利用条件基因剔除技术研究Smad4基因在软骨内成骨过程中的功能
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摘要
TGF-β超家族分子在调节细胞增殖、谱系分化、迁移、黏附和凋亡过程中具有
重要作用。Smad4分子是TGF-β超家族分子信号通路的中心介导者。为了进一步深
入研究TGF-β超家族分子在软骨内成骨过程中的功能，我们利用Cre-loxP系统研制
了软骨细胞特异性Smad4基因剔除小鼠。
我们首先利用ROSA26报告基因小鼠研究了软骨细胞特异性Cre转基因小鼠中
Cre重组酶表达的组织特异性。结果显示，在间充质细胞分化为软骨细胞时，便开
始表达Cre重组酶；Cre重组酶表达于脊椎骨、肋骨、四肢骨等所有由软骨内成骨
形成的骨骼软骨组织部位；胫骨切片可以看到Cre重组酶表达于所有软骨细胞。这
些结果表明Cre转基因小鼠能够在软骨细胞特异性表达Cre重组酶，可以作为在软
骨细胞进行基因剔除的工具小鼠。
通过将软骨细胞特异性Cre转基因小鼠和Smad4条件基因打靶小鼠交配，可以
得到在软骨细胞特异性剔除Smad4基因的小鼠。我们使用了我们建立的不同软骨细
胞特异性Cre转基因小鼠，发现产生的软骨细胞特异性Smad4基因剔除小鼠的表型
不同，分别表现为：胚胎期死亡、新生期死亡和出生后存活。利用剖宫产术观察新
生期死亡小鼠的死亡原因，发现死亡的基因剔除小鼠气管软骨环狭窄，肺不张，表
明小鼠死亡的原因主要是Smad4基因剔除后，气管软骨环狭窄，气体不能通过，致
使肺泡不能扩张，因为缺氧而死亡。
我们主要分析了出生后存活的软骨细胞特异性Smad4基因剔除小鼠的表型，揭
示了Smad4分子介导的BMP和TGF-β信号在软骨内成骨过程中的功能。Smad4基
因剔除小鼠体型矮小，骨骼骨化延迟。从出生后第四天开始，基因剔除小鼠的胫骨
生长板组织结构紊乱，表现为软骨细胞静止区增宽，增殖区和肥大区缩窄，软骨细
胞提前肥大分化。利用Brdu掺入法测量出生后4天小鼠软骨细胞的增殖程度，发
现基因剔除小鼠中增殖区软骨细胞的增殖能力降低。出生16人、22天后基因剔除
小鼠胫骨生长板部位的软骨细胞排列极度紊乱，骨髓腔内残留有片状的静止软骨细
胞。利用原位杂交检测生长板各区软骨细胞的标志分子，发现在软骨细胞增殖区有
一些细胞表达胶原X分子，表明增殖软骨细胞的肥大分化提前。这些结果表明Smad4
摘要
分子可以促进静止软骨细胞分化，刺激增殖软骨细胞增殖，抑制软骨细胞肥大分化，
并提示Smad4介导的信号可能作为调控生长板软骨细胞极性排列的形态发生素维持
生长板软骨的正常组织结构。
我们还研究了阻断Smad4介导的BMP、TGF一p信号对其它调控骨骼发育过程的
关键信号通路的影响。原位杂交和免疫组化结果显示，Smad4基因剔除导致Ihh、
Ptc、PTHrP、PPR基因的表达显著降低，提示基因剔除小鼠中Jh川PTHrP信号通路
的功能减弱。研究还发现Smad4基因剔除导致了FGF信号的功能相对增强，生长板
软骨细胞pZI分子表达水平升高以及STAT一1分子主要位于细胞核内。利用
Von一Kossa染色观察小鼠骨骼矿化情况，发现基因剔除小鼠的骨领位置较高，紧邻
增殖区软骨细胞。原位杂交检测成骨细胞的标志分子骨桥素和骨钙素，发现它们的
表达位置比它们在对照小鼠的表达位置升高。这些结果表明基因剔除小鼠中软骨膜
细胞提前分化为成骨细胞。为了验证Smad4介导的信号是否直接通过调节Ih树PTHrP
信号通路控制软骨细胞的肥大分化，我们进行了胚胎期拓骨体外培养实验。结果显
示Smad4基因剔除的踢骨失去了对BMP和TGF一p信号的反应，但仍能应答Ihh替
代分子Shh的作用，对照和基因剔除踌骨软骨细胞的肥大分化均受到Shh的明显抑
制。这些结果表明基因剔除小鼠中th讨PTHrP信号通路的功能正常，Smad4抑制软
骨细胞肥大分化的作用不依赖于thll/PTHrP信号通路。
我们的实验结果首次提供体内的遗传学证据证明Smad4分子是维持生长板软骨
正常组织结构所必需的。Smed4介导的信号促进静止软骨细胞分化，刺激增殖软骨
细胞增殖，不依赖于Ih可PTHrP信号通路抑制软骨细胞肥大分化。软骨组织特异性
Smad4基因剔除小鼠的成功研制为进一步深入研究smad4介导的TGF一p信号在软骨
内成骨过程中的作用和机制奠定了基础。
TGF-β superfamily molecules play important roles in regulating cell proliferation，
1ineage determination，motility， adhesion and apoptosis.Smad4 is the eentral mediator of
the TGF-β superfamily signalings.In order to comprehensively study the funetion of
TGF-β superfalllily moleeules in the endoehondral ossification，we generated the
chondrocyte specfic Smad4 gene knockout mice using the Cre-loxP system.
