【作者】 史瑞新；

【导师】 周延民；

【作者基本信息】 吉林大学 ， 口腔临床医学， 2011， 博士

【摘要】 口腔正畸治疗与牙齿所受的矫治力的大小方向密切相关,力施加并作用于牙齿后传递到其他相关结构,引起各部位的骨改建,包括牙槽骨、颌骨甚至颞下颌关节区域,从而促使牙齿移动,达到矫治目的。成骨细胞是骨的形成及重建过程的主要的效应细胞,同时成骨细胞在破骨细胞的分化和成熟的调控中起到重要作用,尤其是位于牵张侧的成骨细胞更是研究的关键点,因此有必要进一步深入研究成骨细胞在张力作用下的生物学应答及力学传导机制。目前,较多的研究是针对成骨细胞的后期生物力学应答,其中包括多种基因、第二信使系统以及各种因子在张力作用下发生的改变及规律,但各种结论并不完全一致,有的甚至完全相反,分析其中的原因有多种,可能是力学装置不同,也可能是力值大小的差异等等。究其根本原因,在于对牵张力的力学信号转导机制的具体过程研究仍不详尽,未能发现根本机制机理。低能量激光（Low Level Laser,LLL）,通常也被称做弱激光或者软激光等,工作波长在600～950 nm之间,输出功率通常小于250 mW,其特点是单色性好。激光有多种效应,不仅有简单的热效应,还具有生物化学效应,照射后产生生物刺激效应,与超声波、针灸等物理因子所获得的效应有相似之处,现也被称为激光的光生物调节作用,将其应用到临床,称之为低能量激光治疗（Low-Level-Laser-Therapy,LLLT）。在医学领域,LLLT的光生物调节效应在很多学科占有重要的地位。LLLT对骨重建及形成有积极作用,这一观点已有大量体外和体内的研究支持和证实,但也有认为LLLT对成骨细胞及相应的骨组织修复没有作用的报道,不过这样的负面结果报道很少,LLLT还不被人们普遍接受,有待进一步的理论及实验支持。许多国外的研究报道低能量激光可活化成骨细胞,并促进骨组织愈合及重建。在口腔正畸领域,已将弱激光应用于临床来减少矫治中的疼痛,有文献报道LLLT有加速牙齿移动的作用,但LLLT加速牙齿移动的机制仍不清楚,LLLT加速牙齿移动,应该是通过促进张力侧的骨形成和压力侧的骨吸收来完成的,其中对成骨细胞的作用是关键之一,那LLLT对受张力的成骨细胞到底有什么作用,其机制又是什么呢?他们的叠加不应该是简单的‘1+1=2’的扩大效应,应该有更深层次的对力学作用机制的调节作用。本实验利用四点弯曲细胞加力装置,对成骨样细胞MG-63施加牵张力,同时再施加弱激光照射,观察与成骨细胞功能及力的传导密切相关的几个指标,进一步明确张力与骨重建之间的关系,以及LLLT促进受张力的成骨细胞加速骨重建的机制,为LLLT加速正畸牙齿移动的临床应用及机制研究提供依据和理论基础。1.低能量激光治疗对受张力的MG-63细胞生物学特性的影响目的:采用四点弯曲细胞加力装置,对成骨样细胞MG-63细胞施加牵张力,同时再施加弱激光照射（LLLT）,评价LLLT对受力的MG-63细胞的生物学特性的影响。方法:人成骨样细胞MG-63细胞接种于特制的加力板上,置于含10%胎牛血清的高糖DMEM的培养皿中。随机设置3组:对照组（组Ⅰ）、加力组（组Ⅱ）、激光加力组（组Ⅲ）。加力组力值为3000μstrain,频率0.5 Hz,加力时间1h,激光加力组在加力1h后激光照射1min,波长808 nm,能量密度为3 J·cm- 2。3组细胞均在12h后收集,用流式细胞术检测细胞周期和细胞凋亡率,用分光光度计测量细胞内碱性磷酸酶（alkalinephosphatase, ALP）活性。结果:①采用四点弯曲体外细胞力学加载装置,它可对成骨样细胞施加张应力和压应力,本实验主要研究了张应力。生理水平张应力（3000μstrain）加载前后细胞生长状态良好,但是加载过大的应力（8000μstrain）不利于细胞贴附生长,细胞易脱落甚至引起细胞死亡。②MG-63细胞加载张应力（ 3000μstrain,0.5Hz）后,细胞增殖指数及细胞周期的各期细胞所占百分比发生变化。张应力促使更多的MG-63细胞进入增殖的状态S期,细胞增殖指数有所增加,而LLLT使成骨细胞进入S期的同时,又使更多的细胞暂时停留在G2/M期,增殖指数进一步提高,促进成骨细胞增殖。③张应力使成骨细胞凋亡率降低,同时施加LLLT后凋亡率进一步降低,细胞生长到一定阶段,或增殖或凋亡,牵张力以及LLLT都是使更多的细胞在“岔口”进入增殖的途径,使更少的细胞进入凋亡的途径,促进成骨细胞增殖上调,为骨形成准备了充足的细胞资源。④2个实验组ALP活性值与对照组相比,显著增加（P<0.01）,组Ⅲ与组Ⅱ之间相比,组Ⅲ的细胞ALP活性值增高,且有显著性差异（P<0.05）,说明在受到牵张应力的成骨细胞受LLLT照射时,牵张力对细胞ALP活性的有促进作用,LLLT在此基础上进一步促进细胞ALP活性增高,从而促进骨基质钙化,达到促进成骨的目的。2.低能量激光治疗对受张应力的MG-63细胞外基质蛋白的影响目的:观察LLLT对受张应力的MG-63细胞分泌OPN、coll I的mRNA的表达量和蛋白表达量的变化规律的影响,探讨LLLT对受张应力的MG-63细胞的ECM基因和蛋白的影响。方法:人成骨样细胞MG-63细胞接种于加力板。实验随机分为2组:张力组和激光张力组,力值和激光参数同实验1。张力组细胞加力时间分别为0 h,l h,3 h,6 h,继续培养48 h后收取细胞。激光张力组细胞分别加力0 h,l h,3 h,6 h后,再LLLT照射1min,继续培养48h后收取细胞。分别用半定量逆转录聚合酶链式反应（reverse transcriptase polymerase chain reaction,RT-PCR）和Western Blot的方法检测骨桥蛋白（osteopontin,OPN）、I型胶原（collagen I,coll I）的mRNA的表达量和蛋白表达量,绘制表达量随加力时间的变化图。结果:张力刺激早期可以引起MG-63细胞的I型胶原和OPN的基因和蛋白水平表达的变化,且随加力时间的增加,呈现不同的增加趋势,说明牵张力对成骨细胞基质分泌、成熟和钙化有促进作用。蛋白增加水平稍滞后基因水平,可能是因为蛋白需要翻译和修饰过程。激光张力组细胞的I型胶原和OPN基因和蛋白水平表达的变化趋势与单纯张力组类似,但整体的基因和蛋白水平要高于单纯张力组（ P < 0.05）,说明LLLT对受张力的成骨细胞基质分泌和成熟有进一步的促进作用。3.