種植體微納米形貌對(duì)成骨細(xì)胞行為影響的分子機(jī)制研究
發(fā)布時(shí)間：2018-09-15 19:40
【摘要】：純鈦表面微納米形貌能夠很好地模擬人體骨組織和細(xì)胞外微環(huán)境的結(jié)構(gòu)，因此可能具有更好成骨細(xì)胞生物學(xué)效應(yīng)，但材料形貌對(duì)細(xì)胞調(diào)控的具體分子機(jī)制尚不清楚。本研究采用酸蝕和陽(yáng)極氧化的方法，在純鈦表面構(gòu)建微納米形貌，以人成骨肉瘤細(xì)胞系MG63為研究對(duì)象，全面系統(tǒng)評(píng)價(jià)了微納米形貌對(duì)成骨細(xì)胞生物學(xué)行為的影響，并采用分子生物學(xué)方法，深入研究參與材料形貌調(diào)控細(xì)胞行為的信號(hào)通路途經(jīng)，以闡明骨植入材料的表面形貌對(duì)骨組織形成的影響的分子生物學(xué)機(jī)制。本研究的方法及結(jié)果不僅為骨植入材料的表面結(jié)構(gòu)的優(yōu)化設(shè)計(jì)及基因修飾提供依據(jù)以達(dá)到提高骨植入材料的骨結(jié)合效率的目的，而且將為探明其它材料表面因素如表面化學(xué)成分或其它骨植入材料與細(xì)胞及組織相互作用的分子機(jī)制研究提供參考，從而更加全面指導(dǎo)骨植入材料的表面設(shè)計(jì)。 第一部分 純鈦表面微納米形貌的制備 【目的】采用課題組前期實(shí)驗(yàn)中已建立的純鈦表面微納米形貌的制備方案，制備含有不同管徑TiO2納米管的微納米復(fù)合梯度形貌，為后續(xù)的實(shí)驗(yàn)提供研究模型。 【方法】將純鈦試樣拋光至鏡面，采用5%的氫氟酸酸蝕試樣30min，試樣超聲清洗后，采用穩(wěn)壓直流陽(yáng)極氧化電源在5V和20V兩個(gè)電壓下分別對(duì)試樣進(jìn)行陽(yáng)極氧化處理1h，采用場(chǎng)發(fā)射掃描電鏡觀察試樣表面形貌。 【結(jié)果】純鈦表面經(jīng)酸蝕和陽(yáng)極氧化處理后形成微米坑表面復(fù)合不同管徑TiO2納米管的微納米復(fù)合結(jié)構(gòu)。TiO2納米管管徑的大小與陽(yáng)極氧化電壓成正比，5V和20V電壓下分別形成管徑約為30nm和100nm的納米管陣列。 【結(jié)論】酸蝕和陽(yáng)極氧化方法能夠在純鈦表面形成微米坑復(fù)合不同管徑TiO2納米管的微納米形貌。 第二部分 微納米形貌對(duì)成骨細(xì)胞生物學(xué)行為的影響 【目的】評(píng)價(jià)微納米形貌對(duì)成骨細(xì)胞生物學(xué)行為的影響。 【方法】將人骨肉瘤細(xì)胞系MG63細(xì)胞接種于材料表面，采用掃描電鏡觀察試樣表面細(xì)胞的伸展形態(tài)，MTT方法觀察細(xì)胞增殖水平，實(shí)時(shí)定量PCR方法檢測(cè)細(xì)胞在材料表面成骨相關(guān)基因的表達(dá)水平，采用天狼星紅染色觀察試樣表面成骨細(xì)胞的膠原分泌能力，采用茜蘇紅染色觀察材料表面成骨細(xì)胞的細(xì)胞外基質(zhì)礦化能力。 【結(jié)果】成骨細(xì)胞MG63在微納米表面充分伸展，細(xì)胞隨著TiO2納米管管徑的增大而呈現(xiàn)逐漸伸長(zhǎng)的形態(tài)。微納米形貌對(duì)細(xì)胞的增殖沒有顯著性影響，在30nm管徑納米管表面，增殖能力稍有下降。微納米形貌顯著上調(diào)了細(xì)胞成骨相關(guān)基因的表達(dá)水平，促進(jìn)了成骨細(xì)胞的膠原分泌能力和細(xì)胞外基質(zhì)礦化能力，并且這種促進(jìn)作用在含有100nm納米管的微納米形貌表面更加明顯。 【結(jié)論】微納米形貌能夠促進(jìn)成骨細(xì)胞MG63的成骨分化功能，含有大管徑納米管（100nm）的微納米形貌對(duì)成骨分化的促進(jìn)作用更加明顯。 第三部分 微納米形貌表面Wnt/-catenin信號(hào)通路對(duì)成骨細(xì)胞的影響 【目的】檢測(cè)Wnt/-catenin信號(hào)通路是否參與材料對(duì)細(xì)胞行為的影響，評(píng)價(jià)Wnt/-catenin信號(hào)通路在微納米形貌調(diào)控成骨細(xì)胞功能中的作用。 