Firstly，we investigated the temporal and spatial distribution of the Cre recombinase in
chondrocyte specific Cre transgenic mousc using the ROSA26 reproter mice.The results
showed that the Cre recombinase began to express when the mesenehymal cells
differentiate into the chondroeytes.The aetivity of Cre recombinase can be detected in all
the cartilaginous tissues of vertebra，ribs，and limb bones et al，which were formed
through the cndoehondral ossifieation.All these data indicated that the chondrocyte
specifie Cre transgenic mice spceifically expressed the Cre recombinase in the cartilage
tissues，and was a good tool for making chondrocyte specific gene knoekout mice.
The chondrocyte specific Smad4 knockout mice were obtained by crossing the
chondrocyte specific Cre transgenic mice with the Smad4 conditional knockout mice.The
chondrocyte specific Smad4 knockout mice cxhibited various phenotypes because the
different chondrocyte specific Cre transgenie founder mice were used.The mice either
died at the cmbryonic stages，or died right after birth or survive after birth.we found that
the alveolus of the Smad4 knockout mice could not be distended.They died of lacking of
oxygen since the narrowed trachea prevented the air from entering the lung.
We diselosed the roles of BMP and TGF-β signaling mediated by Smad4 molecule in
the endochondral ossification through studying the chondrocyte specific Smad4 knockout
mice that survived after birth.Mutant mice were smaller than their littermate countrols.
The caleification of their long bones was delayed.At P4，the growth plates of the mutant
mice became disorganized characterized by significantly expanded resting zone of
chondroeytes，reduced proliferating and hypertrophic zones of chondrocytes，and
premature hypertrophy of proliferating chondroeytes.BrdU ineorporation assay showed
that the Proliferation rate ofProliferating ehondroeytes was low inthemutantmiee.Inthe
growth Plates of P 1 6 and P22 mutant miee，the disorganization of ehondroeytes beeame
more aPParent.There were still areas of resting ehondroeytes in the bone marrow eavity.
We further detected the moleeule markersofehondroeyteswithinsituhybridization.The
results showed that there were some chondroeytes exPressing eollagen X loeating in the
Proliferating zone of ehondroeytes，indicating that some Proliferating ehondroeytes
hyPertroPhied Prematurely.Together， these data suggested that Smad4 moleeule Promoted
the differentiation of the resting ehondroeytes，stimulated the Proliferation of the
Proliferating ehondroeytes，and inhibited the hyPertroPhie differentiation of ehondroeytes.
In addition，these data also suggested that Smad4 mediated signals eould funetion as the
morphogen to maintain the Polarity of arrangement and differentiation ofehondrocytes.
