胰蛋白酶和尿素灭活敏感，但对胶原酶、20%丁醇或1%巯基乙醇不敏感。它还能抵抗热灭活（在75 ℃下稳定15 min）和极端pH值灭活（在4 ℃下从pH 2.5至10.0稳定30 min）。通过选择性加热和酸沉淀、分子筛柱层析和疏水聚丙烯酰胺凝胶电泳纯化活性因子，提供了一种根据高压液相色谱、聚丙烯酰胺凝胶电泳和等电聚焦标准均质的材料。表观分子量为83000。纯化因子在与其他促有丝分裂剂相当的剂量下增加骨细胞增殖：0.3 μ g/mL（3.6 nM）显著增加DNA合成至对照的231%（p< 0.001）。纯化的因子在培养的鸡胚骨上也是活性的，如分别通过增加的干重（对照的185%，p< 0.025）和[3 H]脯氨酸掺入（对照的164%，p< 0.001）所测量的，提高了胫骨和股骨的生长速率。骨形成与再吸收的偶联首先由临床观察提出（Harris & Heaney，1969），随后由大鼠的体内研究证实（Thompson等，1975; Baylink & Liu，1979）。我们的概念是，这种骨骼耦合为骨体积的局部控制提供了反调节机制，并且错误的耦合可能导致骨质疏松症（Ivey & Baylink，1981）。来自该实验室的先前研究确定了（a）偶联取决于局部机制-它发生在体外（霍华德等人，1980），（B）偶联由成骨细胞数量的增加介导（Baylink等，1980），（c）通过在体外吸收骨而调节的培养基中的因子将增加骨细胞增殖的速率和胚胎骨的生长（Drivdahl等，1980 a），和（d）可以从鸡胚骨中提取类似的活性因子（Drivdahl等，1980年b）。后两种来源的因子被认为是一种假定的偶联因子，存在于骨基质中，通过骨吸收释放，增加成骨细胞数量，从而增加骨形成率。在目前的研究中，我们试图确定成人骨基质中是否存在类似的假定偶联因子。
John R. Farley and David J. Baylink* abstract: A skeletal growth factor was isolated and purified from demineralized human bone matrix. A dose of 6^ g/mL of the purified factor significantly increased the proliferation rate of embryonic chick bone cells in serum-free culture (292% of controls, p< 0.0001) but had no effect on embryonic chick skin cells plated at the same initial density. The factor is sensitive to inactivation by trypsin and urea, but not by collagenase, 20% butanol, or 1% mercaptoethanol. It is also resistant to inactivation by heat (stable for 15 min at 75 C) and extremes of pH (stable for 30 min at 4 C from pH 2.5 to 10.0). Purification of the active factor byselective heat and acid precipitations, molecular sieve column chromatography, and preparativepolyacrylamide gel electrophoresis provided a material that was homogeneous by the criteria of high-pressure liquid chromatography, polyacrylamide gel electrophoresis, and isoelectric focusing. The apparent molecular weight is 83 000. The purified factor increases bone cell proliferation at doses comparable to other mitogens: 0.3 Mg/mL (3.6 nM) significantly increases DNA synthesis to 231% of controls (p< 0.001). The purified factor was also active on cultured embryonic chick bones, enhancing the growth rate of tibiae and femurs, as measured by increased dry weight (185% of controls, p< 0.025) and [3H] proline incorporation (164% of control, p< 0.001), respectively. e coupling of bone formation to resorption was first suggested by clinical observations (Harris & Heaney, 1969) and subsequently verified by in vivo studies with rats (Thompson et al., 1975; Baylink & Liu, 1979). It is our concept that this skeletal coupling provides a counterregulatory mechanism for the local control of bone volume and that faulty coupling may lead to osteoporosis (Ivey & Baylink, 1981). Previous studies from this laboratory established that (a) coupling depends on a local mechanism—it occurs in vitro (Howard et al., 1980),(b) coupling is mediated by an increase in osteoblast number (Baylink et al., 1980),(c) a factor in culture medium that has been conditioned by resorbing bone in vitro will increase both the rate of bone cell proliferation and the growth of embryonic bone (Drivdahl et al., 1980a), and (d) a similarly active factor can be extracted from embryonic chick bone (Drivdahl et al., 1980b). The factor from these latter two sources has been interpreted to be a putative coupling factor that is present in bone matrix and released by bone resorption to increase os-teoblast number and thereby increase the bone formation rate. In the present studies we sought to determine whether a similar putative coupling factor was present inadult human bone matrix.