【方法】將MG63細(xì)胞接種于材料表面，采用實(shí)時(shí)定量PCR方法檢測(cè)試樣表面MG63細(xì)胞中Wnt信號(hào)通路中受體、激活因子和抑制因子的表達(dá)水平，采用Westernblot檢測(cè)細(xì)胞核內(nèi)-catenin水平；將外源性Wnt信號(hào)通路激活劑Wnt3a和抑制劑Dkk1分別加入到細(xì)胞培養(yǎng)液中，隨后采用Western blot檢測(cè)細(xì)胞核內(nèi)-catenin水平，并采用實(shí)時(shí)定量PCR方法檢測(cè)試樣表面成骨相關(guān)基因的表達(dá)水平，采用商業(yè)試劑盒檢測(cè)成骨細(xì)胞堿性磷酸酶表達(dá)水平，采用天狼星紅染色檢測(cè)成骨細(xì)胞膠原分泌能力的變化，采用MTT方法檢測(cè)細(xì)胞增殖能力，采用流式細(xì)胞術(shù)檢測(cè)細(xì)胞凋亡的變化。 【結(jié)果】微納米形貌上調(diào)了Wnt信號(hào)通路受體--低密度脂蛋白相關(guān)蛋白6（LRP6）和Wnt信號(hào)通路激活劑Wnt3a的表達(dá)，下調(diào)了Wnt通路抑制因子Dkk1/2和分泌型卷曲蛋白相關(guān)蛋白1/2（sFRP1/2）的表達(dá)，Wnt/-catenin信號(hào)在微納米形貌表面被激活。在光滑表面加入外源性Wnt信號(hào)激活劑Wnt3a上調(diào)Wnt/-catenin信號(hào)活性后，成骨分化相關(guān)指標(biāo)如：成骨基因的表達(dá)、堿性磷酸酶水平和膠原分泌能力都隨之增高。在微納米形貌表面加入外源性Wnt信號(hào)抑制劑Dkk1后，微納米表面原本增高的Wnt/-catenin活性受到明顯的抑制，成骨分化相關(guān)指標(biāo)的表達(dá)水平也隨之受到明顯的抑制。材料表面改變Wnt/-catenin信號(hào)活性后對(duì)細(xì)胞增殖和凋亡沒有明顯的影響。 【結(jié)論】微納米形貌通過(guò)調(diào)控Wnt信號(hào)通路中Wnt因子的表達(dá)，，激活Wnt/-catenin信號(hào)活性，被激活的Wnt/-catenin信號(hào)促進(jìn)了成骨細(xì)胞在微納米形貌表面的成骨分化。 第四部分 微納米形貌表面ILK/-catenin信號(hào)通路對(duì)成骨細(xì)胞的影響 【目的】檢測(cè)整合素連接酶/-catenin（ILK/-catenin）信號(hào)通路在微納米形貌調(diào)控成骨細(xì)胞生物學(xué)行為過(guò)程中的作用。 【方法】將MG63細(xì)胞接種于試樣表面，采用實(shí)時(shí)定量PCR方法檢測(cè)-catenin、ILK、整合素1和3亞基的表達(dá)水平，采用western blot方法檢測(cè)胞漿和胞核內(nèi)-catenin、胞漿內(nèi)磷酸化糖原合成激酶3（p-GSK3）和ILK的表達(dá)水平；采用小干擾RNA轉(zhuǎn)染方法沉默ILK的表達(dá)后，檢測(cè)試樣表面細(xì)胞成骨基因表達(dá)水平的改變、膠原分泌能力和細(xì)胞外基質(zhì)礦化水平的變化，并再一次采用western blot檢測(cè)胞漿和胞核內(nèi)-catenin、胞漿內(nèi)GSK3和p-GSK3表達(dá)水平的變化。 【結(jié)果】微納米形貌促進(jìn)了整合素1和3亞基和ILK的表達(dá)。在微納米表面高表達(dá)的ILK一方面通過(guò)促進(jìn)-catenin mRNA的表達(dá)，另一方面通過(guò)磷酸化GSK3進(jìn)而抑制-catenin在胞漿內(nèi)的降解，兩方面共同作用激活了-catenin信號(hào)活性。采用小干擾RNA下調(diào)ILK表達(dá)后，微納米形貌表面的成骨相關(guān)基因的表達(dá)水平、膠原分泌能力和細(xì)胞外基質(zhì)礦化水平也隨之顯著下降。 【結(jié)論】ILK/-catenin信號(hào)通路參與調(diào)控微納米形貌對(duì)成骨分化功能的影響。 第五部分 微納米形貌表面ILK/ERK1/2信號(hào)通路對(duì)成骨細(xì)胞的影響 【目的】檢測(cè)整合素連接酶/細(xì)胞外調(diào)節(jié)蛋白激酶1/2（ILK/ERK1/2）信號(hào)通路在微納米形貌調(diào)控成骨細(xì)胞生物學(xué)行為過(guò)程中的作用。 