0.5Gy X线通过多重信号通路促进成骨细胞增殖与分化
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摘要
骨的生长、发育及内稳态依赖于成骨细胞的骨形成与破骨细胞的骨吸收之间平衡。成骨细胞的主要功能是在骨形成及骨折愈合过程合成分泌大量细胞外基质。众所周知，高剂量辐照对骨组织常具有不良影响，包括放射性骨坏死、骨硬化、骨折及骨矿丢失。然而，低剂量电离辐射对骨组织的生物学效应及分子机制目前仍缺乏相关研究。骨科病人更经常接受到电离辐射，如X线摄影、计算机断层扫描或术中透视，辐射暴露通常为低剂量水平。我们课题组前期研究表明，低剂量X线照射促进大鼠骨折模型愈合过程骨痂的矿化，同时不同水平的低剂量X线在体外对成骨细胞的增殖和分化具有不同影响。但是，目前对于低剂量X线对骨组织刺激兴奋效应的潜在机制仍不清楚。在本研究中，我们进一步探讨低剂量X线辐照对成骨细胞增殖、分化及骨折愈合的影响特征，并在此基础上对相关信号通路的调控作用深入研究，以阐述低剂量X线对骨组织刺激兴奋效应的分子机制，为更好的理解低剂量X线对骨组织的生物学效应提供一定理论基础。
一、0.5Gy X线照射促进成骨细胞增殖、分化及骨折愈合
电离辐射对成骨细胞的影响目前仍存很大争议，且具体分子机制仍不是很清楚。为了进一步研究不同剂量X线对成骨细胞的影响，本部分研究分别应用0.5Gy和5GyX线照射成骨细胞和骨折模型。通过CCK-8，Brdu掺入实验及Sub-G1实验研究对成骨细胞增殖的影响，同时应用Real-time PCR及Western-blot检查骨分化相关基因的表达情况。组织学检查评估骨痂的形成及改建过程。结果显示0.5Gy X线明显促进成骨细胞增殖、分化及骨折愈合。具体表现为，0.5Gy X线增加MC3T3-E1细胞活性及DNA合成，而5GyX线明显抑制细胞活性及增殖，增加细胞的凋亡。Western-blot检测显示MC3T3-E1细胞在电离辐照后骨分化相关标志物蛋白表达水平发生动态变化，包括Col1、ALP、OCN、Runx2、Osterix。在大鼠股骨闭合骨折模型中，0.5GyX线能促进骨折愈合过程，骨痂中PCNA阳性细胞数量、软骨形成及骨痂面积比对照组明显增加，膜内成骨及软骨内成骨进程加速，同时骨形成标志基因Col1α1、Col2α、ALP、OCN、Runx2、Osterix的mRNA表达水平增高。虽然在5Gy照射组骨痂组织中一些骨形成标志物基因的表达也有增高，但是骨痂形成及骨折愈合时间较对照组及0.5Gy组明显延迟，可观察到胶原纤维老化。因此，低剂量的X线照射对成骨细胞具有一定刺激兴奋效应，可增加成骨细胞的增殖、分化及骨折愈合。
二、低剂量X线照射对MC3T3-E1细胞基因表达谱的影响
为了探讨低剂量X射线对成骨细胞基因表达谱的影响及相关分子机制。MC3T3-E1细胞在低剂量X线照射后，继续诱导成骨培养7天，收集样本后采用罗氏NimbleGen小鼠表达谱芯片进行检测。经过RNA抽提纯化和完整性检测后，取5μgRNA进行标记杂交，GenePix Pro6.0软件扫描获取图像并应用NimbleScan2.5软件进行数据分析。应用T检验筛选出p<0.05，两组之间差异表达在1.3倍以上的基因为判定差异基因。结果显示，成骨细胞在低剂量X射线照射后，共发现1412个差异基因，其中559个上调，853个下调。差异基因中促进成骨细胞分化、钙化，及一些生长因子及受体基因上调，同时调控细胞外基质、局部粘附、细胞骨架和血管形成的基因也明显上调。GO和Pathway分析结果显示细胞粘附、细胞周期及调节、Wnt信号、NF-κB信号、细胞骨架、TGF-信号、血小板生长因子受体信号基因明显富集。由此可见，低剂量的X线辐照对成骨细胞的生物学效应的复杂性，其促进成骨细胞分化和矿化可能与细胞外基质-局部粘附-细胞骨架途径、Wnt信号通路及生长因子相关信号通路有关。
三、0.5Gy X线照射通过Wnt/-Catenin信号通路促进成骨细胞分化
为了进一步研究Wnt信号通路在0.5GyX线促进成骨细胞分化中扮演的重要作用，以明确0.5Gy X线照射促进成骨细胞分化的分子机制。成骨细胞接受0.5Gy线照射后流式细胞仪检测细胞凋亡，通过检测骨分化标志物Col1α、ALP、OCN基因蛋白表达水平及ALP活性染色评估成骨细胞分化情况，应用Real-time PCR及Western-blot检测Wnt信号通路中关键分子的表达情况，同时检测Wnt/-Catenin特异性抑制剂XAV939对0.5Gy X线促进成骨细胞分化效应的影响。结果显示，0.5Gy X线可以抑制MC3T3-E1细胞凋亡，照射后4天Col1α、ALP、OCN表达减少，而在分化的中、晚期表达有增加趋势。同时，0.5Gy X射线照射可以激活成骨细胞Wnt/GSK-3/-Catenin信号，应用特异性抑制剂XAV939可以消除0.5Gy X射线照射导致的ALP表达升高。因此，低剂量X线照射成骨细胞后早期可以促进细胞存活，及中、晚期骨性分化标志物表达水平增高，这与低剂量X线照射对成骨细胞Wnt信号的影响密切相关。
四、TGF-1/Smad2、3信号通路在0.5Gy X射线促进成骨细胞分化过程的作用
验证TGF-1/Smads通路在0.5Gy X线照射在促进成骨细胞增殖和分化过程的重要作用。克隆形成实验及细胞周期检测0.5Gy X线照射后成骨细胞的分裂增殖能力，同时评估X线照射对成骨细胞分化、矿化的影响。Real-time PCR检测X线照射后成骨细胞周期调控因子、TGF-1及受体ALK5的mRNA的表达水平，ELISA检测X线照射后TGF-1分泌活性，Western-blot检测TGF-1信号通路关键调控分子Smad2、3的表达水平。结果显示，0.5GyX线照射能增加成骨细胞克隆形成率及S+G2/M细胞比例，增加CyclinD1和Id2的mRNA表达水平，而且导致骨性标志物表达动态变化及促进成骨细胞分化、矿化，同时0.5GyX线照射可以激活TGF-1/Smad2、3信号通路。因此，低剂量X线照射可以促进成骨细胞增殖和表达骨分化相关基因，增加成骨细胞分化成熟、矿化及骨形成能力，在这一过程TGF-1/Smad2,3/Osterix通路可能参与了重要分子调控机制。