低能量激光治疗对受牵张力的MG-63细胞内Ca²⁺浓度的影响目的:观察LLLT对受张力的MG-63细胞内Ca²⁺浓度的变化规律和Ca²⁺阳性细胞百分比的影响,探讨LLLT对受张力的MG-63细胞的影响机制。方法:人成骨样细胞MG-63细胞接种于加力板。实验随机分为2组:张力组和激光张力组,力值和激光参数同实验1。张力细胞分别加力0 min、5 min、15 min、30 min、60 min后,立即收取细胞。激光张力组细胞分别加力0 min、5 min、15 min、30 min、60 min后,再LLLT照射1 min,立即收取细胞。用流式细胞术进行活性成骨样细胞内Ca²⁺浓度和Ca²⁺阳性细胞百分比的检测。结果:①周期性拉伸应变短时间（5min）内可以引起成骨细胞内Ca²⁺浓度增加,随时间加力时间变化规律呈‘波浪形’,此变化早于细胞增殖活性的变化,推测Ca²⁺作为第二信使,可能是张应力早期促进成骨细胞增殖的的信号传导的枢纽。②激光加力组0min（即单纯激光组）与加力组0min（即空白对照组）相比,单纯激光组比对照组,细胞内Ca²⁺浓度和Ca²⁺阳性细胞百分比均有显著差异（P<0.05）,说明成骨细胞对LLLT的响应,也是通过了第二信使Ca²⁺来发挥作用。③张力可以引起成骨细胞内Ca²⁺浓度增加,呈“波浪形”起伏较大,LLLT使此变化曲线变得相对平缓,且整体水平得到提高,同时Ca²⁺阳性细胞百分比也有明显提高,由此推测LLLT极有可能是通过调节胞内Ca²⁺浓度的变化节奏和提高Ca²⁺阳性细胞百分比,这一“信号通路”来达到进一步促进成骨细胞增殖、基质合成及矿化的目的。综上所述,MG-63细胞受张力早期适合剂量的LLLT处理可以其产生正性的作用。如促进增殖和分化,减少凋亡等。周期性拉伸应变短时间（5min）内可以引起成骨细胞内Ca²⁺浓度增加,随时间加力时间变化规律呈‘波浪形’,此变化早于细胞增殖活性的变化,推测Ca²⁺作为第二信使,胞内Ca²⁺浓度的变化可能是张应力早期促进成骨细胞增殖的的信号传导的枢纽。激光张力组胞内Ca²⁺浓度的变化曲线比张力组变的相对平缓,且整体水平得到提高,由此推测LLLT极有可能是通过胞内Ca²⁺浓度的变化这一“信号通路”来达到进一步促进成骨细胞增殖、基质合成及矿化的目的。本研究为临床LLLT应用于加速正畸牙齿移动提供理论支持。更多还原

【Abstract】 Orthodontic treatment is related to the forces acting on the teeth and other structures. Within the dentoalveolar systems the principle changes resulting from the forces are seen,but the forces can also influence other structure,such as temporomnadibular and joint area sutures. Both modeling and remodeling are modulated by the interaction of metabolic and mechanical signals. It is important to study the biological response and mechanism of the osteoblast to the mechanical stretching strain because these changes in mechanical stimulus modulate bone mass and architecture mainly through the osteoblast. There are many studies about the late biological responses of osteoblast to stretching stress , but the results are different. In order to understand the initial responses involved in mechnao-transduction,it is necessary for us to examine the effect of stretching mechanical strain.Low level laser(LLL) is a kind of visible light with the wavelength between 600~950 nm,sometimes it is also called weak laser,soft laser,cold laser, and so on. LLL can offer the output power below 250 mW and is characterized by its monochromatism.Furthermore it not only has heat effect,but also biochemical effects, which could not result in irreversible damage to bio-tissue. It just likes the effect obtained by physical factors such as a puncture and moxibustion,so that is called“biostimulation”, or“photo-biomodulation”. The corresponding therapy is called low-level-laser-therapy (LLLT). There are many areas where LLLT could play an important role on our medical workers. There are many study about the positive effects of LLLT on bone fomation and rebuilding,but there are still several studies confirmed that LLLT has no effect on osteoblast and bone rebuilding.Now, LLLT is still not a widely acceptable therapy, therefore,it is necessary to verify the efficacy to osteoblast of LLLT more experiments.Osteoblastic cells MG-63 were subjected to the mechanical strain by a four-point bending system(ZL01256849.X) at 0.5 Hz. The cells were loaded with tension stress at 3000μstrain and then subjected to LLLT.