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安全有效地加快正畸牙移动速率是医患的共同愿望。增加破骨细胞活性,可导致压力侧牙槽骨吸收加快,从而达到提高牙移动速率的目的。如果成骨活动不能相应增加,则导致骨稳态失衡,牙槽骨骨量减少,进而增加牙齿松动、牙槽骨吸收、牙根吸收以及正畸治疗后复发的风险。因此,同时提高成骨、破骨活动是安全有效地加快正畸牙移动的可行方法。信号转导与转录激活因子3（signal transducer and activatorof transcription 3,STAT3）与骨代谢关系密切。有研究显示,成骨细胞特异性敲除STAT3的小鼠表现为骨形态异常、骨质疏松[1];敲除或抑制破骨前体细胞内STAT3导致破骨分化下降[2];STAT3也介导了骨细胞对于机械应力加载产生的成骨效应[3]。以上研究表明,STAT3在成骨、破骨分化中均发挥作用,并对机械信号产生反应,由此推断STAT3可能参与调控正畸牙移动骨塑建与骨重建。本研究探究了STAT3在调控正畸牙槽骨塑建速率及骨稳态方面发挥的作用,为安全有效提高正畸牙移动速率提供理论基础和潜在靶点。
1 材料和方法
1.1 主要试剂
8周龄雄性Wistar大鼠（四川大学实验动物中心,SYXK（川）2018-185）。小鼠成骨前体细胞MC3T3-e1（亚克隆14）和小鼠单核巨噬细胞白血病细胞RAW264.7（均购自中国科学院昆明细胞库）。
DMEM培养基（Gibco,美国）、胎牛血清（Corning,美国）,stattic（Selleck,美国）,抗坏血酸（百灵威,中国）,β-甘油磷酸钠（Sigma-aldrich,美国）,BCIP/NBT碱性磷酸酯酶显色试剂盒（碧云天,中国）,抗酒石酸酸性磷酸酶（tartrate-resistant acid phosphatase,TRAP）染液试剂盒（凯基生物,中国）,TRIzol（Invitrogen,美国）,TB Green® PrimeScriptTM RT reagent Kit with gDNA Eraser（Takara,日本）。
1.2 实验动物分组
8周龄雄性Wistar大鼠40只,体重（250 ± 20）g,随机平均分为2组：对照组（牙移动）,实验组（牙移动加局部注射STAT3抑制剂stattic）（图1）。
图1
图1
大鼠牙移动装置图及局部注射位点口内照
Figure 1
Intra-oral picture of the device for orthodontic tooth movement and location of the local injection in rats
以镍钛拉簧拉双侧上颌第一磨牙向近中移动建立牙移动模型,力值40 g。实验组于第一磨牙近中腭侧黏骨膜下注射10 μL 50 μM stattic溶液,每隔1日注射1次,相邻两次注射间隔48 h,在建立牙移动的前两天开始局部注射;对照组注射等体积生理盐水。在牙移动的第7天、第14天每组分别随机处死10只大鼠,收集双侧上颌骨。
1.3 Micro-CT扫描及分析
标本经4%多聚甲醛在4 ℃固定48 h后,储存于4 ℃ 0.5%多聚甲醛溶液中等待扫描。扫描精度10 μm。在正中矢状向截面测量第一磨牙与第二磨牙牙冠之间的最小距离为牙移动距离,每个样本重复测3次,取平均值。在第一磨牙根分叉处选取400 μm× 400 μm × 500 μm的长方体作为研究对象,分析该区域牙槽骨的骨体积分数（bone volume/tissue volume,BV/TV）、骨小梁数目（trabecular number,Tb.N）、骨小梁厚度（trabecular thickness,Tb.Th）、骨小梁分离度（trabecular separation,Tb.Sp）、骨密度（bone mineral density,BMD）。
1.4 细胞培养
小鼠成骨前体细胞MC3T3-e1（亚克隆14）和小鼠单核巨噬细胞白血病细胞RAW264.7扩增于含10%胎牛血清、1%双抗的DMEM培养基;加入50 μg/mL抗坏血酸、10 mM β-甘油磷酸钠后成为成骨诱导培养基。两种细胞共培养于Transwell®培养板,下层接种MC3T3-e1,密度2.5 × 105个/孔;上层小室接种RAW264.7,密度5 × 105个/孔。共培养体系分为对照组和实验组。对照组使用普通成骨诱导培养基,实验组使用含5 μM stattic的成骨诱导培养基。
1.5 ALP染色
MC3T3-e1经共培养3 d后,以BCIP/NBT碱性磷酸酯酶显色试剂盒按说明书进行染色,显示成骨细胞内ALP活性。
1.6 TRAP染色
RAW264.7经共培养3 d后,以TRAP染液试剂盒按说明书进行染色,显示TRAP阳性细胞。
1.7 qRT-PCR
用TRIzol法提取细胞总RNA,用PrimeScriptTM RT reagent Kit with gDNA Eraser合成cDNA,TB Green® Premix Ex TaqTM II进行qRT-PCR检测,内参为GAPDH,所使用核因子κB受体活化因子配体（receptor activator of nuclear factor-κB ligand,RANKL）、骨保护素（osteoprotegerin,OPG）引物序列见表1。
表1 qRT-PCR引物序列
Table 1
| Genes | Primer sequences（5′→3′） |
|---|---|
| RANKL | F: CAGCATCGCTCTGTTCCTGTA R: CTGCGTTTTCATGGAGTCTCA |
| OPG | F: GAGGAGTCTGGTAGTGGTTCC R: TGTTCACCCTGGTTGAGTAAGT |
