【作者】 罗进勇；

【导师】 尹一兵；

【作者基本信息】 重庆医科大学 ， 临床检验诊断学， 2008， 博士

【摘要】 骨形态发生蛋白(Bone Morphogenetic Proteins,BMPs)是转化生长因子β(Transforming growth factorβ, TGFβ)超家族的成员之一,最初因为其能诱导骨和软骨的形成而得名,目前认为,BMPs与系统发育中的细胞增殖和分化有关,BMPs可以调控间充质干细胞向骨、软骨、肌腱和脂肪分化。至今共分离和鉴定了20余种BMPs,主要已报道的具有诱导成骨活性的BMPs为BMP2、5、7等,其中BMP2和BMP7已经被用于临床治疗,但在临床实际应用上效果还不能令人满意。因此,寻找更强的诱导细胞成骨分化的细胞因子成为目前共同关注的课题,也是临床的迫切需要。在实验室的前期研究工作中,我们通过重组腺病毒介导的方法,系统研究了BMP2～BMP15共14种BMPs在诱导骨形成中的的作用。采用间充质干细胞C3H10细胞系,分别在体外和裸鼠体内进行研究。发现除传统BMP2、7外,BMP4、6、9也具有诱导成骨作用,其中BMP9诱导间充质干细胞C3H10成骨的作用,远强于传统应用的BMP2和BMP7。BMP9是目前已知的BMPs(包括BMP2、BMP7)中诱导成骨能力最强的因子,有希望作为一种更强的促进细胞成骨分化的细胞因子替代品,因此具有广阔的潜在的临床应用价值。但是,BMP9诱导成骨的机制目前并不十分清楚。作为TGFβ家族成员,所有的BMPs(包括BMP9)都主要通过TGFβ信号转导通路传递信号,发挥其诱导成骨的功能。首先BMPs与具有丝氨酸/苏氨酸激酶活性的跨膜TGFβⅡ型和Ⅰ型受体结合,启动信号转导,随后,磷酸化的Ⅰ型受体激活转录因子Smads,由激活的Smads调控细胞核内相关基因的表达。因此,在BMPs信号转导过程中,TGFβ受体起着承上(与BMPs结合)和启下(活化Smads)的作用,是BMPs信号转导的关键节点,是BMPs早期信号转导的最重要的分子之一,与BMPs发挥诱导成骨的活性关系密切。BMP2和BMP7等成骨性BMPs,其相关的TGFβ受体已经被鉴定和分析清楚,但是与BMP9诱导成骨有关的TGFβ受体,目前了解很少,这也限制了对于BMP9诱导成骨机制的揭示。基于以上分析,本研究综合应用分子生物学、细胞生物学、实验动物学等多项技术,如细胞培养、基因克隆、基因转染、重组腺病毒、细胞分化实验技术、real time PCR、动物模型、Micro-CT、组织化学染色等,主要鉴定分析与BMP9诱导成骨有关的TGFβ受体,为BMP9诱导成骨的机制,在受体水平上提供证据。整个研究分为两大部分,主要研究结果如下:第一部分研究中,我们分析已知的七种TGFβⅠ型受体和四种TGFβⅡ型受体的核苷酸序列,PCR扩增含受体胞外结构域和跨膜结构域部分的片段,并将片段克隆入腺病毒穿梭质粒载体pAdtrace-TO4,通过菌落PCR、限制性内切酶酶切和DNA序列测定对阳性克隆进行鉴定,得到共11种TGFβ受体相应的显性负性突变体(dn-receptor)质粒;随后,在BJAdeasy细菌中同源重组dn-receptor腺病毒质粒,重组dn-receptor腺病毒质粒转染293细胞,使腺病毒在其中包装和扩增,最后得到11种dn-receptor重组腺病毒。将制备的dn-receptor腺病毒感染目标细胞C3H10,在感染24小时后,荧光显微镜观察发现11种dn-receptor腺病毒均可在C3H10内表达RFP红色荧光蛋白,产生红色荧光;在病毒感染C3H10细胞48小时后,提取细胞RNA,经过逆转录制备cDNA,PCR扩增,结果显示,所有11种dn-receptor均可在C3H10细胞内表达。通过这部分实验,我们最终获得了11种TGFβ受体相应的显性负性突变体腺病毒,并验证了其在细胞内的表达。显性负性突变的TGFβ受体可以与细胞内的野生型受体竞争相应配体,抑制配体功能的发挥。该部分制备的腺病毒用于第二部分实验,而且也可以用于TGFβ信号通路中与TGFβ受体有关的相关研究。第二部分研究中,首先在体外实验中,我们利用显性负性突变型TGFβ受体腺病毒和BMP9共同作用间充质干细胞C3H10、骨髓基质细胞、小鼠胚胎成纤维细胞(mouse embryonic fibroblasts, MEFs)和鼠成肌细胞C2C12,通过ALP定性和定量试验以及荧光素酶报告基因试验,初步筛选出了5种dn-receptor可以抑制BMP9诱导的ALP和荧光素酶活性,它们分别是dnALK1、dnALK2、dn-BMPRⅡ、dn-ActRⅡ和dnActRⅡB;进一步研究发现,它们对于ALP表达的抑制存在剂量依赖性,而且还可以抑制C3H10和MEFs细胞的钙盐沉积,抑制细胞的成骨分化;通过Real-time PCR发现dnALK1、dnALK2、dn-BMPRⅡ、dn-ActRⅡ和dnActRⅡB这5dn-receptor可以抑制BMP9靶基因(Smad6,Smad7和Id1)的表达。将dnALK1、dnALK2、dn-BMPRⅡ、dn-ActRⅡ和dnActRⅡB和BMP9共感染C3H10细胞,在裸鼠皮下进行注射,5周后观察并剥离皮下包块,Micro-CT扫描和影像重建;石蜡包埋、切片,组织化学染色观察包块内细胞成骨情况,结果显示与对照相比,dnALK1、dnALK2、dn-BMPRⅡ、dn-ActRⅡ和dnActRⅡB处理组成骨包块偏小,而且其包块内成骨不活跃,表明dnALK1、dnALK2、dn-BMPRⅡ、dn-ActRⅡ和dnActRⅡB可以抑制BMP9诱导的C3H10在裸鼠皮下的成骨。从以上结果看这五种dn-recepotr相应的野生型受体(ALK1、ALK2、BMPRⅡ、ActRⅡ和ActRⅡB)很可能与BMP9诱导成骨有关。因此,我们利用pSOS系统筛选了能抑制相应受体表达的siRNA(首先测试了ALK1和ALK2),再利用RNAi抑制ALK1和ALK2的表达,发现抑制ALK1和ALK2表达后,BMP9诱导的荧光素酶活性可相应被抑制。综上所述,本研究从细胞水平到动物整体水平,全面鉴定分析了可能与BMP9诱导成骨有关的TGFβ受体,从整个研究中我们得到以下研究结论:1. TGFβ受体与BMP9诱导成骨活性密切相关,相应dn-receptor可以抑制BMP9诱导的细胞成骨分化。2.构建和制备的dn-receptor腺病毒可以有效地在目标细胞中表达相应的dn-recepotr,因此,它们不但可以用于BMP9相关TGFβ受体的研究和分析,也可以用于TGFβ家族其它分子的研究(如BMP13, BMP14,它们对于软骨形成有重要作用,其相关的TGFβ受体未知)。