①The cell cycle, apoptosis of MG-63 were measured by flow cytometry (FCM)and the ALP of MG-63 were measured by spectro photometer.②To investigate the effect of LLLT on mRNA and protein expression of col I and OPN of MG-63 undering tension stimulus in vitro and to detect the effect of LLLT on ECMP and the mechanism of LLLT.③To investigate the changes of intracelluler calcium concentration when osteoblasts undering stretching responded to LLLT.Result:1. The Four-point Bending System can exert a physiologic magnitude tension strain on osteoblastic cells MG-63 in vitro. Before and after physiological stresses,the osteoblast adhesion and growth status are very well, But after loading over physiological level,the cells fell off and even go to death.2. Compared to a control group, stretching strain groups present less G1-stage cells ,more S-stage cells and higher proliferation index (PI,PI=S+G2/ G1+S+G2) (P<0.05). Laser-strain groups present more G2-stage cells than the other two groups(P<0.05). Compared to stretching strain group, the apoptosis rates in laser-strain groups are lower (P<0.05). The expression of ALP in laser-strain groups and stretching strain group are higher than that in control group(P<0.01).and the The expression of ALP in laser-strain groups is higer than that in stretching strain group (P< 0.05).3. With RT-PCR method and western blot ,the expression of collagen I and OPN in laser-strain group and stretching strain group are higher than that in control group(P<0.05).And the expression of collagen I and OPN in laser-strain group are higher than that in stretching strain group(P<0.05).4. Concentration of the intracellular calcium ion in laser-strain groups are higher than that in strain group(P < 0.05) . Concentration of the intracellular calcium ion in laser-strain groups increase gently in stead of‘jump’in stretching strain group.In conclusion,LLLT may have a positive effect on MG-63 undering stretching strain in vitro, for instance, promoting proliferation and differentiation and expressing col I and OPN. The intracelluler calcium plays an important role in cell mechaotransduction and responses to LLLT . LLLT could regulate the rhythm of increasing concentration of the intracellular calcium ion and make it gently.These results obtained by this paper can provide a theory supporting for clinical applications of LLLT.更多还原