重组人骨形成蛋白成骨分化及其定量测活的研究
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摘要
骨形成蛋白（bone morphogenetic protein，BMP）的发现证实了骨形成的诱
导学说，为难治性骨折和骨缺损的治疗提供新的希望。从1988年Wozney首次
克隆人BMP cDNA基因，至今已发现近20种人BMP。BMP属于TGF-β超家族
成员，不仅在骨骼的发育和再生修复中发挥关键作用，而且在中胚层的诱导
和分化、造血组织的形成和发育、神经系统的发育和修复等方面都起着重要
作用。因而对BMP的研究不仅有重要的理论意义，而且还具有广阔的临床应
用前景。从骨组织提取BMP，方法烦琐、收获量少、纯度低、重复性差，因
此，用基因工程制备BMP就成为将BMP推向应用的必然途径。国内外在COS、
CHO、昆虫等真核细胞中已经表达得到不同种人BMP，但表达量低，成本昂
贵；我研究组率先在大肠杆菌中高表达获得具诱骨活性的人BMP-2和-3羧基
端肽，为BMP的研究和应用提供了新来源。
然而BMP的研究和应用遇到了一大难题——至今还没有理想的定量测
定BMP生物学活性的方法，这大大限制了BMP临床应用的进度。目前国内
外对BMP的测活，基本上还是沿用Urist最早采用的体内植入法，即将BMP
第 四 军 医 大 学 博 土 论 义
植入动物骨以外的组织（最常用是肌肉）中，植入后不同时间作组织学切片、
X－线照像等检查，以观察其有无诱导骨组织生成的活性。该方法能从整体观
察BMP的真实活性，但实验周期长（一般要2－3周）、只能定性而不能定量，
而且结果受动物品种、植入状况等因素影响，仅看BMP有无活性，难以比
较不同来源、不同批次等BMP的活性，这使BW的研究工作和生产应用缺
乏量的依据。为此国内外在BW定量测活研究上作过很多努力，包括在整
体植人法（常用植入肌肉中的方法称为肌袋实验）的基础上发展的方法。近
年来又发展了一些体外测活方法，如测定BMP刺激后培养细胞的ALP活性、
蛋白聚糖水平、钙盐沉积的量和速度等。但进展都不大，BMP的定量测活
仍未解决。
BMP定量测活困难的原因在于它是个分化因子。一般促进细胞生长繁
殖的生长因于可以用同位素前体参入DNA来观察促生长的效应，从而定义
其活性的单位。而BMP与这些细胞生长因子不同，它的活性表现在使细胞
分化上，促进细胞生长不是BMP活性的主要表现，有时反而抑制细胞繁殖；
故不能用DNA合成和细胞分裂繁殖的速度来衡量其活性。而且BMP促细胞
分化的表现又是多方面的，因此造成定量测活的困难。不过，因BMP最初
是作为诱骨分于被发现，所以其活性检测基本还是围绕BMP这一基本特性，
即促成骨分化来进行。
为此我们也从BMP分化功能的角度探讨了BMP的定量测活，包括测定
BMP刺激后ALP活性、合成蛋白量等的变化，除骨髓细胞外，其余细胞系
nP活性并无明显变化。另外我们还用放免法测定了BMP－2作用不同细胞
后骨钙素（OC）的含量变化，该指标虽然特异，但反应幅度不够大，最大
增加约1倍多，且该方法要用同位素，检测步骤烦琐。所以说BMP的研究，
特别是重组人BMP的研究和临床应用，迫切需要建立一种可快速定量的活
性测定方法。
近年克隆发现的Cbfal是成骨分化特异性转录因子，对成骨分化发挥极
其关键的转录激活作用，Cbfal的基因敲除或突变将导致全身骨骼发育障碍。
Cbfal 在非成骨细胞或成骨前体细胞均可上调成骨分化相关基因——一骨钙
一
第 四 军 医 大 学 博 士 论 文
素、碱性磷酸酶（Alkaline phosphatase，ALPL骨桥素及 1型胶原基因的表达，
而且证实这些基因的启动子内均有与 Cbfa 结合的作用元件。研究发现，
某些正常情况下并无成骨细胞表型的细胞（如间充质细胞C3H10TI／2等），
在BMP诱导其成骨分化过程中，首先出现Cbfal 的高表达，然后出现碱性
磷酸酶（ALP）、骨钙素（OC）、骨桥素的表达。这说明Cbfal是BMP作用
信号的下游靶分子，BMP诱导成骨分化是通过Cbfal的中介完成，即Cbfal
接到BMP刺激信号后，结合到成骨分化相关基因启动子内的Cbfal作用元
件，从而启动、激活这些基因的转录、表达。
这些研究结果使我们产生定量测定BMP活性的新思路。因骨钙素是成骨
分化特异性相关标志，所以我们选定骨钙素作为BMP诱骨分化的靶向指标。
而荧光素酶报告基因系统是近年发展起来、广泛应用的基因转录报告系统，
它是通过上游启动子启动Luciferase基因表达，然后检测荧光素酶分解底物产
Bone morphogenetic protein (BMP) was first discovered as a osteoinductive molecule, which proved substantial morphogenetic evidence for the theory of induction. Nearly 20 cDNAs of human BMP have been cloned since Wozney etal reported the successful cloning of BMP-1 through BMP-4 in 1988. On the basis of characteristics that include amino acid similarity, the