【方法】將MG63細(xì)胞接種到材料表面，采用western blot方法檢測(cè)試樣表面總ERK1/2、磷酸化ERK1/2和ILK的表達(dá)水平；采用小干擾RNA轉(zhuǎn)染的方法沉默ILK表達(dá)后，再次采用western blot檢測(cè)試樣表面總ERK1/2和磷酸化ERK1/2表達(dá)水平的變化。將ERK1/2信號(hào)通路抑制劑U0126加入到細(xì)胞培養(yǎng)液中處理細(xì)胞后，采用MTT方法檢測(cè)試樣表面細(xì)胞增殖能力的變化，采用實(shí)時(shí)定量PCR方法檢測(cè)細(xì)胞成骨分化相關(guān)基因表達(dá)水平的變化，采用商業(yè)試劑盒染色檢測(cè)成骨細(xì)胞堿性磷酸酶分泌水平的變化，采用天狼星紅染色檢測(cè)細(xì)胞膠原分泌能力的變化，采用茜蘇紅染色檢測(cè)成骨細(xì)胞細(xì)胞外基質(zhì)礦化水平的變化。 【結(jié)果】微納米形貌顯著性上調(diào)了ILK和磷酸化ERK1/2的活性。通過(guò)小干擾RNA轉(zhuǎn)染下調(diào)ILK表達(dá)之后，微納米形貌表面磷酸化ERK1/2的活性也顯著降低。U0126顯著性抑制了微納米形貌表面細(xì)胞的增殖水平、成骨基因表達(dá)水平、膠原分泌能力和細(xì)胞外基質(zhì)礦化水平。 【結(jié)論】ILK/ERK1/2信號(hào)通路參與調(diào)控微納米形貌對(duì)成骨增殖和分化的影響。
[Abstract]:Pure titanium surface micro-and nano-morphology can well simulate the structure of human bone tissue and extracellular microenvironment, so it may have better biological effect on osteoblasts, but the specific molecular mechanism of cell regulation by material morphology is still unclear. Osteosarcoma cell line MG63 was studied. The effects of micro-and nano-morphology on the biological behavior of osteoblasts were comprehensively and systematically evaluated. The signal pathways involved in the morphological regulation of osteosarcoma cells were studied by molecular biological methods to clarify the effects of surface morphology of bone implants on bone formation. The methods and results of this study not only provide the basis for optimizing the surface structure and gene modification of bone implant materials to improve the bone-binding efficiency of bone implant materials, but also reveal the surface factors of other materials such as surface chemical composition or the interaction between other bone implant materials and cells and tissues. Sub mechanism research provides a reference for more comprehensive guidance on the surface design of bone implant materials.