| GAPDH | F: GACATCAAGAAGGTGGTGAAGC R: GAAGGTGGAAGAGTGGGAGTT |
1.8 统计学分析
以SPSS 19.0进行分析,计量资料以x ± s表示。经检验,骨小梁形态学各项参数、牙移动距离、基因表达情况符合正态分布,各组方差齐;骨小梁形态学参数采用双因素方差分析及Bonferroni校正进行组间比较;牙移动距离和基因表达分析采用独立样本t检验。P < 0.05为差异有统计学意义。
2 结 果
2.1 抑制STAT3激活对正畸牙移动区松质骨微结构的影响
图2
图2
7 d、14 d时2组大鼠上颌第一磨牙根分叉处牙槽骨micro-CT矢状向截面图
Figure 2
Micro-CT sagittal images of alveolar bone of the upper first molars at the bifurcation on day 7 and day 14 in two groups of rats
a： the trabecular morphology of interradicular alveolar bone of the upper first molars in the control group on day 7; b： the trabecular morphology of interradicular alveolar bone of the upper first molars in the test group on day 7; c： the trabecular morphology of interradicular alveolar bone of the upper first molars in the control group on day 14; d： the trabecular morphology of interradicular alveolar bone of the upper first molars in the test group on day 14
表2 2组大鼠7 d、14 d上颌第一磨牙根分叉处牙槽骨微结构micro-CT测量值
Table 2
| Parameters | Days | Control group | Test group | P | t |
|---|---|---|---|---|---|
| BV/TV（%） | 7 | 0.597 ± 0.032 | 0.575 ± 0.063 | 0.490 | 0.72 |
| 14 | 0.533 ± 0.028 | 0.455 ± 0.030 | 0.003 | 3.81 | |
| Tb.N（1/mm） | 7 | 8.290 ± 0.599 | 7.920 ± 0.443 | 0.286 | 1.13 |
| 14 | 7.970 ± 0.591 | 7.090 ± 0.491 | 0.028 | 2.57 | |
| Tb.Th（mm） | 7 | 0.095 ± 0.012 | 0.095 ± 0.010 | 0.982 | 0.02 |
| 14 | 0.085 ± 0.006 | 0.069 ± 0.008 | 0.006 | 3.44 | |
| Tb.Sp（mm） | 7 | 0.090 ± 0.015 | 0.098 ± 0.013 | 0.421 | 0.84 |
| 14 | 0.092 ± 0.016 | 0.116 ± 0.012 | 0.025 | 2.63 | |
| BMD（g/cc） | 7 | 1.100 ± 0.085 | 1.060 ± 0.080 | 0.442 | 0.80 |
| 14 | 1.030 ± 0.075 | 0.902 ± 0.067 | 0.016 | 2.89 |
表3 Micro-CT测量值方差分析表
Table 3
| Parameters | Source of variation | F | P |
|---|---|---|---|
| BV/TV（%） | Interaction | 2.70 | 0.131 |
| Time | 30.20 | <0.001 | |
| Inhibitor | 6.06 | 0.034 | |
| Tb.N（1/mm） | Interaction | 19.73 | 0.001 |
| Time | 101.90 | < 0.001 | |
| Inhibitor | 3.55 | 0.089 | |
| Tb.Th（mm） | Interaction | 10.69 | 0.008 |
| Time | 53.60 | < 0.001 | |
| Inhibitor | 2.09 | 0.179 | |
| Tb.Sp（mm） | Interaction | 23.29 | 0.001 |
| Time | 39.65 | < 0.001 | |
| Inhibitor | 3.09 | 0.110 | |
| BMD（g/cc） | Interaction | 4.48 | 0.060 |
| Time | 28.15 | <0.001 | |
| Inhibitor | 3.79 | 0.080 |
2.2 抑制STAT3激活对正畸牙移动速率的影响
第7天时,实验组牙移动量明显低于对照组（P=0.005）;在第14天时,两组牙移动量差异无统计学意义（P=0.307）（表4）。
表4 2组牙移动量随时间变化情况
Table 4
| Days | Control group | Test group | Mean | t | P |