BMPs grouped as a family within the TGF-B superfamily. BMPs not only play a key role in skeletogenesis and bone repair but also involve in dorsal/ventral pattern and extraskeletal organogenesis such as hematopoietic and nervous system during embryonic development. Natural BMPs can be extracted from animal or human bones, but the extraction is labor- and time-consuming process and the amounts of BMPs obtained from bones are often less productive. Fortunately, different recombinant human BMPs (rhBMP) have been expressed in mammalian cells, insects cells or E.coli and all of them can induce cartilage and bone formation in in vivo assay,
so the recombinant DNA technology will allow a supply of almost unlimited amounts of rhBMP for further
Department of Biochemistry and Molecular Biology. FMMU
- 7 -
study and clinical applications. Especially the use of bacterial expression system for the production of rhBMPs might facilitate the introduction of them for clinical applications, as this system offers an extremely high protein output at relatively low cost. In the near future, rhBMPs can potentially replace conventional autogenous bone grafts in the repair of nonunions, large bone fractures, and bone defects.
But there is not an ideal quantitative bioassay method for BMP so far, for BMPs are cell differentiation factors. Their activities are always judged in vivo in mouse or other animals by a standard if they can induce ectopic bone formation. Although this in vivo rodent assay system is reliable and classical, it needs a long time and is affected by many factors. So it is necessary to develop a new in vitro assay method for BMP. A lot of efforts have been applied, including detection of ALP, osteocalcin (OC) and glycogan etc, but the results are not satisfied, for they are not specific or sensitive for BMP's effects. We also analyzed the changes of ALP, OC and synthesis of protein in bone morrow stromal cells (MSCs), NIH3T3 fibroblast and C2C12 myoblast cells stimulated by rhBMP-2. The results showed that concentrations of OC and proteins were gently added in all 3 cells, while the level of ALP was only gone up in the MSCs in dose-dependant effect. Therefore, the direct detection of ALP or OC is not suitable for the quantitative assay of rhBMPs' biological abilities.