五、IGF-1/Akt信号通路在0.5Gy X射线促成骨细胞分化过程的调控作用
研究低剂量X线照射对成骨细胞IGF-1信号的影响，同时探讨IGF-1信号与Wnt信号、TGF-信号间的相互作用，以期深入阐述低剂量X线对成骨细胞的生物学效应及相关分子机制。首先构建IRS-1shRNA慢病毒载体，感染MC3T3-E1细胞后检测IRS-1基因沉默情况。成骨细胞在接受0.5GyX线照射后，Real-time PCR检测IGF-1及膜受体IGF-1R的mRNA表达水平，Western-blot检测IGF-1信号通路关键调控分子Akt的活性。通过RNA干扰阻断IGF-1/Akt信号通路，同时检测其对成骨细胞分化及Wnt/-Catenin和TGF-1/Smad2，3信号的影响。结果显示，成功构建了IRS-1shRNA慢病毒载体和建立了稳转细胞克隆。低剂量X线促进成骨细胞分化的同时可激活IGF-1/Akt信号通路，RNA干扰阻断IRS-1基因可抑制IGF-1/Akt信号，也可消除低剂量X线促进成骨细胞分化成熟及矿化的效应。在低剂量X线照后早期，IGF-1信号可以调节Wnt信号通路中关键分子GSK-3活性，而对TGF-1通路中Smad2的表达水平无显著影响。因此可以看出，0.5GyX线照射通过激活IGF-1/Akt信号通路促进成骨细胞分化和矿化过程。同时，IGF-1/Akt信号可能参与了Wnt信号及TGF-1信号的相互作用。
Bone development and homeostasis are maintained through the balance betweenbone-forming osteoblasts and bone-resorbing osteoclasts. Osteoblasts are the chiefbone-making cells that are responsible for the production of bone extracellular matrixduring the remodeling or healing of bone. It is well known that high-dose irradiationdelivers deleterious effects to bone tissue, including osteoradionecrosis, sclerosis, loss ofbone mass and bone fracture, in a dose-and time-dependent manner. However, the effectsof low-dose irradiation on bone responses and healing have rarely been described in theliterature. Orthopedic patients are more often subjected to radiation exposure, such asradiography, computed tomography or fluoroscopy during surgery, where the exposure toionizing radiation is usually at low-dose levels. Our preliminary studies surprisinglydemonstrated that low dose X-irradiation promoted callus formation and mineralization ina rat model, and this type of radiation was also shown to have different effects on theproliferation and differentiation of osteoblasts in vitro. However, the underlyingmechanism has not been evaluated in osteoblasts exposed to low-dose irradiation. In thisstudy, we investigated how low-dose X-ray irradiation influences the proliferation anddifferentiation of osteoblasts and promotes fracture healing. The expression patterns ofosteogenic genes were evaluated to explore the possible mechanisms involved inirradiation-stimulated osteoblast differentiation. Our findings provide a greaterunderstanding of the biologic responses of osteoblasts exposed to low dose X-irradiationand highlight the potential positive effects of such treatment.