|---|---|---|---|---|---|
| 7 | 0.343 ± 0.041 | 0.273 ± 0.044 | 0.308 ± 0.055 | 3.280 | 0.005 |
| 14 | 0.406 ± 0.038 | 0.423 ± 0.029 | 0.414 ± 0.035 | 1.056 | 0.307 |
2.3 抑制STAT3激活对MC3T3-e1与RAW264.7共培养体系成骨、破骨向分化的影响
与对照组相比,实验组处理3 d后,共培养体系中MC3T3-e1 ALP染色明显较浅,说明抑制共培养体系中STAT3激活降低了ALP活性,抑制成骨分化;RAW264.7 TRAP阳性细胞数目明显减少,说明抑制共培养体系中STAT3激活抑制破骨向分化（图3）。
图3
图3
2组MC3T3-e1成骨分化及RAW264.7破骨分化情况
Figure 3
The osteoblastic differentiation of MC3T3-e1 cells and the osteoclastic differentiation of RAW264.7 cells in the two groups
a： ALP staining of MC3T3-e1 cells in the control group （× 40）; b： ALP staining of MC3T3-e1 cells in the test group （× 40）; c： TRAP staining of RAW264.7 cells in the control group （× 40）; d： TRAP staining of RAW264.7 cells in the test group （× 40）; ALP： alkaline phosphatase; TRAP： tartrate-resistant acid phosphatase
qRT-PCR结果表明,stattic抑制MC3T3-e1 RANKL、OPG的mRNA表达,且对OPG的抑制作用更明显,RANKL/OPG比值升高（表5）。
表5 2组MC3T3-e1 RANKL和OPG mRNA表达水平及RANKL/OPG mRNA比值
Table 5
| Measurements | Control group | Test group | P | t |
|---|---|---|---|---|
| RANKL | 1.000 ± 0.093 | 0.677 ± 0.041 | 0.011 | 4.510 |
| OPG | 1.030 ± 0.065 | 0.604 ± 0.077 | 0.004 | 5.990 |
| RANKL/OPG ratio | 0.971 ± 0.042 | 1.241 ± 0.089 | 0.038 | 3.058 |
Supplementary information can be obtained by scanning the OSID code
3 讨 论
3.1 研究意义
3.2 结果分析
炎症因子IL-6已被证实参与正畸牙移动及牙周炎发展进程[8,9]。作为IL-6重要的下游信号转导因子,STAT3具有促进成骨和破骨的双重作用[10]。破骨前体细胞中,抑制STAT3激活直接削弱RANKL诱导下的破骨分化[11,12]。成骨分化依赖于STAT3的激活,上调成骨细胞及间充质干细胞STAT3激活水平增加其成骨能力及增殖活性[13,14]。STAT3同时介导成骨活动与破骨活动的特性在本研究中也得到了证实。STAT3对骨量的宏观影响取决于其对成骨与破骨的相对调控。本研究中,在正畸牙移动模型中发现抑制STAT3激活导致骨量降低。文献表明,在人类个体中,STAT3功能丧失突变导致高免疫球蛋白E综合征（hyper-immunoglobulin E syndrome,HIES）,其体征包括骨质疏松及复发性微小骨折,也提示STAT3与骨量呈正相关[15]。然而在骨关节炎等病理性情况下,STAT3被过度激活,破骨活动亢进,此时抑制STAT3激活则削弱破骨活动,引起骨密度的增加[16],与本研究涉及的生理性牙移动模型有所区别。这提示STAT3在不同的生理、病理性骨代谢过程中发挥不同作用。
本研究的共培养体系中,没有添加外源性RANKL促进破骨前体细胞分化,破骨细胞细胞分化依赖于成骨前体细胞所分泌的RANKL。共培养结果表明,抑制STAT3激活对成骨和破骨分化均有抑制,与原有研究一致[2]。在成骨细胞中,抑制STAT3提高RANKL/OPG mRNA 比值,促进破骨分化,而在共培养体系中的宏观表现为破骨分化受抑制,提示STAT3抑制剂可能作用于破骨细胞本身抑制其破骨分化。
在体内实验中,正常大鼠牙移动第7天时破骨活跃、成骨活动较弱[17]。本实验STAT3抑制剂使牙移动速率下降,结合本研究体外实验结果,推测STAT3抑制剂可能通过抑制破骨活动降低了牙移动速率。正常大鼠在牙移动第14天时,成骨活动达到高峰,而破骨活动有所减缓,牙移动处于缓慢期[17]。Micro-CT结果显示,实验组骨质较为疏松,说明STAT3抑制剂可能抑制了成骨活动,使得实验组与对照组相比,牙移动阻力减小,在7~14 d牙移动速率加快,到第14天时牙移动量和对照组相比没有明显差异。实验组在破骨活动受抑制的情况下,骨量呈减少趋势,说明STAT3对成骨活动调控的作用更强,STAT3的激活状态与骨量呈正相关。
本实验通过STAT3功能抑制实验初步证实了STAT3在正常大鼠正畸牙移动中对牙移动速率和牙槽骨骨量可能发挥正向调控的作用。未来需要开展更多的研究以深入阐明STAT3在不同生理、病理状态下对牙槽骨塑建及骨稳态的调控作用。
参考文献