Meanwhile the mechanism of osteoblast differentiation is well progressed and the Cbfal (Core binding factor al) is cloned as an essential transcription factor required for osteoblast differentiation. The gene knockout or mutation of Cbfal will lead to a total lack of bone or abnormal skeleton. Cbfal stim Dlates osteoblast-specific gene expression, such as ALP, osteocalcin (OC), type I collagen, osteopontin, and bone sialoprotein. The consensus DNA binding sequence for Cbfal is found in the promoters of these genes as PuACCPuCA, which is regarded as osteoblast-specific element (OSE). More importantly, BMPs' up-regulation of the expression of ALP and OC is associated with the expression of Cbfal, which is prior to the expression of osteoblastic phenotype. It is suggested that Cbfal is one of BMPs targeting key transcriptors which then activate the expression of osteoblast-
Department of Biochemistry and Molecular Biology, FMMU
- 8 -
specific genes. So we can take advantage of the findings that effects of BMPs on osteoblast differentiation are partly mediated by Cbfal, and augment the effects of BMPs by increasing numbers of OSE (Cbfal activating osteoblast-specific element) in order to quantitatively assay the biological abilities of rhBMP. Then we focus on the OC promoter and Luciferase reporter gene as OC is one of osteoblastic phenotype and Luciferase reporter gen
导学说，为难治性骨折和骨缺损的治疗提供新的希望。从1988年Wozney首次
克隆人BMP cDNA基因，至今已发现近20种人BMP。BMP属于TGF-β超家族
成员，不仅在骨骼的发育和再生修复中发挥关键作用，而且在中胚层的诱导
和分化、造血组织的形成和发育、神经系统的发育和修复等方面都起着重要
作用。因而对BMP的研究不仅有重要的理论意义，而且还具有广阔的临床应
用前景。从骨组织提取BMP，方法烦琐、收获量少、纯度低、重复性差，因
此，用基因工程制备BMP就成为将BMP推向应用的必然途径。国内外在COS、
CHO、昆虫等真核细胞中已经表达得到不同种人BMP，但表达量低，成本昂
贵；我研究组率先在大肠杆菌中高表达获得具诱骨活性的人BMP-2和-3羧基
端肽，为BMP的研究和应用提供了新来源。
然而BMP的研究和应用遇到了一大难题——至今还没有理想的定量测
定BMP生物学活性的方法，这大大限制了BMP临床应用的进度。目前国内
外对BMP的测活，基本上还是沿用Urist最早采用的体内植入法，即将BMP
第 四 军 医 大 学 博 土 论 义
植入动物骨以外的组织（最常用是肌肉）中，植入后不同时间作组织学切片、
X－线照像等检查，以观察其有无诱导骨组织生成的活性。该方法能从整体观
察BMP的真实活性，但实验周期长（一般要2－3周）、只能定性而不能定量，
而且结果受动物品种、植入状况等因素影响，仅看BMP有无活性，难以比
较不同来源、不同批次等BMP的活性，这使BW的研究工作和生产应用缺
乏量的依据。为此国内外在BW定量测活研究上作过很多努力，包括在整
体植人法（常用植入肌肉中的方法称为肌袋实验）的基础上发展的方法。近
年来又发展了一些体外测活方法，如测定BMP刺激后培养细胞的ALP活性、
蛋白聚糖水平、钙盐沉积的量和速度等。但进展都不大，BMP的定量测活
仍未解决。
BMP定量测活困难的原因在于它是个分化因子。一般促进细胞生长繁
殖的生长因于可以用同位素前体参入DNA来观察促生长的效应，从而定义
其活性的单位。而BMP与这些细胞生长因子不同，它的活性表现在使细胞
分化上，促进细胞生长不是BMP活性的主要表现，有时反而抑制细胞繁殖；
故不能用DNA合成和细胞分裂繁殖的速度来衡量其活性。而且BMP促细胞
分化的表现又是多方面的，因此造成定量测活的困难。不过，因BMP最初
是作为诱骨分于被发现，所以其活性检测基本还是围绕BMP这一基本特性，
即促成骨分化来进行。
为此我们也从BMP分化功能的角度探讨了BMP的定量测活，包括测定
BMP刺激后ALP活性、合成蛋白量等的变化，除骨髓细胞外，其余细胞系
nP活性并无明显变化。另外我们还用放免法测定了BMP－2作用不同细胞
后骨钙素（OC）的含量变化，该指标虽然特异，但反应幅度不够大，最大
增加约1倍多，且该方法要用同位素，检测步骤烦琐。所以说BMP的研究，
特别是重组人BMP的研究和临床应用，迫切需要建立一种可快速定量的活
性测定方法。
近年克隆发现的Cbfal是成骨分化特异性转录因子，对成骨分化发挥极
其关键的转录激活作用，Cbfal的基因敲除或突变将导致全身骨骼发育障碍。
Cbfal 在非成骨细胞或成骨前体细胞均可上调成骨分化相关基因——一骨钙
一
第 四 军 医 大 学 博 士 论 文
素、碱性磷酸酶（Alkaline phosphatase，ALPL骨桥素及 1型胶原基因的表达，
而且证实这些基因的启动子内均有与 Cbfa 结合的作用元件。研究发现，
某些正常情况下并无成骨细胞表型的细胞（如间充质细胞C3H10TI／2等），
在BMP诱导其成骨分化过程中，首先出现Cbfal 的高表达，然后出现碱性
磷酸酶（ALP）、骨钙素（OC）、骨桥素的表达。这说明Cbfal是BMP作用
信号的下游靶分子，BMP诱导成骨分化是通过Cbfal的中介完成，即Cbfal
接到BMP刺激信号后，结合到成骨分化相关基因启动子内的Cbfal作用元
件，从而启动、激活这些基因的转录、表达。
这些研究结果使我们产生定量测定BMP活性的新思路。因骨钙素是成骨
分化特异性相关标志，所以我们选定骨钙素作为BMP诱骨分化的靶向指标。
而荧光素酶报告基因系统是近年发展起来、广泛应用的基因转录报告系统，
它是通过上游启动子启动Luciferase基因表达，然后检测荧光素酶分解底物产
