软骨发生过程中基因表达谱的测定及生物信息学分析
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摘要
目的建立软骨发育模型,运用基因芯片检测软骨发育过程的基因表达谱,探讨软骨发育相关基因的功能和相互作用关系,为软骨发生(形成)机制的阐明奠定基础,为软骨组织工程提供实验依据。
方法建立小鼠胚胎肢芽发育模型,观察软骨发生的全过程;采用基因芯片技术进行大规模、高通量的基因表达谱分析;运用生物信息学分析技术筛选软骨发生的相关基因;利用体内、体外建模、基因转染和分子生物学技术等对基因芯片检测结果进行验证。
结果成功建立了小鼠胚胎肢芽软骨发育模型,运用Affymetrix mouse 430 2.0全基因组芯片检测包括软骨发育基因在内的小鼠全部45000个基因,检测出的发育各期的差异表达基因数均在2998个,运用聚类分析和表达趋势分析方法从中筛选出17个基因表达组群的表达趋势,其中包括有99个既往研究显示与软骨及骨发育相关的热点基因,同时有已经命名但功能尚未探明的基因107个,尚未命名的未知转录本890个。
结论软骨发育的关键时期应该在E10(间质干细胞)～E14(软骨)阶段。软骨的发育是一个涉及多种酶活性调节、生长分化调节因子,与组织架构密切相关的细胞骨架、细胞凋亡(Apoptosis),以及细胞周期(Cell cycle)、RNA转录、DNA复制等多个类别和基因族的复杂调控过程。这一复杂的调控网络既包括目前研究结果所关注的已知与软骨发育相关的基因,也包括许多功能尚未清楚甚至尚未命名的转录本,同时与血管、肌肉、神经等多种组织器官的发育存在着复杂的相关交差和关联。
Objective: The aim of the present study was to investigate gene expression during the critical period of mice embryonal limb bud chondrogenesis in order to better understand the complexities mechanisms that control this process and its relationship to the cell cycle changes that occur in the critical period.
Method: Mice embryonal anterior limb buds was cut from mice embryo during 10.5～14.5 days of the prenatal development by micromanipulation, poly(A+) RNA was isolated from the five anterior limb buds cell populations and subjected to a gene expression analysis using a microarray containing～34,000 known mice genes and～39,000 expressed sequence tags (ESTs). The critical period of mice embryonal limb bud development was investigated by biochemistry, histology, immunohistochemistry and microarray analysis.
Results: A≥2-fold difference in the expression of 2998 known genes or ESTs was observed in the five days. The differences in expression of several of the genes detected by the microarray hybridization were confirmed by Real-time PCR analyses. There were obvious increases in mRNAs for TIFs (transcription initiation factors) and RNA polymerases during the critical period. Such as TFⅡA(Gtf2a2), TFⅡB(Gtf2b),TFDⅡD(Taf5, -6, -7, -9, -10, -12, -13), TFⅡE(Gtf2e1, -2e2), TFⅡF(Gtf2f2), TFⅡH(cdk7, Gtf2h1, -2, -3, -4, Ercc3, Ccnh, Mant1); RNA polymerasesⅠ(Rpo1-1, -1-2, -1-3, -1-4), RNA polymerasesⅡ(Pol2a, -b, -c, -e, -g, -k, -I, -j)and RNA polymerasesⅢ(Sin, Rpc8, Polr3b, -d, -k). Ccnh, Cdk7 and Mnat1 were also observed in cell cycle gens changes, this finding revealed a complex pattern of gene expression during the critical period of mice embryonal limb bud development, including many genes not yet reported to be expressed by mice embryonal limb bud chondrogenesis.
Conclusion: The 10.5～14.5 days of the prenatal development is the critical period of mice embryonal limb bud development. Changes in gene expression associated with the critical period were observed in a wide variety of genes , including genes encoding extracellular matrix proteins , transcription initiation factors, and growth factors. At least 99 of the genes that were regulated in response to chondrogenesis were also found to be expressed in several pathways. Our results indicate that the simplicity of the system makes it possible to explain the complexities mechanisms partially during the critical period of mice embryonal limb bud chondrogenesis.