Cell-specific paracrine actions of IL-6 family cytokines from bone, marrow and muscle that control bone formation and resorption
[J].
STAT3 controls osteoclast differentiation and bone homeostasis by regulating NFATc1 transcription
[J].
STAT3 in osteocytes mediates osteogenic response to loading
[J].
Study of bone remodeling in corticotomy-assisted orthodontic tooth movement in rats
[J].
Effects of low-level laser irradiation on the rate of orthodontic tooth movement and associated pain with self-ligating brackets
[J].
Effect of high-frequency vibration on orthodontic tooth movement and bone density
[J].
Interactive effects of periodontitis and orthodontic tooth movement on dental root resorption, tooth movement velocity and alveolar bone loss in a rat model
[J].
Protein and mRNA expressions of IL-6 and its key signaling factors under orthodontic forces in mice: an in-vivo study
[J].
Analysis of IL-6, IL-10 and NF-κB gene polymorphisms in aggressive and chronic periodontitis
[J].
A novel microRNA regulates osteoclast differentiation via targeting protein inhibitor of activated STAT3 (PIAS3)
[J].
MicroRNAs (miRNAs) involve in the regulation of a wide range of physiological processes. Recent studies suggested that miRNAs might play a role in osteoclast differentiation. Here, we identify a new miRNA (miR-9718) in primary mouse osteoclasts that promotes osteoclast differentiation by repressing protein inhibitor of activated STAT3 (PIAS3) at the post-transcriptional level. MiR-9718 was found to be transcribed during osteoclastogenesis, which was induced by macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor-kappa B ligand (RANKL). Overexpression of miR-9718 in RAW 264.7 cells promoted M-CSF and RANKL-induced osteoclastogenesis, whereas inhibition of miR-9718 attenuated it. PIAS3 was predicted to be a target of miR-9718. Luciferase reporter gene validated the prediction. Transfection of pre-miR-9718 in RAW 264.7 cells induced by both M-CSF and RANKL inhibited expression of PIAS3 protein, while the mRNA levels of PIAS3 were not attenuated. In vivo, our study showed that silencing of miR-9718 using a specific antagomir inhibited bone resorption and increased bone mass in mice receiving ovariectomy (OVX) and in sham-operated control mice. Thus, our study showed that miR-9718 played an important role in osteoclast differentiation via targeting PIAS3 both in vitro and in vivo. (C) 2014 Elsevier Inc.