Bone morphogenetic protein (BMP) was first discovered as a osteoinductive molecule, which proved substantial morphogenetic evidence for the theory of induction. Nearly 20 cDNAs of human BMP have been cloned since Wozney etal reported the successful cloning of BMP-1 through BMP-4 in 1988. On the basis of characteristics that include amino acid similarity, the BMPs grouped as a family within the TGF-B superfamily. BMPs not only play a key role in skeletogenesis and bone repair but also involve in dorsal/ventral pattern and extraskeletal organogenesis such as hematopoietic and nervous system during embryonic development. Natural BMPs can be extracted from animal or human bones, but the extraction is labor- and time-consuming process and the amounts of BMPs obtained from bones are often less productive. Fortunately, different recombinant human BMPs (rhBMP) have been expressed in mammalian cells, insects cells or E.coli and all of them can induce cartilage and bone formation in in vivo assay,
so the recombinant DNA technology will allow a supply of almost unlimited amounts of rhBMP for further
Department of Biochemistry and Molecular Biology. FMMU
- 7 -
study and clinical applications. Especially the use of bacterial expression system for the production of rhBMPs might facilitate the introduction of them for clinical applications, as this system offers an extremely high protein output at relatively low cost. In the near future, rhBMPs can potentially replace conventional autogenous bone grafts in the repair of nonunions, large bone fractures, and bone defects.
But there is not an ideal quantitative bioassay method for BMP so far, for BMPs are cell differentiation factors. Their activities are always judged in vivo in mouse or other animals by a standard if they can induce ectopic bone formation. Although this in vivo rodent assay system is reliable and classical, it needs a long time and is affected by many factors. So it is necessary to develop a new in vitro assay method for BMP. A lot of efforts have been applied, including detection of ALP, osteocalcin (OC) and glycogan etc, but the results are not satisfied, for they are not specific or sensitive for BMP's effects. We also analyzed the changes of ALP, OC and synthesis of protein in bone morrow stromal cells (MSCs), NIH3T3 fibroblast and C2C12 myoblast cells stimulated by rhBMP-2. The results showed that concentrations of OC and proteins were gently added in all 3 cells, while the level of ALP was only gone up in the MSCs in dose-dependant effect. Therefore, the direct detection of ALP or OC is not suitable for the quantitative assay of rhBMPs' biological abilities.
Meanwhile the mechanism of osteoblast differentiation is well progressed and the Cbfal (Core binding factor al) is cloned as an essential transcription factor required for osteoblast differentiation. The gene knockout or mutation of Cbfal will lead to a total lack of bone or abnormal skeleton. Cbfal stim Dlates osteoblast-specific gene expression, such as ALP, osteocalcin (OC), type I collagen, osteopontin, and bone sialoprotein. The consensus DNA binding sequence for Cbfal is found in the promoters of these genes as PuACCPuCA, which is regarded as osteoblast-specific element (OSE). More importantly, BMPs' up-regulation of the expression of ALP and OC is associated with the expression of Cbfal, which is prior to the expression of osteoblastic phenotype. It is suggested that Cbfal is one of BMPs targeting key transcriptors which then activate the expression of osteoblast-
Department of Biochemistry and Molecular Biology, FMMU
- 8 -
specific genes. So we can take advantage of the findings that effects of BMPs on osteoblast differentiation are partly mediated by Cbfal, and augment the effects of BMPs by increasing numbers of OSE (Cbfal activating osteoblast-specific element) in order to quantitatively assay the biological abilities of rhBMP. Then we focus on the OC promoter and Luciferase reporter gene as OC is one of osteoblastic phenotype and Luciferase reporter gen
引文
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