Part I Low-dose X-irradiation promotes osteoblast proliferation, differentiation andfracture healing in vitro and in vivo
There is a great controversy regarding the biologic responses of osteoblasts toX-irradiation, and the mechanisms are poorly understood. In this study, the biologicaleffects of low-dose radiation in stimulating osteoblast proliferation, differentiation andfracture healing were identified using in vitro cell culture and in vivo animal studies. First, low-dose (0.5Gy) X-irradiation induced significant increases in the cell viability andproliferation of MC3T3-E1cells. However, high-dose (5Gy) X-radiation inhibited theviability and proliferation of osteoblasts. In addition, dynamic variations in osteoblastdifferentiation markers, including type I collagen, alkaline phosphatase, Runx2, Osterixand osteocalcin, were observed after both low-dose and high-dose irradiation by Westernblot analysis. Second, fracture healing was evaluated via histology and gene expressionafter single-dose X-irradiation, and low-dose X-irradiation was found to accelerate fracturehealing of closed femoral fractures in rats. In low-dose X-ray-irradiated fractures,increased proliferating cell nuclear antigen (PCNA)-positive cells, cartilage formation, andfracture callus were observed. In addition, we observed more rapid completion ofendochondral and intramembranous ossification, which was accompanied by alteredexpression of gene involved in bone remodeling and fracture callus mineralization.Although several osteoblast differentiation gene expression levels were increased in thefracture callus of high-dose irradiated rats, the callus formation and fracture union weredelayed compared to the control and low-dose irradiated fractures. These results revealbeneficial effects of low-dose irradiation, including the stimulation of osteoblastproliferation, differentiation and fractures healing, and highlight its potential translationalapplication in novel therapies against bone-related diseases.
Part II The effects of low-dose X-ray radiation on the profile of gene expression inMC3T3-E1cells
In order to evaluate global differences of expression between low-dose irradiationgroup and the control, we conducted microarray analyses using mouse Roche NimbleGen12x135K chips. MC3T3-E1cells were irradiated0.5Gy X-irradiation. Cells withoutirradiation were regarded as control group. Cells were cultured for7days after irradiationand collected. Total RNA were extracted and RNA integrity was assessed by standarddenaturing agarosegel electrophoresis. About5μg total RNA of each sample was used forlabeling and array hybridization. The experiment of collecting fluorescent information wasperformed by scanning images. They were then imported into software and expression datawere analyzed. Differentially expressed genes with statistical significance were identifiedthrough Volcano Plot filtering and the threshold was fold change≥1.3. Pathway Analysisand GO analysis were applied to determine the roles of these differentially expressed genes played in these biological pathways or GO terms. Finally, Hierarchical clustering wasperformed to show distinguishable gene expression profiling among samples. We identified1412differentially expressed transcripts in the radiated group compared with the control.Among the identified genes,559transcripts were up-and853transcripts weredown-regulated after irradiation. These identified transcripts were further analyzed byPathway analysis and GO analysis. The overrepresented genes were associated withosteoblast differentiation and ossification. Many genes belonged to focal adhesion,extracellular matrix space, cytoskeletal protein binding and angiogenesis. Some transcriptspossessing growth factor activity such as epidermal growth factor receptor, fibroblastgrowth factor, insulin-like growth factor were statistically overpresented. The studyindicated low-dose X-ray radiation promoted some genes of osteoblast differentiationup-regulated which might be involved extracellular matrix, actin cytoskeleton, focaladhension and growth factor activities.