The prenylflavonoid icaritin enhances osteoblast proliferation and function by signal transducer and activator of transcription factor 3 (STAT-3) regulation of C-X-C chemokine receptor type 4 (CXCR4) expression
[J].
IL-6 and sIL-6R induces STAT3-dependent differentiation of human VSMCs into osteoblast-like cells through JMJD2B-mediated histone demethylation of RUNX2
[J].
Monocytes induce STAT3 activation in human mesenchymal stem cells to promote osteoblast formation
[J].
Reduced bone density in patients with autosomal dominant hyper-IgE syndrome
[J].
Background and Purpose Autosomal dominant hyper-IgE syndrome (AD-HIES) is a rare primary immunodeficiency disorder. It has been recognized as a multisystem disorder and is characterized by both immunologic and non-immunologic manifestations. Possible bone involvement in autosomal dominant HIES include fractures, scoliosis, cystic bone changes, and osteopenia. We sought to evaluate the changes in bone density in adolescents and young adults with AD-HIES, mostly with proven STAT3 mutation, followed in our institute.
Methods We studied eight patients with AD-HIES who attended our immunology clinic. All patients underwent at least one bone mass dual-energy x-ray absorptiometry assessment (dual-energy x-ray absorptiometry scan). These findings were evaluated.
Results The age of the patients at the time of their first bone density scan ranged between 10 and 24 years (mean 16.1 +/- 4.0 years); the duration of follow-up was 4-11 years (mean 5.8 +/- 3.5 years). Four patients had a history of fractures. Mean Z score in these patients was -1.8 +/- 0.7. For three patients, Z score was below -1. The other four patients had no history of fractures. Mean Z score in these patients was -0.9 +/- 0.5. Only one patient in this group had a Z score below -1. Bone density was below average in all patients; mean spinal Z score was -1.6 +/- 0.4. Four patients were followed through the second decade, and all showed progressive deterioration in bone density. Three were treated with alendronate sodium, with improvement in the bone scan results.
Conclusions Bone density decreases considerably over time in adolescents and young adults suffering from AD-HIES. Treatment with alendronate sodium may be effective in alleviating osteopenia.
IL-1β and TNFα-initiated IL-6-STAT3 pathway is critical in mediating inflammatory cytokines and RANKL expression in inflammatory arthritis
[J].Rheumatoid arthritis (RA) is a chronic inflammatory disease that causes irreversible joint damage and significant disability. However, the fundamental mechanisms underlying how inflammation and joint destruction in RA develop and are sustained chronically remain largely unknown. Here, we show that signal transducer and activator of transcription 3 (STAT3) is the key mediator of both chronic inflammation and joint destruction in RA. We found that inflammatory cytokines highly expressed in RA patients, such as IL-1 beta, tumor necrosis factor alpha and IL-6, activated STAT3 either directly or indirectly and in turn induced expression of IL-6 family cytokines, further activating STAT3 in murine osteoblastic and fibroblastic cells. STAT3 activation also induced expression of receptor activator of nuclear factor kappa B ligand (RANKL), a cytokine essential for osteoclastogenesis, and STAT3 deficiency or pharmacological inhibition promoted significant reduction in expression of both IL-6 family cytokines and RANKL in vitro. STAT3 inhibition was also effective in treating an RA model, collagen-induced arthritis, in vivo through significant reduction in expression of IL-6 family cytokines and RANKL, inhibiting both inflammation and joint destruction. Leukemia inhibitory factor expression and STAT3 activation by IL-1 beta were mainly promoted by IL-6 but still induced in IL-6-deficient cells. Thus, our data provide new insight into RA pathogenesis and provide evidence that inflammatory cytokines trigger a cytokine amplification loop via IL-6-STAT3 that promotes sustained inflammation and joint destruction.
Osteoclastogenesis and osteogenesis during tooth movement
[J].