3通过测试C3H10、BMSC、MEFs和C2C12四种细胞发现:在7种Ⅰ型dn-receptor中,dnALK1和dnALK2能有效抑制BMP9诱导的荧光素酶活性和早期成骨指标ALP活性;在4种Ⅱ型dn-receptor中,dn-BMPRⅡ、dn-ActRⅡ、dnActRⅡB可以有效抑制BMP9诱导的荧光素酶活性和ALP活性,而且这种抑制作用具有剂量依赖性。4.进一步研究证实,dnALK1、dnALK2、dn-BMPRⅡ、dn-ActRⅡ、dnActRⅡB可以有效抑制BMP9诱导的钙盐沉积,并且可抑制BMP9调控的相关靶基因基因(Smad6,Smad7,Id1)的表达。因此,在11种TGFβ受体中,ALK1、ALK2、BMPRⅡ、ActRⅡ、ActRⅡB可能与BMP9诱导成骨有关。5.通过pSOS系统筛选了能有效抑制ALK1、ALK2表达的小分子干扰RNA(siRNA),发现在利用RNA干扰(RNAi)技术抑制C3H10细胞种ALK1、ALK2表达后,继而可抑制BMP9诱导的荧光素酶活性,初步显示在抑制目标细胞相关TGFβ受体表达后,BMP9成骨活性受到了抑制。这提示,继续研究细胞内ALK1、ALK2、BMPRⅡ、ActRⅡ、ActRⅡB等受体表达抑制后,对于BMP9诱导成骨效应的影响,将有助于阐明BMP9诱导成骨的机制。更多还原

【Abstract】 BMPs(Bone Morphogenetic Proteins) belong to the transforming growth factorb (TGFβ) superfamily, Originally isolated as proteins that induce bone and cartilage formation in vivo, BMPs are multifunctional regulators of proliferation and differentiation during development.. BMPs can regulate the differentiation of mesenchymal stem cells into cartilage, bone, tendon/ligament, and fat lineages. More than 20 BMPs have been identified, Prior work show that BMP2, BMP5, BMP7 has potent osteogenic activity, and are currently being investigated in human clinical trials of fracture healing and spine fusion,but the result is not satisfied. Therefore, it very important to look for more efficacious factor which can stimulate osgenetic defferentiation of cells,.In our lab’s previous study, we elucidate the distinct ability of 14 BMPs (BMP2~BMP15) to induce osteogenic differentiation of osteoblast progenitor cells by using adenovirus that eapress these 14 BMPs. Using mesenchymal stem cell line C3H10, we have demonstrated that, besides BMP2 and BMP7, BMP4, BMP6, BMP9 exhibit the osteogenic activity both in vitro cell experiment and in vivo nude models, and osteogenic activity of BMP9 are more than BMP2 and BMP7 which have been used in clinial trails, so BMP9 are the most strong factor to induce bone formation. BMP9 are the most efficacious factor for potentail clinical applications involving bone formation.But the mechanism under the bone formation induced by BMP9 reamins unclear. Belong to the TGFβsuperfamily, all BMPs mainly transduct their signal by TGFβsignal traduction patheay, and then control bone formation. In the first step,BMPs initiate their signaling cascade by binding to a dimeric complex of two transmembrane serine–threonine kinase receptors, termed TGFβtype I and type II. receptor,and then, the activated receptor subsequently phosphorylate transcriptional factors, called Smads, which activate the expression of target genes in concert with coactivators. Therefore, in this signal pathway, TGFβreceptor act as a key point which can bind BMPs and activate Smads, TGFβreceptor are most