Part III0.5Gy X-ray radiation promoted osteoblast proliferation and differentiationby Wnt/-Catenin signaling
In order to investigate the important role of Wnt signaling in the processes of0.5GyX-ray promote osteoblast differentiation, and make clear the molecular mechanismsinvolved. Flow cytometry was employed to detect the apoptosis after osteoblast exposureto0.5Gy X-ray radiation. The protein expression level of osteoblast differentiationmarkers, such as Col1α, ALP, OCN, were detected and ALP activity staining wasperformed. Real-time PCR and Western blot were utilized to evaluate the variations of keyfactors in Wnt signaling pathways, while specific inhibitor of Wnt/-Catenin, XAV939wasused to block the Wnt signaling. Our results showed that the apoptosis of MC3T3-E1significantly declined at three days after0.5Gy X-ray radiation. The protein levels ofCol1α, ALP and OCN had increase during osteoblast differentiation except a temporary fallat four days of radiation exposure. At the same time,0.5Gy X-ray radiation can activateWnt/GSK-3/-Catenin signaling, and specific inhibitor XAV939completely abrogatedthe increase in ALP expression and activaty induced by0.5Gy X-ray radiation. This studydemonstrated that low dose X-ray radiation promoted osteoblast early survival, andstimulated middle and late osteoblast differentiation, in which Wnt signaling participatedthe regulation processes.
Part IV0.5Gy X-ray radiation stimulates TGF-1/Smad2/3signaling, osteoblastproliferation and differentiation in vitro
To investigate the changes of TGF-1/Smad2/3signaling pathway in osteoblastproliferation and differentiation induced by0.5Gy X-ray radiation. Clone formationefficiency and cell cycle analysis were performed to detect the ability osteoblast divisionand proliferation after radiation exposure. The effects of irradiation on osteoblastdifferentiation and mineralization were assessed at different times. Real-time PCR wasused to detect the mRNA expression level of cyclin D1, Id2, TGF-1, ALK5afterirradiation, and TGF-1secretory activity was evaluated by ELISA. Western blot wasutilized to detect the expression of phosphorylated Smad2, Runx2and Osterix that wereimportant signaling molecules in TGF-1signaling pathways. The results revealed that0.5Gy X-ray radiation increased clone formation rate and G2/M proportion in osteoblast. Theincreases in mRNA expression levels of CyclinD1, Id2, TGF-1and ALK5were found inosteoblast after radiation exposure. Osteoblast differentiation markers presented dynamicchanges after0.5Gy X-ray radiation. And an accelerated mineralization was detected at thesame time.0.5GyX-ray radiation also activated TGF-1/Smad2/3/Osterix signalingpathwaysin osteoblast. Thus, low dose X-ray radiation can increase the expression ofrelated osteogenesis genes, and promote osteoblast differentiation, mature, mineralizationand bone formation ability. In this process, TGF-1/Smad2/3/Osterix may be involved inthe important regulation mechanism.
Part V IGF-1/Akt signaling involves in the processes of differentiation regulationinduced by0.5Gy X-ray radiation
This part highlighted the effects of low dose X-ray radiation on IGF-1signaling inosteoblast, and the cross-talk between IGF-1signaling, and Wnt signaling and TGF-1signaling. Our purpose was to understand the biological effects of low dose irradiation onosteoblast and the related mechanism more deeply. We constructed IRS-1shRNAlentivirus firstly. Then the shRNA lentivirus infected MC3T3-E1in ordor to silence IRS-1gene. After osteoblast exposure to0.5Gy X-ray radiation, real-time PCR detected themRNA expression of IGF-1and membrane receptor IGF-1R. Western blot was used toinvestigate activity of Akt which was a key regulation molecular in IGF-1signalingpathway. Moreover, after irradiation, the changes in osteoblast differentiation, Wnt/-Catenin signaling, TGF-1/Smad2/3signaling were detected after IGF-1/Aktsignaling pathway was blocked by IGF-1R RNA interference. We successfully constructedIRS-1mRNA interference vector and established stable transfection clones. The resultsshowed that low dose X-ray acradiation activated IGF-1/Akt signaling pathway andpromoted the osteoblast differentiation at the same time. IRS-1gene silencing blockedIGF-1/Akt signaling. That also eliminated the effects of low dose X-ray radiation onosteoblast differentiation, mature and mineralization. At early stage after low dose X-rayradiation, IGF-1signaling adjusted Wnt signaling pathways by activating GSK-3, andthere had no significant effects on the expression level of phosphorylated Smad2whichwas an important molecular mediator in TGF-1signaling pathway. Thus it can be seenthat0.5Gy X-ray radiation promoted osteoblast differentiation and mineralization byactivating IGF-1/Akt signal pathway, and IGF-1/Akt signaling might be cross-talk withWnt signaling and TGF-1after low dose radiation.
