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

【作者】 张继帅；

【导师】 杨晓；

【作者基本信息】 中国人民解放军军事医学科学院 ， 遗传学， 2004， 博士

【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。更多还原

【Abstract】 TGF-β superfamily molecules play important roles in regulating cell proliferation，1ineage determination，motility， adhesion and apoptosis.Smad4 is the eentral mediator ofthe TGF-β superfamily signalings.In order to comprehensively study the funetion ofTGF-β superfalllily moleeules in the endoehondral ossification，we generated thechondrocyte specfic Smad4 gene knockout mice using the Cre-loxP system. Firstly，we investigated the temporal and spatial distribution of the Cre recombinase inchondrocyte specific Cre transgenic mousc using the ROSA26 reproter mice.The resultsshowed that the Cre recombinase began to express when the mesenehymal cellsdifferentiate into the chondroeytes.The aetivity of Cre recombinase can be detected in allthe cartilaginous tissues of vertebra，ribs，and limb bones et al，which were formedthrough the cndoehondral ossifieation.All these data indicated that the chondrocytespecifie Cre transgenic mice spceifically expressed the Cre recombinase in the cartilagetissues，and was a good tool for making chondrocyte specific gene knoekout mice. The chondrocyte specific Smad4 knockout mice were obtained by crossing thechondrocyte specific Cre transgenic mice with the Smad4 conditional knockout mice.Thechondrocyte specific Smad4 knockout mice cxhibited various phenotypes because thedifferent chondrocyte specific Cre transgenie founder mice were used.The mice eitherdied at the cmbryonic stages，or died right after birth or survive after birth.we found thatthe alveolus of the Smad4 knockout mice could not be distended.They died of lacking ofoxygen 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 inthe endochondral ossification through studying the chondrocyte specific Smad4 knockoutmice 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 mutantmice became disorganized characterized by significantly expanded resting zone ofchondroeytes，reduced proliferating and hypertrophic zones of chondrocytes，andpremature hypertrophy of proliferating chondroeytes.BrdU ineorporation assay showedthat the Proliferation rate ofProliferating ehondroeytes was low inthemutantmiee.Inthegrowth Plates of P 1 6 and P22 mutant miee，the disorganization of ehondroeytes beeamemore aPParent.There were still areas of resting ehondroeytes in the bone marrow eavity.We further detected the moleeule markersofehondroeyteswithinsituhybridization.Theresults showed that there were some chondroeytes exPressing eollagen X loeating in theProliferating zone of ehondroeytes，indicating that some Proliferating ehondroeyteshyPertroPhied Prematurely.Together， these data suggested that Smad4 moleeule Promotedthe differentiation of the resting ehondroeytes，stimulated the Proliferation of theProliferating ehondroeytes，and inhibited the hyPertroPhie differentiation of ehondroeytes.In addition，these data also suggested that Smad4 mediated signals eould funetion as themorphogen to maintain the Polarity of arrangement and differentiation ofehondrocytes. We further studied the effeet of Smad4 gene knoekout on other signaling Pathwayswhieh regulate the bone develoPment.In situ hybridization and immunohistoehemistryanalysis revealed that the exPression of Ihh，Pte，PTHrP and PPR moleeules weredeereased in the mutant miee，suggesting thats”Zad4 deletion resulted in redueedlhl PTHrP signaling.Our results also showed thats Zad4 deletion eaused the increasedaetivity of FGF signaling.we found that PZI exPression was inereased，and the Stat-lwas aetivated as revealed by the nuelear loealization of Statl Protein in manyehondroeytes of the mutant miee.Through Von一Kossa staining，we found that the loeationof bone eollar of mutant miee was higher than that of eontrol miee.In the mutant miee，the bo-le eollar was abutted to thetlletllatexPressi()一1()f更多还原