important molecular in early signal transduction of BMPs, and involve in osteogenic activity of BMPs. Unlike other osteogenic BMPs such as BMP2 and BMP7 whose TGFβreceptor involve bone formation have been characterized, TGFβreceptor of BMP9 in osteogenesis are unknow, this also is a limited factor for elucidating mechenism of BMP9 induced bone formation.Based on this background, in this research, we applied comprehensive techniques in molecular biology, cell biology and experimental zoology such as cell culture, gene clone, gene transfection, recominant adenovirus techniques, cell differantiation techniques, real time PCR, xenograft animal model, Micro-CT, histology techniques, and mainly focu on chracterization of TGFβreceptor in BMP9 induced bone formation, from this research, theoreetic evdence can been find to elucidate the mechanism under BMP9 induced osteogenesis. The total research is divided into two parts and the main research results are as follows:In the first part, we use PCR to amplify fragment containing extracellular domain and transmemrane domain of TGFβreceptor after analyising nucleotide of 7 TGFβtypeⅠreceptors and 4 TGFβtypeⅡreceptors, and then clone these fragments into adenovural shuutle vector pAdtrace-TO4. By using colony PCR, Restriction Endonucleases and DNA sequencing, dominant negative mutant (dn-receptor) of total 11 TGFβreceptors were constructed successfully, then, recombinant adenovirus vectors of 11 dn-receptors were generated in Bjadeasy competent cells. Recombinant adenovirus vectors were transfected into 293 packaging cell. Recombinat adenovirus were packaged and amplified in 193 packaging cell, and finally we got 11 dn-receptor adenovirus. In orde to validate expression of dn-receptor adenovirus in target cell, mesenchymal stem cell C3H10 were infected by dn-receptor virus, after 24hour all 11 dn-receptor virus can infect C3H10 and express RFP in C3H10, after 48hour, cell total RNA was purified using trizol reagent and cDNA was produced by RT reaction. All 11 dn-receptors can express in C3H10 by using PCR to validate. In this part of research, we finally got 11 dn-receptor adenovirus and validated their expression in C3H10 cell. The dn-receptors can competed with wild receptors by binding to ligand, as a consequence, inhibit function of ligand. dn-receptor adenovirus constructed in this part can be used not only in second part of this reseach but also in other related TGFβreceptor research in TGFβsignal pathway.In the second part, we use dn-receptor virus and BMP9 to co-stimulate meshchymal stem cell C3H10, bone marrow stromal cells (BMSC), mouse embryonic fibroblasts (MEFs) and myogenic cell C2C12. Totally, 5 of 11 dn-rceptors who can inhibit BMP9 induced ALP and luciferase acitivity