Together, our experiments revealed dynamic phenotypic expression changes inosteoblastic cells after X-ray radiation. Low-dose (0.5Gy) X-ray radiation had stimulatoryeffects on osteoblast proliferation, differentiation and fracture healing in vitro and vivo.Multiple signaling pathways including Wnt/-Catenin signaling, TGF-1/Smad2/3signaling and IGF-1/Akt signaling, mediated these stimulatory effects of radiation onosteoblast. Our findings provide a better understanding of low-dose radiation-inducedbiologic responses during bone formation and may lead to the development of improvedstrategies with translational applications for bone-related diseases in humans.
一、0.5Gy X线照射促进成骨细胞增殖、分化及骨折愈合
电离辐射对成骨细胞的影响目前仍存很大争议，且具体分子机制仍不是很清楚。为了进一步研究不同剂量X线对成骨细胞的影响，本部分研究分别应用0.5Gy和5GyX线照射成骨细胞和骨折模型。通过CCK-8，Brdu掺入实验及Sub-G1实验研究对成骨细胞增殖的影响，同时应用Real-time PCR及Western-blot检查骨分化相关基因的表达情况。组织学检查评估骨痂的形成及改建过程。结果显示0.5Gy X线明显促进成骨细胞增殖、分化及骨折愈合。具体表现为，0.5Gy X线增加MC3T3-E1细胞活性及DNA合成，而5GyX线明显抑制细胞活性及增殖，增加细胞的凋亡。Western-blot检测显示MC3T3-E1细胞在电离辐照后骨分化相关标志物蛋白表达水平发生动态变化，包括Col1、ALP、OCN、Runx2、Osterix。在大鼠股骨闭合骨折模型中，0.5GyX线能促进骨折愈合过程，骨痂中PCNA阳性细胞数量、软骨形成及骨痂面积比对照组明显增加，膜内成骨及软骨内成骨进程加速，同时骨形成标志基因Col1α1、Col2α、ALP、OCN、Runx2、Osterix的mRNA表达水平增高。虽然在5Gy照射组骨痂组织中一些骨形成标志物基因的表达也有增高，但是骨痂形成及骨折愈合时间较对照组及0.5Gy组明显延迟，可观察到胶原纤维老化。因此，低剂量的X线照射对成骨细胞具有一定刺激兴奋效应，可增加成骨细胞的增殖、分化及骨折愈合。
二、低剂量X线照射对MC3T3-E1细胞基因表达谱的影响
为了探讨低剂量X射线对成骨细胞基因表达谱的影响及相关分子机制。MC3T3-E1细胞在低剂量X线照射后，继续诱导成骨培养7天，收集样本后采用罗氏NimbleGen小鼠表达谱芯片进行检测。经过RNA抽提纯化和完整性检测后，取5μgRNA进行标记杂交，GenePix Pro6.0软件扫描获取图像并应用NimbleScan2.5软件进行数据分析。应用T检验筛选出p<0.05，两组之间差异表达在1.3倍以上的基因为判定差异基因。结果显示，成骨细胞在低剂量X射线照射后，共发现1412个差异基因，其中559个上调，853个下调。差异基因中促进成骨细胞分化、钙化，及一些生长因子及受体基因上调，同时调控细胞外基质、局部粘附、细胞骨架和血管形成的基因也明显上调。GO和Pathway分析结果显示细胞粘附、细胞周期及调节、Wnt信号、NF-κB信号、细胞骨架、TGF-信号、血小板生长因子受体信号基因明显富集。由此可见，低剂量的X线辐照对成骨细胞的生物学效应的复杂性，其促进成骨细胞分化和矿化可能与细胞外基质-局部粘附-细胞骨架途径、Wnt信号通路及生长因子相关信号通路有关。
三、0.5Gy X线照射通过Wnt/-Catenin信号通路促进成骨细胞分化
为了进一步研究Wnt信号通路在0.5GyX线促进成骨细胞分化中扮演的重要作用，以明确0.5Gy X线照射促进成骨细胞分化的分子机制。成骨细胞接受0.5Gy线照射后流式细胞仪检测细胞凋亡，通过检测骨分化标志物Col1α、ALP、OCN基因蛋白表达水平及ALP活性染色评估成骨细胞分化情况，应用Real-time PCR及Western-blot检测Wnt信号通路中关键分子的表达情况，同时检测Wnt/-Catenin特异性抑制剂XAV939对0.5Gy X线促进成骨细胞分化效应的影响。结果显示，0.5Gy X线可以抑制MC3T3-E1细胞凋亡，照射后4天Col1α、ALP、OCN表达减少，而在分化的中、晚期表达有增加趋势。同时，0.5Gy X射线照射可以激活成骨细胞Wnt/GSK-3/-Catenin信号，应用特异性抑制剂XAV939可以消除0.5Gy X射线照射导致的ALP表达升高。因此，低剂量X线照射成骨细胞后早期可以促进细胞存活，及中、晚期骨性分化标志物表达水平增高，这与低剂量X线照射对成骨细胞Wnt信号的影响密切相关。
四、TGF-1/Smad2、3信号通路在0.5Gy X射线促进成骨细胞分化过程的作用