Part one
Preparation of micro and nano morphology on pure titanium surface
[Objective] The Gradient Morphology of titanium nanotubes with different diameters was prepared by using the preparation method of the surface micro-nano morphology of pure titanium which had been established in the previous experiment of our research group.
[Methods] The pure titanium sample was polished to the mirror surface, and the sample was etched with 5% hydrofluoric acid for 30 minutes. After ultrasonic cleaning, the samples were anodized for 1 h at 5V and 20V respectively by a voltage regulated DC anodizing power supply. The surface morphology of the samples was observed by field emission scanning electron microscopy.
[Results] The surface of pure titanium was treated by acid etching and anodic oxidation to form micro-and nano-composite structure with different diameter of nanotubes. The diameter of nanotubes was proportional to anodic oxidation voltage, and nanotube arrays with diameter of about 30 nm and 100 nm were formed at 5V and 20V respectively.
[Conclusion] Acid etching and anodic oxidation can form micro-pits on the surface of pure titanium and form micro-and nano-morphologies of TiO2 nanotubes with different diameters.
The second part
Effects of micro and nano morphology on biological behavior of osteoblasts
[Objective] to evaluate the effect of micro and nano morphology on the biological behavior of osteoblasts.
[Methods] Human osteosarcoma cell line MG63 was inoculated on the surface of the material. The morphology of osteoblasts was observed by scanning electron microscopy, the proliferation of osteoblasts was observed by MTT, the expression of osteogenic related genes on the surface of the material was detected by real-time quantitative PCR, and the osteoblasts on the surface of the sample were observed by Sirius red staining. The ability of collagen secretion was observed by Alizarin staining.
[Results] Osteoblasts MG63 fully extended on the surface of micro-nano-tubes and gradually elongated with the increase of the diameter of the nano-tubes. The morphology of micro-nano-tubes had no significant effect on the proliferation of the cells. On the surface of the nano-tubes with 30 nm diameter, the proliferation ability of osteoblasts decreased slightly. At the same level, the collagen secretion ability and extracellular matrix mineralization ability of osteoblasts were promoted, and this effect was more obvious on the surface of the micro-nano morphology containing 100 nm nanotubes.
[Conclusion] Micronano-morphology can promote osteogenic differentiation of osteoblasts MG63, and the effect of micronano-morphology with large diameter nanotubes (100 nm) on osteogenic differentiation is more obvious.
The third part
Effects of Wnt/-catenin signal pathway on osteoblasts on micro and nano morphology surfaces
[Objective] To investigate the role of Wnt /-catenin signaling pathway in the regulation of osteoblast function by micro-and nano-morphology.
[Methods] MG63 cells were seeded on the surface of the material, and the expression levels of Wnt receptor, activator and inhibitor in MG63 cells were detected by real-time quantitative PCR. The levels of intranuclear-catenin were detected by Western blot, and the exogenous Wnt signal pathway activator Wnt3a and inhibitor Dkk1 were added to MG63 cells respectively. In the culture medium, Western blot was used to detect the level of - Catenin in the nucleus, real-time quantitative PCR was used to detect the expression of osteogenic related genes, commercial kit was used to detect the expression of alkaline phosphatase in osteoblasts, and Sirius red staining was used to detect the change of collagen secretion ability of osteoblasts. Cell proliferation was detected by MTT, and apoptosis was detected by flow cytometry.
