BMP9在骨形成中的分子機(jī)制及其信號途徑的研究
發(fā)布時(shí)間：2018-12-18 18:29
【摘要】： BMPs屬于TGF-β超家族的一員,在胚胎發(fā)育及骨骼形成中扮演著重要的角色。雖然如今只有幾種BMP被證實(shí)有促進(jìn)骨形成的作用并應(yīng)用于臨床(主要為BMP2及BMP7),但并未有報(bào)道確其認(rèn)為具有最強(qiáng)成骨作用的BMP。本課題從兩個(gè)方面著手,一方面是從體內(nèi)實(shí)驗(yàn)探索BMP9的特殊的信號轉(zhuǎn)導(dǎo)通路:另一方面從體外實(shí)驗(yàn)去證明BMP9是最強(qiáng)的成骨誘導(dǎo)因子。 第一部分是通過基因芯片、Real-time PCR、Western-Blotting等方法探索BMP9是否遵循傳統(tǒng)經(jīng)典BMP信號傳導(dǎo)通路介導(dǎo)骨形成的理論機(jī)制。比較BMP2、9作用下小鼠多潛能干細(xì)胞C3H10中BMP信號傳導(dǎo)通路核心基因Smads的表達(dá)差異;同時(shí)采用RNA干擾的方法沉默Smad4表達(dá)以減弱經(jīng)典BMP信號傳導(dǎo)系統(tǒng)的作用,比較BMP2、9作用下誘導(dǎo)C3H10體外成骨的差異。結(jié)果是與BMP2作用下C3H10中BMP信號傳導(dǎo)通路核心基因Smads表達(dá)皆升高相比,BMP9刺激下上述基因無明顯表達(dá)上調(diào);Smad4基因表達(dá)沉默后,BMP9誘導(dǎo)干細(xì)胞體外成骨的作用無明顯抑制。BMP9并不完全遵循傳統(tǒng)經(jīng)典BMP信號傳導(dǎo)通路而發(fā)揮誘導(dǎo)成骨的生物學(xué)作用。 第二部分是比較腺病毒介導(dǎo)的人骨形態(tài)發(fā)生蛋白一9(Adv-hBMP9)和Adv-hBMP2納米羥基磷灰石/聚酰胺復(fù)合物在損傷部位的骨缺損重建修復(fù)效果。新西蘭白兔36只,隨機(jī)分成三組,制成雙側(cè)橈骨中段13mm骨缺損模型。A組:植入Adv-hBMP9+納米羥基磷灰石/聚酰胺骨;B組:植入Adv-hBMP2+納米羥基磷灰石/聚酰胺骨;C組:注入Adv-GFP+羥基磷灰石/聚酰胺骨。在2,4,8,12,16周各時(shí)相點(diǎn)分別進(jìn)行大體觀察、x線照片、組織學(xué)切片。結(jié)果是BMP9組骨缺損完全修復(fù),BMP2組骨缺損部分修復(fù),而GFP組骨缺損修復(fù)明顯欠佳。重組腺病毒介導(dǎo)的BMP9對橈骨骨缺損后的成骨修復(fù)作用強(qiáng)于BMP2。我們的研究為BMP9的成骨中的作用機(jī)制及信號途徑打下一定基礎(chǔ),為臨床上利用BMP9基因治療骨骼系統(tǒng)相關(guān)疾病提供更強(qiáng)的骨誘導(dǎo)因子。
[Abstract]:BMPs is a member of TGF- 尾 superfamily and plays an important role in embryonic development and bone formation. Although only a few types of BMP have been proven to promote bone formation and have been used in clinical applications (mainly BMP2 and BMP7), there are no reports of BMP. that it believes has the strongest osteogenic effect. In this study, two aspects are involved, one is to explore the special signal transduction pathway of BMP9 in vivo, and the other is to prove that BMP9 is the strongest osteogenic factor in vitro. The first part is to explore whether BMP9 follows the traditional classical BMP signaling pathway to mediate bone formation through gene chip, Real-time PCR,Western-Blotting and other methods. To compare the expression of Smads, the core gene of BMP signal transduction pathway, in C3H10 of mouse multipotent stem cells induced by BMP2,9. At the same time, RNA interference was used to silence the expression of Smad4 in order to attenuate the role of classical BMP signal transduction system, and to compare the difference of C3H10 osteogenesis induced by BMP2,9 in vitro. The results showed that the expression of core gene Smads of BMP signal transduction pathway in C3H10 was higher than that in C3H10 induced by BMP2, but the expression of these genes was not up-regulated under BMP9 stimulation. After the expression of Smad4 gene was silenced, BMP9 did not inhibit the osteogenesis of stem cells in vitro. BMP9 did not follow the classical BMP signal transduction pathway and played a biological role in inducing osteogenesis. The second part is to compare the effect of adenovirus-mediated human bone morphogenetic protein-9 (Adv-hBMP9) and Adv-hBMP2 nano-hydroxyapatite / polyamide composite in the reconstruction of bone defect. Thirty-six New Zealand white rabbits were randomly divided into three groups to make bilateral radial 13mm bone defect model: group A: implantation of Adv-hBMP9 nano-hydroxyapatite / polyamide bone, group B: implantation of Adv-hBMP2 nano-hydroxyapatite / polyamide bone; Group C: Adv-GFP hydroxyapatite / polyamide bone was injected. Gross observation, X-ray photograph