We further studied the effeet of Smad4 gene knoekout on other signaling Pathways
whieh regulate the bone develoPment.In situ hybridization and immunohistoehemistry
analysis revealed that the exPression of Ihh，Pte，PTHrP and PPR moleeules were
deereased in the mutant miee，suggesting thats”Zad4 deletion resulted in redueed
lhl PTHrP signaling.Our results also showed thats Zad4 deletion eaused the increased
aetivity of FGF signaling.we found that PZI exPression was inereased，and the Stat-l
was aetivated as revealed by the nuelear loealization of Statl Protein in many
ehondroeytes of the mutant miee.Through Von一Kossa staining，we found that the loeation
of bone eollar of mutant miee was higher than that of eontrol miee.In the mutant miee，
the bo-le eollar was abutted to the
tlle
tllat
exPressi()一1()f
重要作用。Smad4分子是TGF-β超家族分子信号通路的中心介导者。为了进一步深
入研究TGF-β超家族分子在软骨内成骨过程中的功能，我们利用Cre-loxP系统研制
了软骨细胞特异性Smad4基因剔除小鼠。
我们首先利用ROSA26报告基因小鼠研究了软骨细胞特异性Cre转基因小鼠中
Cre重组酶表达的组织特异性。结果显示，在间充质细胞分化为软骨细胞时，便开
始表达Cre重组酶；Cre重组酶表达于脊椎骨、肋骨、四肢骨等所有由软骨内成骨
形成的骨骼软骨组织部位；胫骨切片可以看到Cre重组酶表达于所有软骨细胞。这
些结果表明Cre转基因小鼠能够在软骨细胞特异性表达Cre重组酶，可以作为在软
骨细胞进行基因剔除的工具小鼠。
通过将软骨细胞特异性Cre转基因小鼠和Smad4条件基因打靶小鼠交配，可以
得到在软骨细胞特异性剔除Smad4基因的小鼠。我们使用了我们建立的不同软骨细
胞特异性Cre转基因小鼠，发现产生的软骨细胞特异性Smad4基因剔除小鼠的表型
不同，分别表现为：胚胎期死亡、新生期死亡和出生后存活。利用剖宫产术观察新
生期死亡小鼠的死亡原因，发现死亡的基因剔除小鼠气管软骨环狭窄，肺不张，表
明小鼠死亡的原因主要是Smad4基因剔除后，气管软骨环狭窄，气体不能通过，致
使肺泡不能扩张，因为缺氧而死亡。
我们主要分析了出生后存活的软骨细胞特异性Smad4基因剔除小鼠的表型，揭
示了Smad4分子介导的BMP和TGF-β信号在软骨内成骨过程中的功能。Smad4基
因剔除小鼠体型矮小，骨骼骨化延迟。从出生后第四天开始，基因剔除小鼠的胫骨
生长板组织结构紊乱，表现为软骨细胞静止区增宽，增殖区和肥大区缩窄，软骨细
胞提前肥大分化。利用Brdu掺入法测量出生后4天小鼠软骨细胞的增殖程度，发
现基因剔除小鼠中增殖区软骨细胞的增殖能力降低。出生16人、22天后基因剔除
小鼠胫骨生长板部位的软骨细胞排列极度紊乱，骨髓腔内残留有片状的静止软骨细
胞。利用原位杂交检测生长板各区软骨细胞的标志分子，发现在软骨细胞增殖区有
一些细胞表达胶原X分子，表明增殖软骨细胞的肥大分化提前。这些结果表明Smad4
摘要
分子可以促进静止软骨细胞分化，刺激增殖软骨细胞增殖，抑制软骨细胞肥大分化，
并提示Smad4介导的信号可能作为调控生长板软骨细胞极性排列的形态发生素维持
生长板软骨的正常组织结构。
我们还研究了阻断Smad4介导的BMP、TGF一p信号对其它调控骨骼发育过程的
关键信号通路的影响。原位杂交和免疫组化结果显示，Smad4基因剔除导致Ihh、
Ptc、PTHrP、PPR基因的表达显著降低，提示基因剔除小鼠中Jh川PTHrP信号通路
的功能减弱。研究还发现Smad4基因剔除导致了FGF信号的功能相对增强，生长板
软骨细胞pZI分子表达水平升高以及STAT一1分子主要位于细胞核内。利用
Von一Kossa染色观察小鼠骨骼矿化情况，发现基因剔除小鼠的骨领位置较高，紧邻
增殖区软骨细胞。原位杂交检测成骨细胞的标志分子骨桥素和骨钙素，发现它们的
表达位置比它们在对照小鼠的表达位置升高。这些结果表明基因剔除小鼠中软骨膜
细胞提前分化为成骨细胞。为了验证Smad4介导的信号是否直接通过调节Ih树PTHrP
信号通路控制软骨细胞的肥大分化，我们进行了胚胎期拓骨体外培养实验。结果显
示Smad4基因剔除的踢骨失去了对BMP和TGF一p信号的反应，但仍能应答Ihh替
代分子Shh的作用，对照和基因剔除踌骨软骨细胞的肥大分化均受到Shh的明显抑
制。这些结果表明基因剔除小鼠中th讨PTHrP信号通路的功能正常，Smad4抑制软
骨细胞肥大分化的作用不依赖于thll/PTHrP信号通路。
我们的实验结果首次提供体内的遗传学证据证明Smad4分子是维持生长板软骨
正常组织结构所必需的。Smed4介导的信号促进静止软骨细胞分化，刺激增殖软骨
细胞增殖，不依赖于Ih可PTHrP信号通路抑制软骨细胞肥大分化。软骨组织特异性
Smad4基因剔除小鼠的成功研制为进一步深入研究smad4介导的TGF一p信号在软骨
内成骨过程中的作用和机制奠定了基础。
TGF-β superfamily molecules play important roles in regulating cell proliferation，
1ineage determination，motility， adhesion and apoptosis.Smad4 is the eentral mediator of
the TGF-β superfamily signalings.In order to comprehensively study the funetion of
TGF-β superfalllily moleeules in the endoehondral ossification，we generated the
chondrocyte specfic Smad4 gene knockout mice using the Cre-loxP system.