[Results] Micronano-morphology up-regulated the expression of Wnt signaling pathway receptor, low density lipoprotein-related protein 6 (LRP6) and Wnt signaling pathway activator Wnt3a, down-regulated the expression of Wnt pathway inhibitor Dkk1/2 and secretory convolution protein-related protein 1/2 (sFRP1/2), and activated the Wnt/-catenin signal on the surface of micronano-morphology. When the exogenous Wnt signal activator Wnt3a was added to the surface of micronano-particles, the expression of osteogenic genes, the level of alkaline phosphatase and the ability of collagen secretion were increased. The expression levels of osteogenic differentiation-related indicators were also significantly inhibited by the obvious inhibition. There was no significant effect on the proliferation and apoptosis of the cells by altering the Wnt/catenin signal activity on the surface of the material.
[Conclusion] Micron-nano morphology can activate Wnt /-catenin signal activity by regulating the expression of Wnt factor in Wnt signaling pathway, and the activated Wnt /-catenin signal promotes osteogenic differentiation of osteoblasts on the surface of micron-nano morphology.
The fourth part
Effects of ILK/-catenin signal pathway on osteoblasts on micro and nano morphology surfaces
[Objective] To investigate the role of integrin ligase /-catenin (ILK /-catenin) signaling pathway in the regulation of osteoblast biological behavior by micro-and nano-morphology.
[Methods] MG63 cells were inoculated on the surface of the sample, and the expression levels of - catenin, ILK, integrin 1 and 3 subunits were detected by real-time quantitative PCR, and the expression levels of - catenin, phosphorylated glycogen synthase kinase 3 (p-GSK3) and ILK in the cytoplasm and nucleus were detected by Western blot. After expression, the changes of osteogenic gene expression, collagen secretion and extracellular matrix mineralization were detected, and the expressions of intracytoplasmic and nuclear-catenin, GSK3 and p-GSK3 were detected by Western blot.
[Results] Integrin-1 and integrin-3 subunits and IL-K were enhanced by micro-and nano-morphology. High expression of IL-K on the micro-and nano-surface activated the activity of-catenin signal by promoting the expression of-catenin mRNA on the one hand and inhibiting the degradation of-catenin in the cytoplasm by phosphorylating GSK3 on the other. After K expression, the expression level of osteogenesis-related genes, collagen secretion ability and extracellular matrix mineralization on the surface of micro-and nano-morphology decreased significantly.
[Conclusion] ILK/-catenin signaling pathway is involved in regulating the effect of micro and nano morphology on osteogenic differentiation.
The fifth part
Effects of ILK/ERK1/2 signal pathway on osteoblasts on micro and nano morphology surfaces
[Objective] To investigate the role of integrin ligase/extracellular regulated protein kinase 1/2 (ILK/ERK1/2) signaling pathway in the morphological regulation of osteoblasts.
[Methods] MG63 cells were seeded onto the surface of the material, and the total ERK1/2, phosphorylated ERK1/2 and ILK expression levels were detected by Western blot. After the ILK expression was silenced by small interfering RNA transfection, the total ERK1/2 and phosphorylated ERK1/2 expression levels were detected by Western blot. After the cells were treated with U0126, MTT was used to detect the changes of cell proliferation, real-time quantitative PCR was used to detect the expression of osteogenic differentiation-related genes, and commercial kit staining was used to detect the changes of alkaline phosphatase secretion in osteoblasts. Sirius red staining was used to detect the changes of collagen secretion and alizarin staining was used to detect the mineralization of extracellular matrix of osteoblasts.
[Results] The activity of ILK and phosphorylated ERK1/2 were significantly up-regulated by micro-and nano-morphology. The activity of phosphorylated ERK1/2 on the surface of micro-and nano-morphology was also significantly down-regulated by small interfering RNA transfection. U0126 significantly inhibited the proliferation level of cells on the surface of micro-and nano-morphology, osteogenic gene expression level, collagen secretion ability and fineness. Extracellular matrix mineralization level.