were identified by using ALP staing, ALP quantitative reading, luciferase reportor assay. They are dnALK1、dnALK2、dn-BMPRⅡ、dn-ActRⅡ和dnActRⅡB. The further study showed that the inhibition of dnALK1、dnALK2、dn-BMPRⅡ、dn-ActRⅡ和dnActRⅡB in BMP9 induced ALP repression was dose dependent, furthermore, dnALK1、dnALK2、dn-BMPRⅡ、dn-ActRⅡ和dnActRⅡB can also inhibit Calcification of C3H10 and MEFs induced by BMP9, and inhibit BMP9 target gene exression in C3H10 cell. C3H10 were co-infected by dnALK1、dnALK2、dn-BMPRⅡ、dn-ActRⅡ, dnActRⅡB and BMP9 adenovirus, and then were subcutaneous injieced into nude mice, the bulb’s size was measured Micro-CT , osteogenetic process in bulb was anlysised by histologic staining. We found that dnALK1、dnALK2、dn-BMPRⅡ、dn-ActRⅡ和dnActRⅡB can inhibit bone formation of C3H10 induced by BMP9 in nude mice. Therefore, 5 wild receptors (ALK1, ALK2, BMPRⅡ, ActRⅡ, ActRⅡB) may involve bone formation induced by BMP9. We use pSOS system to screen efficacious siRNA for thse wild receptor knock down (we try ALK1/ALK2 first), and found that ALK1/ALK2 knock down can inhibit BMP9 induced luciferase activity.In all, we characterized the potential TGFβreceptor in BMP9 induced osteogenesis from molecular cell level to whole animal level comprehensively and the research conclusions are as follows:1. TGFβreceptor is correlative to osteogenetic activity of BMP9. Dominant negative mutant of BMP9 related TGFβreceptor inhibit cell osteogensis induced by BMP9.2. The dn-receptor adenovirus we constructed can efficaciously express in target cell, they can be used not only in research of BMP9 related receptor, but also in other research of TGFβsuperfamily.3. After test 4 cell line (C3H10, BMSC, MEFs, C2C12), we found that 2 of 7 typeⅠdn-receptor and 3 of 4 typeⅡdn-receptor can efficaciously inhibit BMP9 induced ALP and luciferase activity. The inhibition of BMP9 induced ALP activity by these dn-receptor are dose-dependent.4. Further study confirmed that dnALK1, dnALK2, dn-BMPRⅡ, dn-ActRⅡ, dnActRⅡB can efficaciously inhibit calcification induced by BMP9, and inhibit target gene expression controlled by BMP9. Therefore, the wild receptor of these 5 dn-receptors (ALK1, ALK2, BMPRⅡ, ActRⅡ, ActRⅡB) may play key role in BMP9 induced osteogensis.5. The efficacious siRNA for ALK1/ALK2 are screened by pSOS system, we found that ALK1/ALK2 knock-down by RNAi can inhibit BMP9 induced luciferase activity, these result implied that osteogentic activity of BMP9 can be inhibit by BMP9 related receptor knock down. It is meaningful to keep probing the effect of BMP9 related TGFβreceptor (ALK1, ALK2, BMPRⅡ, ActRⅡ, ActRⅡB) knock down on BMP9 induced bone formation, thses further study will be helpful to elucidate mechanism behind BMP9 induced osteogenesis.更多还原