方法建立小鼠胚胎肢芽发育模型,观察软骨发生的全过程;采用基因芯片技术进行大规模、高通量的基因表达谱分析;运用生物信息学分析技术筛选软骨发生的相关基因;利用体内、体外建模、基因转染和分子生物学技术等对基因芯片检测结果进行验证。
结果成功建立了小鼠胚胎肢芽软骨发育模型,运用Affymetrix mouse 430 2.0全基因组芯片检测包括软骨发育基因在内的小鼠全部45000个基因,检测出的发育各期的差异表达基因数均在2998个,运用聚类分析和表达趋势分析方法从中筛选出17个基因表达组群的表达趋势,其中包括有99个既往研究显示与软骨及骨发育相关的热点基因,同时有已经命名但功能尚未探明的基因107个,尚未命名的未知转录本890个。
结论软骨发育的关键时期应该在E10(间质干细胞)～E14(软骨)阶段。软骨的发育是一个涉及多种酶活性调节、生长分化调节因子,与组织架构密切相关的细胞骨架、细胞凋亡(Apoptosis),以及细胞周期(Cell cycle)、RNA转录、DNA复制等多个类别和基因族的复杂调控过程。这一复杂的调控网络既包括目前研究结果所关注的已知与软骨发育相关的基因,也包括许多功能尚未清楚甚至尚未命名的转录本,同时与血管、肌肉、神经等多种组织器官的发育存在着复杂的相关交差和关联。
Objective: The aim of the present study was to investigate gene expression during the critical period of mice embryonal limb bud chondrogenesis in order to better understand the complexities mechanisms that control this process and its relationship to the cell cycle changes that occur in the critical period.
Method: Mice embryonal anterior limb buds was cut from mice embryo during 10.5～14.5 days of the prenatal development by micromanipulation, poly(A+) RNA was isolated from the five anterior limb buds cell populations and subjected to a gene expression analysis using a microarray containing～34,000 known mice genes and～39,000 expressed sequence tags (ESTs). The critical period of mice embryonal limb bud development was investigated by biochemistry, histology, immunohistochemistry and microarray analysis.
Results: A≥2-fold difference in the expression of 2998 known genes or ESTs was observed in the five days. The differences in expression of several of the genes detected by the microarray hybridization were confirmed by Real-time PCR analyses. There were obvious increases in mRNAs for TIFs (transcription initiation factors) and RNA polymerases during the critical period. Such as TFⅡA(Gtf2a2), TFⅡB(Gtf2b),TFDⅡD(Taf5, -6, -7, -9, -10, -12, -13), TFⅡE(Gtf2e1, -2e2), TFⅡF(Gtf2f2), TFⅡH(cdk7, Gtf2h1, -2, -3, -4, Ercc3, Ccnh, Mant1); RNA polymerasesⅠ(Rpo1-1, -1-2, -1-3, -1-4), RNA polymerasesⅡ(Pol2a, -b, -c, -e, -g, -k, -I, -j)and RNA polymerasesⅢ(Sin, Rpc8, Polr3b, -d, -k). Ccnh, Cdk7 and Mnat1 were also observed in cell cycle gens changes, this finding revealed a complex pattern of gene expression during the critical period of mice embryonal limb bud development, including many genes not yet reported to be expressed by mice embryonal limb bud chondrogenesis.
Conclusion: The 10.5～14.5 days of the prenatal development is the critical period of mice embryonal limb bud development. Changes in gene expression associated with the critical period were observed in a wide variety of genes , including genes encoding extracellular matrix proteins , transcription initiation factors, and growth factors. At least 99 of the genes that were regulated in response to chondrogenesis were also found to be expressed in several pathways. Our results indicate that the simplicity of the system makes it possible to explain the complexities mechanisms partially during the critical period of mice embryonal limb bud chondrogenesis.