[Conclusion] ILK/ERK1/2 signaling pathway is involved in regulating the effect of micro and nano morphology on the proliferation and differentiation of osteoblasts.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2013
【分類號(hào)】：R783.1
本文編號(hào)：2244308
[Abstract]:Pure titanium surface micro-and nano-morphology can well simulate the structure of human bone tissue and extracellular microenvironment, so it may have better biological effect on osteoblasts, but the specific molecular mechanism of cell regulation by material morphology is still unclear. Osteosarcoma cell line MG63 was studied. The effects of micro-and nano-morphology on the biological behavior of osteoblasts were comprehensively and systematically evaluated. The signal pathways involved in the morphological regulation of osteosarcoma cells were studied by molecular biological methods to clarify the effects of surface morphology of bone implants on bone formation. The methods and results of this study not only provide the basis for optimizing the surface structure and gene modification of bone implant materials to improve the bone-binding efficiency of bone implant materials, but also reveal the surface factors of other materials such as surface chemical composition or the interaction between other bone implant materials and cells and tissues. Sub mechanism research provides a reference for more comprehensive guidance on the surface design of bone implant materials.
Part one
Preparation of micro and nano morphology on pure titanium surface
[Objective] The Gradient Morphology of titanium nanotubes with different diameters was prepared by using the preparation method of the surface micro-nano morphology of pure titanium which had been established in the previous experiment of our research group.
[Methods] The pure titanium sample was polished to the mirror surface, and the sample was etched with 5% hydrofluoric acid for 30 minutes. After ultrasonic cleaning, the samples were anodized for 1 h at 5V and 20V respectively by a voltage regulated DC anodizing power supply. The surface morphology of the samples was observed by field emission scanning electron microscopy.
[Results] The surface of pure titanium was treated by acid etching and anodic oxidation to form micro-and nano-composite structure with different diameter of nanotubes. The diameter of nanotubes was proportional to anodic oxidation voltage, and nanotube arrays with diameter of about 30 nm and 100 nm were formed at 5V and 20V respectively.
[Conclusion] Acid etching and anodic oxidation can form micro-pits on the surface of pure titanium and form micro-and nano-morphologies of TiO2 nanotubes with different diameters.
The second part
Effects of micro and nano morphology on biological behavior of osteoblasts
[Objective] to evaluate the effect of micro and nano morphology on the biological behavior of osteoblasts.
[Methods] Human osteosarcoma cell line MG63 was inoculated on the surface of the material. The morphology of osteoblasts was observed by scanning electron microscopy, the proliferation of osteoblasts was observed by MTT, the expression of osteogenic related genes on the surface of the material was detected by real-time quantitative PCR, and the osteoblasts on the surface of the sample were observed by Sirius red staining. The ability of collagen secretion was observed by Alizarin staining.
[Results] Osteoblasts MG63 fully extended on the surface of micro-nano-tubes and gradually elongated with the increase of the diameter of the nano-tubes. The morphology of micro-nano-tubes had no significant effect on the proliferation of the cells. On the surface of the nano-tubes with 30 nm diameter, the proliferation ability of osteoblasts decreased slightly. At the same level, the collagen secretion ability and extracellular matrix mineralization ability of osteoblasts were promoted, and this effect was more obvious on the surface of the micro-nano morphology containing 100 nm nanotubes.
[Conclusion] Micronano-morphology can promote osteogenic differentiation of osteoblasts MG63, and the effect of micronano-morphology with large diameter nanotubes (100 nm) on osteogenic differentiation is more obvious.
The third part
Effects of Wnt/-catenin signal pathway on osteoblasts on micro and nano morphology surfaces
[Objective] To investigate the role of Wnt /-catenin signaling pathway in the regulation of osteoblast function by micro-and nano-morphology.
[Methods] MG63 cells were seeded on the surface of the material, and the expression levels of Wnt receptor, activator and inhibitor in MG63 cells were detected by real-time quantitative PCR. The levels of intranuclear-catenin were detected by Western blot, and the exogenous Wnt signal pathway activator Wnt3a and inhibitor Dkk1 were added to MG63 cells respectively. In the culture medium, Western blot was used to detect the level of - Catenin in the nucleus, real-time quantitative PCR was used to detect the expression of osteogenic related genes, commercial kit was used to detect the expression of alkaline phosphatase in osteoblasts, and Sirius red staining was used to detect the change of collagen secretion ability of osteoblasts. Cell proliferation was detected by MTT, and apoptosis was detected by flow cytometry.