and histological section were carried out at each time point of 2 ~ 4 ~ 8 ~ 12 ~ 12 ~ (th) weeks. The results showed that the bone defects were repaired completely in BMP9 group, partially in BMP2 group, but not in GFP group. The effect of recombinant adenovirus-mediated BMP9 on bone repair after radial bone defect is stronger than that of BMP2.. Our study lays a foundation for the mechanism and signal pathway of BMP9 osteogenesis and provides a stronger bone inducer for the clinical treatment of skeletal system related diseases with BMP9 gene.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R346
[Abstract]:BMPs is a member of TGF- 尾 superfamily and plays an important role in embryonic development and bone formation. Although only a few types of BMP have been proven to promote bone formation and have been used in clinical applications (mainly BMP2 and BMP7), there are no reports of BMP. that it believes has the strongest osteogenic effect. In this study, two aspects are involved, one is to explore the special signal transduction pathway of BMP9 in vivo, and the other is to prove that BMP9 is the strongest osteogenic factor in vitro. The first part is to explore whether BMP9 follows the traditional classical BMP signaling pathway to mediate bone formation through gene chip, Real-time PCR,Western-Blotting and other methods. To compare the expression of Smads, the core gene of BMP signal transduction pathway, in C3H10 of mouse multipotent stem cells induced by BMP2,9. At the same time, RNA interference was used to silence the expression of Smad4 in order to attenuate the role of classical BMP signal transduction system, and to compare the difference of C3H10 osteogenesis induced by BMP2,9 in vitro. The results showed that the expression of core gene Smads of BMP signal transduction pathway in C3H10 was higher than that in C3H10 induced by BMP2, but the expression of these genes was not up-regulated under BMP9 stimulation. After the expression of Smad4 gene was silenced, BMP9 did not inhibit the osteogenesis of stem cells in vitro. BMP9 did not follow the classical BMP signal transduction pathway and played a biological role in inducing osteogenesis. The second part is to compare the effect of adenovirus-mediated human bone morphogenetic protein-9 (Adv-hBMP9) and Adv-hBMP2 nano-hydroxyapatite / polyamide composite in the reconstruction of bone defect. Thirty-six New Zealand white rabbits were randomly divided into three groups to make bilateral radial 13mm bone defect model: group A: implantation of Adv-hBMP9 nano-hydroxyapatite / polyamide bone, group B: implantation of Adv-hBMP2 nano-hydroxyapatite / polyamide bone; Group C: Adv-GFP hydroxyapatite / polyamide bone was injected. Gross observation, X-ray photograph and histological section were carried out at each time point of 2 ~ 4 ~ 8 ~ 12 ~ 12 ~ (th) weeks. The results showed that the bone defects were repaired completely in BMP9 group, partially in BMP2 group, but not in GFP group. The effect of recombinant adenovirus-mediated BMP9 on bone repair after radial bone defect is stronger than that of BMP2.. Our study lays a foundation for the mechanism and signal pathway of BMP9 osteogenesis and provides a stronger bone inducer for the clinical treatment of skeletal system related diseases with BMP9 gene.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R346
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