引文
1 Rosen V. BMP and BMP inhibitors in bone. Ann N Y Acad Sci, 2006, 1068:19-25.
2 Farhadi J, Jaquiery C, Barbero A, et al. Differentiation-dependent up-regulation of BMP-2, TGF-beta1, and VEGF expression by FGF-2 in human bone marrow stromal cells. Plast Reconstr Surg. 2005, 116:1379-1386.
3 Yeh LC, Lee JC. Osteogenic protein - 1 increases gene expression of vascular endothelial growth factor in primary cultures of fetal rat calvaria cells. Mol Cell Endocrinol, 1999,153: 113 - 124.
4 Saadeh PB, Mehrara BJ, Steinbrech DS, et al. Transforming growth factor– beta1 modulates the expression of vascular endothelial growth factor by osteoblasts. Am J Physiol, 1999, 277: C628 - C637.
5 Higashikawa A, Kawaguchi H, Nakamura K, et al. Interactions of chondrocytes and osteoblasts during endochondral bone formation. Clin Calcium, 2006,16:109-116.
6 Derner R, Anderson AC. The bone morphogenic protein. Clin Podiatr Med Surg, 2005, 22:607-618, vii.
7 Sato K, Urist MR.Bone morphogenetic protein—induced cartilage development in tissue culture.Clin Orthop,1984,(183):180—187
8孙梁,胡蕴玉,熊卓,等.三种快速成型制作的聚酯/钙磷盐人工骨修复兔桡骨缺损的实验研究.中华外科杂志, 2005, 43:535 - 539.
9 Wan C, He Q, Li G. Osteoclastogenesis in the nonadherent cell population of human bone marrow is inhibited by rhBMP-2 alone or together with rhVEGF. J Orthop Res, 2006, 24:29-36.
10 Peng H, Usas A, Olshanski A, et al. VEGF improves, whereas sFlt1 inhibits,BMP2-induced bone formation and bone healing through modulation of angiogenesis. J Bone Miner Res, 2005, 20:2017-2127.
11 Hall AP, Westwood FR, Wadsworth PF. Review of the effects of anti-angiogenic compounds on the epiphyseal growth plate. Toxicol Pathol, 2006, 34:131-147.
12 Weiss S, Zimmermann G, Baumgart R, et al. Systemic regulation of angiogenesis and matrix degradation in bone regeneration--distraction osteogenesis compared to rigid fracture healing. Bone, 2005, 37:781-790.
13 Orlandini M, Spreafico A, Bardelli M, et al. Vascular endothelial growth factor-D activates VEGFR-3 expressed in osteoblasts inducing their differentiation. J Biol Chem, 2006, 281:17961-17967.
14章军辉,陈永强,汤亭亭,等.透明质酸复合BMP一2转染的兔骨髓基质干细胞在裸鼠肌内成骨的实验研究.中华外科杂志, 2004, 42:1198-1199.
15 Kaito T, Myoui A, Takaoka K, et al. Potentiation of the activity of bone morphogenetic protein - 2 in bone regeneration by a PLA - PEG/hydroxyapatite composite. Biomaterials, 2005, 26: 73 - 79.
16孙明林,胡蕴玉,吕荣,等.一种新型生物活性人工骨的制备及成骨活性的研究.中华外科杂志, 2002, 40:932-935.
17 Sucato DJ, Hedequist D, Zhang H, et al. Recombinant human bone morphogenetic protein - 2 enhances anterior spinal fusion in a thoracoscop ically instrumented animal model. J Bone Joint Surg (Am ) , 2004, 86: 752 - 762.
18 Ohyama T, Kubo Y, Iwata H, et al. Beta -tricalcium phosphate combined with recombinant human bone morphogenetic protein -2: a substitute for autograft, used for packing interbody fusion cages in the canine lumbar spine. Neurol Med Chir ( Tokyo ) ,2004, 44: 234 - 240.