验证TGF-1/Smads通路在0.5Gy X线照射在促进成骨细胞增殖和分化过程的重要作用。克隆形成实验及细胞周期检测0.5Gy X线照射后成骨细胞的分裂增殖能力，同时评估X线照射对成骨细胞分化、矿化的影响。Real-time PCR检测X线照射后成骨细胞周期调控因子、TGF-1及受体ALK5的mRNA的表达水平，ELISA检测X线照射后TGF-1分泌活性，Western-blot检测TGF-1信号通路关键调控分子Smad2、3的表达水平。结果显示，0.5GyX线照射能增加成骨细胞克隆形成率及S+G2/M细胞比例，增加CyclinD1和Id2的mRNA表达水平，而且导致骨性标志物表达动态变化及促进成骨细胞分化、矿化，同时0.5GyX线照射可以激活TGF-1/Smad2、3信号通路。因此，低剂量X线照射可以促进成骨细胞增殖和表达骨分化相关基因，增加成骨细胞分化成熟、矿化及骨形成能力，在这一过程TGF-1/Smad2,3/Osterix通路可能参与了重要分子调控机制。
五、IGF-1/Akt信号通路在0.5Gy X射线促成骨细胞分化过程的调控作用
研究低剂量X线照射对成骨细胞IGF-1信号的影响，同时探讨IGF-1信号与Wnt信号、TGF-信号间的相互作用，以期深入阐述低剂量X线对成骨细胞的生物学效应及相关分子机制。首先构建IRS-1shRNA慢病毒载体，感染MC3T3-E1细胞后检测IRS-1基因沉默情况。成骨细胞在接受0.5GyX线照射后，Real-time PCR检测IGF-1及膜受体IGF-1R的mRNA表达水平，Western-blot检测IGF-1信号通路关键调控分子Akt的活性。通过RNA干扰阻断IGF-1/Akt信号通路，同时检测其对成骨细胞分化及Wnt/-Catenin和TGF-1/Smad2，3信号的影响。结果显示，成功构建了IRS-1shRNA慢病毒载体和建立了稳转细胞克隆。低剂量X线促进成骨细胞分化的同时可激活IGF-1/Akt信号通路，RNA干扰阻断IRS-1基因可抑制IGF-1/Akt信号，也可消除低剂量X线促进成骨细胞分化成熟及矿化的效应。在低剂量X线照后早期，IGF-1信号可以调节Wnt信号通路中关键分子GSK-3活性，而对TGF-1通路中Smad2的表达水平无显著影响。因此可以看出，0.5GyX线照射通过激活IGF-1/Akt信号通路促进成骨细胞分化和矿化过程。同时，IGF-1/Akt信号可能参与了Wnt信号及TGF-1信号的相互作用。
Bone development and homeostasis are maintained through the balance betweenbone-forming osteoblasts and bone-resorbing osteoclasts. Osteoblasts are the chiefbone-making cells that are responsible for the production of bone extracellular matrixduring the remodeling or healing of bone. It is well known that high-dose irradiationdelivers deleterious effects to bone tissue, including osteoradionecrosis, sclerosis, loss ofbone mass and bone fracture, in a dose-and time-dependent manner. However, the effectsof low-dose irradiation on bone responses and healing have rarely been described in theliterature. Orthopedic patients are more often subjected to radiation exposure, such asradiography, computed tomography or fluoroscopy during surgery, where the exposure toionizing radiation is usually at low-dose levels. Our preliminary studies surprisinglydemonstrated that low dose X-irradiation promoted callus formation and mineralization ina rat model, and this type of radiation was also shown to have different effects on theproliferation and differentiation of osteoblasts in vitro. However, the underlyingmechanism has not been evaluated in osteoblasts exposed to low-dose irradiation. In thisstudy, we investigated how low-dose X-ray irradiation influences the proliferation anddifferentiation of osteoblasts and promotes fracture healing. The expression patterns ofosteogenic genes were evaluated to explore the possible mechanisms involved inirradiation-stimulated osteoblast differentiation. Our findings provide a greaterunderstanding of the biologic responses of osteoblasts exposed to low dose X-irradiationand highlight the potential positive effects of such treatment.