Firstly，we investigated the temporal and spatial distribution of the Cre recombinase in
chondrocyte specific Cre transgenic mousc using the ROSA26 reproter mice.The results
showed that the Cre recombinase began to express when the mesenehymal cells
differentiate into the chondroeytes.The aetivity of Cre recombinase can be detected in all
the cartilaginous tissues of vertebra，ribs，and limb bones et al，which were formed
through the cndoehondral ossifieation.All these data indicated that the chondrocyte
specifie Cre transgenic mice spceifically expressed the Cre recombinase in the cartilage
tissues，and was a good tool for making chondrocyte specific gene knoekout mice.
The chondrocyte specific Smad4 knockout mice were obtained by crossing the
chondrocyte specific Cre transgenic mice with the Smad4 conditional knockout mice.The
chondrocyte specific Smad4 knockout mice cxhibited various phenotypes because the
different chondrocyte specific Cre transgenie founder mice were used.The mice either
died at the cmbryonic stages，or died right after birth or survive after birth.we found that
the alveolus of the Smad4 knockout mice could not be distended.They died of lacking of
oxygen since the narrowed trachea prevented the air from entering the lung.
We diselosed the roles of BMP and TGF-β signaling mediated by Smad4 molecule in
the endochondral ossification through studying the chondrocyte specific Smad4 knockout
mice that survived after birth.Mutant mice were smaller than their littermate countrols.
The caleification of their long bones was delayed.At P4，the growth plates of the mutant
mice became disorganized characterized by significantly expanded resting zone of
chondroeytes，reduced proliferating and hypertrophic zones of chondrocytes，and
premature hypertrophy of proliferating chondroeytes.BrdU ineorporation assay showed
that the Proliferation rate ofProliferating ehondroeytes was low inthemutantmiee.Inthe
growth Plates of P 1 6 and P22 mutant miee，the disorganization of ehondroeytes beeame
more aPParent.There were still areas of resting ehondroeytes in the bone marrow eavity.
We further detected the moleeule markersofehondroeyteswithinsituhybridization.The
results showed that there were some chondroeytes exPressing eollagen X loeating in the
Proliferating zone of ehondroeytes，indicating that some Proliferating ehondroeytes
hyPertroPhied Prematurely.Together， these data suggested that Smad4 moleeule Promoted
the differentiation of the resting ehondroeytes，stimulated the Proliferation of the
Proliferating ehondroeytes，and inhibited the hyPertroPhie differentiation of ehondroeytes.
In addition，these data also suggested that Smad4 mediated signals eould funetion as the
morphogen to maintain the Polarity of arrangement and differentiation ofehondrocytes.
We further studied the effeet of Smad4 gene knoekout on other signaling Pathways
whieh regulate the bone develoPment.In situ hybridization and immunohistoehemistry
analysis revealed that the exPression of Ihh，Pte，PTHrP and PPR moleeules were
deereased in the mutant miee，suggesting thats”Zad4 deletion resulted in redueed
lhl PTHrP signaling.Our results also showed thats Zad4 deletion eaused the increased
aetivity of FGF signaling.we found that PZI exPression was inereased，and the Stat-l
was aetivated as revealed by the nuelear loealization of Statl Protein in many
ehondroeytes of the mutant miee.Through Von一Kossa staining，we found that the loeation
of bone eollar of mutant miee was higher than that of eontrol miee.In the mutant miee，
the bo-le eollar was abutted to the
tlle
tllat
exPressi()一1()f
引文
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