2 Farhadi J, Jaquiery C, Barbero A, et al. Differentiation-dependent up-regulation of BMP-2, TGF-beta1, and VEGF expression by FGF-2 in human bone marrow stromal cells. Plast Reconstr Surg. 2005, 116:1379-1386.
3 Yeh LC, Lee JC. Osteogenic protein - 1 increases gene expression of vascular endothelial growth factor in primary cultures of fetal rat calvaria cells. Mol Cell Endocrinol, 1999,153: 113 - 124.
4 Saadeh PB, Mehrara BJ, Steinbrech DS, et al. Transforming growth factor– beta1 modulates the expression of vascular endothelial growth factor by osteoblasts. Am J Physiol, 1999, 277: C628 - C637.
5 Higashikawa A, Kawaguchi H, Nakamura K, et al. Interactions of chondrocytes and osteoblasts during endochondral bone formation. Clin Calcium, 2006,16:109-116.
6 Derner R, Anderson AC. The bone morphogenic protein. Clin Podiatr Med Surg, 2005, 22:607-618, vii.
7 Sato K, Urist MR.Bone morphogenetic protein—induced cartilage development in tissue culture.Clin Orthop,1984,(183):180—187
8孙梁,胡蕴玉,熊卓,等.三种快速成型制作的聚酯/钙磷盐人工骨修复兔桡骨缺损的实验研究.中华外科杂志, 2005, 43:535 - 539.
9 Wan C, He Q, Li G. Osteoclastogenesis in the nonadherent cell population of human bone marrow is inhibited by rhBMP-2 alone or together with rhVEGF. J Orthop Res, 2006, 24:29-36.
10 Peng H, Usas A, Olshanski A, et al. VEGF improves, whereas sFlt1 inhibits,BMP2-induced bone formation and bone healing through modulation of angiogenesis. J Bone Miner Res, 2005, 20:2017-2127.
11 Hall AP, Westwood FR, Wadsworth PF. Review of the effects of anti-angiogenic compounds on the epiphyseal growth plate. Toxicol Pathol, 2006, 34:131-147.
12 Weiss S, Zimmermann G, Baumgart R, et al. Systemic regulation of angiogenesis and matrix degradation in bone regeneration--distraction osteogenesis compared to rigid fracture healing. Bone, 2005, 37:781-790.
13 Orlandini M, Spreafico A, Bardelli M, et al. Vascular endothelial growth factor-D activates VEGFR-3 expressed in osteoblasts inducing their differentiation. J Biol Chem, 2006, 281:17961-17967.
14章军辉,陈永强,汤亭亭,等.透明质酸复合BMP一2转染的兔骨髓基质干细胞在裸鼠肌内成骨的实验研究.中华外科杂志, 2004, 42:1198-1199.
15 Kaito T, Myoui A, Takaoka K, et al. Potentiation of the activity of bone morphogenetic protein - 2 in bone regeneration by a PLA - PEG/hydroxyapatite composite. Biomaterials, 2005, 26: 73 - 79.
16孙明林,胡蕴玉,吕荣,等.一种新型生物活性人工骨的制备及成骨活性的研究.中华外科杂志, 2002, 40:932-935.
17 Sucato DJ, Hedequist D, Zhang H, et al. Recombinant human bone morphogenetic protein - 2 enhances anterior spinal fusion in a thoracoscop ically instrumented animal model. J Bone Joint Surg (Am ) , 2004, 86: 752 - 762.
18 Ohyama T, Kubo Y, Iwata H, et al. Beta -tricalcium phosphate combined with recombinant human bone morphogenetic protein -2: a substitute for autograft, used for packing interbody fusion cages in the canine lumbar spine. Neurol Med Chir ( Tokyo ) ,2004, 44: 234 - 240.