Part I Low-dose X-irradiation promotes osteoblast proliferation, differentiation andfracture healing in vitro and in vivo
There is a great controversy regarding the biologic responses of osteoblasts toX-irradiation, and the mechanisms are poorly understood. In this study, the biologicaleffects of low-dose radiation in stimulating osteoblast proliferation, differentiation andfracture healing were identified using in vitro cell culture and in vivo animal studies. First, low-dose (0.5Gy) X-irradiation induced significant increases in the cell viability andproliferation of MC3T3-E1cells. However, high-dose (5Gy) X-radiation inhibited theviability and proliferation of osteoblasts. In addition, dynamic variations in osteoblastdifferentiation markers, including type I collagen, alkaline phosphatase, Runx2, Osterixand osteocalcin, were observed after both low-dose and high-dose irradiation by Westernblot analysis. Second, fracture healing was evaluated via histology and gene expressionafter single-dose X-irradiation, and low-dose X-irradiation was found to accelerate fracturehealing of closed femoral fractures in rats. In low-dose X-ray-irradiated fractures,increased proliferating cell nuclear antigen (PCNA)-positive cells, cartilage formation, andfracture callus were observed. In addition, we observed more rapid completion ofendochondral and intramembranous ossification, which was accompanied by alteredexpression of gene involved in bone remodeling and fracture callus mineralization.Although several osteoblast differentiation gene expression levels were increased in thefracture callus of high-dose irradiated rats, the callus formation and fracture union weredelayed compared to the control and low-dose irradiated fractures. These results revealbeneficial effects of low-dose irradiation, including the stimulation of osteoblastproliferation, differentiation and fractures healing, and highlight its potential translationalapplication in novel therapies against bone-related diseases.
Part II The effects of low-dose X-ray radiation on the profile of gene expression inMC3T3-E1cells
In order to evaluate global differences of expression between low-dose irradiationgroup and the control, we conducted microarray analyses using mouse Roche NimbleGen12x135K chips. MC3T3-E1cells were irradiated0.5Gy X-irradiation. Cells withoutirradiation were regarded as control group. Cells were cultured for7days after irradiationand collected. Total RNA were extracted and RNA integrity was assessed by standarddenaturing agarosegel electrophoresis. About5μg total RNA of each sample was used forlabeling and array hybridization. The experiment of collecting fluorescent information wasperformed by scanning images. They were then imported into software and expression datawere analyzed. Differentially expressed genes with statistical significance were identifiedthrough Volcano Plot filtering and the threshold was fold change≥1.3. Pathway Analysisand GO analysis were applied to determine the roles of these differentially expressed genes played in these biological pathways or GO terms. Finally, Hierarchical clustering wasperformed to show distinguishable gene expression profiling among samples. We identified1412differentially expressed transcripts in the radiated group compared with the control.Among the identified genes,559transcripts were up-and853transcripts weredown-regulated after irradiation. These identified transcripts were further analyzed byPathway analysis and GO analysis. The overrepresented genes were associated withosteoblast differentiation and ossification. Many genes belonged to focal adhesion,extracellular matrix space, cytoskeletal protein binding and angiogenesis. Some transcriptspossessing growth factor activity such as epidermal growth factor receptor, fibroblastgrowth factor, insulin-like growth factor were statistically overpresented. The studyindicated low-dose X-ray radiation promoted some genes of osteoblast differentiationup-regulated which might be involved extracellular matrix, actin cytoskeleton, focaladhension and growth factor activities.
Part III0.5Gy X-ray radiation promoted osteoblast proliferation and differentiationby Wnt/-Catenin signaling
In order to investigate the important role of Wnt signaling in the processes of0.5GyX-ray promote osteoblast differentiation, and make clear the molecular mechanismsinvolved. Flow cytometry was employed to detect the apoptosis after osteoblast exposureto0.5Gy X-ray radiation. The protein expression level of osteoblast differentiationmarkers, such as Col1α, ALP, OCN, were detected and ALP activity staining wasperformed. Real-time PCR and Western blot were utilized to evaluate the variations of keyfactors in Wnt signaling pathways, while specific inhibitor of Wnt/-Catenin, XAV939wasused to block the Wnt signaling. Our results showed that the apoptosis of MC3T3-E1significantly declined at three days after0.5Gy X-ray radiation. The protein levels ofCol1α, ALP and OCN had increase during osteoblast differentiation except a temporary fallat four days of radiation exposure. At the same time,0.5Gy X-ray radiation can activateWnt/GSK-3/-Catenin signaling, and specific inhibitor XAV939completely abrogatedthe increase in ALP expression and activaty induced by0.5Gy X-ray radiation. This studydemonstrated that low dose X-ray radiation promoted osteoblast early survival, andstimulated middle and late osteoblast differentiation, in which Wnt signaling participatedthe regulation processes.