[Results] Micronano-morphology up-regulated the expression of Wnt signaling pathway receptor, low density lipoprotein-related protein 6 (LRP6) and Wnt signaling pathway activator Wnt3a, down-regulated the expression of Wnt pathway inhibitor Dkk1/2 and secretory convolution protein-related protein 1/2 (sFRP1/2), and activated the Wnt/-catenin signal on the surface of micronano-morphology. When the exogenous Wnt signal activator Wnt3a was added to the surface of micronano-particles, the expression of osteogenic genes, the level of alkaline phosphatase and the ability of collagen secretion were increased. The expression levels of osteogenic differentiation-related indicators were also significantly inhibited by the obvious inhibition. There was no significant effect on the proliferation and apoptosis of the cells by altering the Wnt/catenin signal activity on the surface of the material.
[Conclusion] Micron-nano morphology can activate Wnt /-catenin signal activity by regulating the expression of Wnt factor in Wnt signaling pathway, and the activated Wnt /-catenin signal promotes osteogenic differentiation of osteoblasts on the surface of micron-nano morphology.
The fourth part
Effects of ILK/-catenin signal pathway on osteoblasts on micro and nano morphology surfaces
[Objective] To investigate the role of integrin ligase /-catenin (ILK /-catenin) signaling pathway in the regulation of osteoblast biological behavior by micro-and nano-morphology.
[Methods] MG63 cells were inoculated on the surface of the sample, and the expression levels of - catenin, ILK, integrin 1 and 3 subunits were detected by real-time quantitative PCR, and the expression levels of - catenin, phosphorylated glycogen synthase kinase 3 (p-GSK3) and ILK in the cytoplasm and nucleus were detected by Western blot. After expression, the changes of osteogenic gene expression, collagen secretion and extracellular matrix mineralization were detected, and the expressions of intracytoplasmic and nuclear-catenin, GSK3 and p-GSK3 were detected by Western blot.
[Results] Integrin-1 and integrin-3 subunits and IL-K were enhanced by micro-and nano-morphology. High expression of IL-K on the micro-and nano-surface activated the activity of-catenin signal by promoting the expression of-catenin mRNA on the one hand and inhibiting the degradation of-catenin in the cytoplasm by phosphorylating GSK3 on the other. After K expression, the expression level of osteogenesis-related genes, collagen secretion ability and extracellular matrix mineralization on the surface of micro-and nano-morphology decreased significantly.
[Conclusion] ILK/-catenin signaling pathway is involved in regulating the effect of micro and nano morphology on osteogenic differentiation.
The fifth part
Effects of ILK/ERK1/2 signal pathway on osteoblasts on micro and nano morphology surfaces
[Objective] To investigate the role of integrin ligase/extracellular regulated protein kinase 1/2 (ILK/ERK1/2) signaling pathway in the morphological regulation of osteoblasts.
[Methods] MG63 cells were seeded onto the surface of the material, and the total ERK1/2, phosphorylated ERK1/2 and ILK expression levels were detected by Western blot. After the ILK expression was silenced by small interfering RNA transfection, the total ERK1/2 and phosphorylated ERK1/2 expression levels were detected by Western blot. After the cells were treated with U0126, MTT was used to detect the changes of cell proliferation, real-time quantitative PCR was used to detect the expression of osteogenic differentiation-related genes, and commercial kit staining was used to detect the changes of alkaline phosphatase secretion in osteoblasts. Sirius red staining was used to detect the changes of collagen secretion and alizarin staining was used to detect the mineralization of extracellular matrix of osteoblasts.
[Results] The activity of ILK and phosphorylated ERK1/2 were significantly up-regulated by micro-and nano-morphology. The activity of phosphorylated ERK1/2 on the surface of micro-and nano-morphology was also significantly down-regulated by small interfering RNA transfection. U0126 significantly inhibited the proliferation level of cells on the surface of micro-and nano-morphology, osteogenic gene expression level, collagen secretion ability and fineness. Extracellular matrix mineralization level.
[Conclusion] ILK/ERK1/2 signaling pathway is involved in regulating the effect of micro and nano morphology on the proliferation and differentiation of osteoblasts.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2013
【分類號(hào)】：R783.1
【共引文獻(xiàn)】
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