Part IV0.5Gy X-ray radiation stimulates TGF-1/Smad2/3signaling, osteoblastproliferation and differentiation in vitro
To investigate the changes of TGF-1/Smad2/3signaling pathway in osteoblastproliferation and differentiation induced by0.5Gy X-ray radiation. Clone formationefficiency and cell cycle analysis were performed to detect the ability osteoblast divisionand proliferation after radiation exposure. The effects of irradiation on osteoblastdifferentiation and mineralization were assessed at different times. Real-time PCR wasused to detect the mRNA expression level of cyclin D1, Id2, TGF-1, ALK5afterirradiation, and TGF-1secretory activity was evaluated by ELISA. Western blot wasutilized to detect the expression of phosphorylated Smad2, Runx2and Osterix that wereimportant signaling molecules in TGF-1signaling pathways. The results revealed that0.5Gy X-ray radiation increased clone formation rate and G2/M proportion in osteoblast. Theincreases in mRNA expression levels of CyclinD1, Id2, TGF-1and ALK5were found inosteoblast after radiation exposure. Osteoblast differentiation markers presented dynamicchanges after0.5Gy X-ray radiation. And an accelerated mineralization was detected at thesame time.0.5GyX-ray radiation also activated TGF-1/Smad2/3/Osterix signalingpathwaysin osteoblast. Thus, low dose X-ray radiation can increase the expression ofrelated osteogenesis genes, and promote osteoblast differentiation, mature, mineralizationand bone formation ability. In this process, TGF-1/Smad2/3/Osterix may be involved inthe important regulation mechanism.
Part V IGF-1/Akt signaling involves in the processes of differentiation regulationinduced by0.5Gy X-ray radiation
This part highlighted the effects of low dose X-ray radiation on IGF-1signaling inosteoblast, and the cross-talk between IGF-1signaling, and Wnt signaling and TGF-1signaling. Our purpose was to understand the biological effects of low dose irradiation onosteoblast and the related mechanism more deeply. We constructed IRS-1shRNAlentivirus firstly. Then the shRNA lentivirus infected MC3T3-E1in ordor to silence IRS-1gene. After osteoblast exposure to0.5Gy X-ray radiation, real-time PCR detected themRNA expression of IGF-1and membrane receptor IGF-1R. Western blot was used toinvestigate activity of Akt which was a key regulation molecular in IGF-1signalingpathway. Moreover, after irradiation, the changes in osteoblast differentiation, Wnt/-Catenin signaling, TGF-1/Smad2/3signaling were detected after IGF-1/Aktsignaling pathway was blocked by IGF-1R RNA interference. We successfully constructedIRS-1mRNA interference vector and established stable transfection clones. The resultsshowed that low dose X-ray acradiation activated IGF-1/Akt signaling pathway andpromoted the osteoblast differentiation at the same time. IRS-1gene silencing blockedIGF-1/Akt signaling. That also eliminated the effects of low dose X-ray radiation onosteoblast differentiation, mature and mineralization. At early stage after low dose X-rayradiation, IGF-1signaling adjusted Wnt signaling pathways by activating GSK-3, andthere had no significant effects on the expression level of phosphorylated Smad2whichwas an important molecular mediator in TGF-1signaling pathway. Thus it can be seenthat0.5Gy X-ray radiation promoted osteoblast differentiation and mineralization byactivating IGF-1/Akt signal pathway, and IGF-1/Akt signaling might be cross-talk withWnt signaling and TGF-1after low dose radiation.
Together, our experiments revealed dynamic phenotypic expression changes inosteoblastic cells after X-ray radiation. Low-dose (0.5Gy) X-ray radiation had stimulatoryeffects on osteoblast proliferation, differentiation and fracture healing in vitro and vivo.Multiple signaling pathways including Wnt/-Catenin signaling, TGF-1/Smad2/3signaling and IGF-1/Akt signaling, mediated these stimulatory effects of radiation onosteoblast. Our findings provide a better understanding of low-dose radiation-inducedbiologic responses during bone formation and may lead to the development of improvedstrategies with translational applications for bone-related diseases in humans.
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