寡核苷酸（ODN）促成骨细胞增殖活化的作用及机制研究
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摘要
正畸过程中的牙齿移动与牙槽骨组织的改建关系密切,牙槽骨改建包括骨吸收和骨重建两方面。生理条件下,牙槽骨组织的再生重建是一个由生长因子诱导骨髓间充质干细胞或牙周膜干细胞向成骨细胞分化,而后成骨细胞成熟活化,合成骨基质并最终矿化成骨的复杂生物学过程。在牙槽骨组织再生重建的过程中,体内外各种因素均可影响成骨细胞的增殖和成熟活化。为满足医学临床的应用需求,寻找安全稳定、高效低毒、成本低廉的新型药剂,以促进成骨细胞的增殖和成熟活化进而调控牙槽骨再生的过程正越来越成为口腔医学研究领域的热点。
寡核苷酸(oligodeoxynucleotides, ODN)是指含有50个以下核苷酸单体的多核苷酸链。以往的研究表明,ODN可通过受体介导的形式被细胞摄取,内吞进入胞内发挥作用；经磷酸硫代化修饰可增强ODN被细胞摄取与抵抗体内核酸酶消化的能力,增加胞内摄取率并有利于其在体内的稳定。此外,ODN可通过TLR9介导的分子通路调控成骨-破骨平衡,提示ODN可能对成骨细胞分化过程有重要作用。本研究组在前期的研究中已从人工合成的12条不同序列的ODN中成功筛选出2条对骨髓间充质干细胞的增殖及向成骨分化均有作用的ODN。前期研究初步证实特定序列的ODN可以促骨髓间充质干细胞向成骨细胞分化,为进一步研究ODN对成骨细胞增殖与成熟活化的影响提供了重要的理论依据并奠定了实验基础。
本研究以前期研究工作为基础,提出了特定序列的ODN能够对成骨细胞增殖和成熟活化发挥调控作用这一科学假设。本研究中所采用的11条ODN均由吉林大学基础医学院分子生物学教研室设计并合成,按其免疫学作用特点可分成三类：(1)具有免疫刺激性的ODN,包括BW001、FC001、BW006、FC002、YW002、FC004和YW001；(2)具有免疫抑制性的ODN,包括SAT05f、MT01和FC003；(3)免疫惰性ODN,比如MS19。本研究通过一系列的体内外生物学实验展开探讨：首先应用体外细胞培养技术获取鼠源性及人源性成骨细胞；采用MTT法和碱性磷酸酶检测等细胞生物学技术,筛选促成骨细胞增殖及活化的寡核苷酸；观察大鼠实验性牙移动模型局部应用特定序列ODN后牙槽骨组织学结构的变化,测量比较实验组和对照组正畸牙移动的距离,采用免疫组织化学染色的方法观察比较骨改建相关蛋白表达的情况：采用流式细胞仪检测特定序列ODN作用于人源性成骨样细胞MG 63后对细胞周期的影响；进一步通过Real Time-PCR、Western blot等分子生物学技术,从转录和翻译水平研究特定序列ODN对体外培养的MG 63细胞骨改建相关因子表达的影响,阐明特定序列的ODN对成骨细胞增殖及成熟活化的影响与作用机制,为ODN用于调控正畸牙移动牙槽骨重建的研究奠定理论基础。
本研究具体内容分五部分实验展开：
实验1不同ODN对大鼠成骨细胞增殖活性的影响
采用Wistar大鼠作为研究对象,原代培养大鼠成骨细胞,茜素红染色鉴定细胞来源。确定细胞最佳铺板浓度后,在实验组细胞中分别加入11条不同序列的ODN,对照组细胞中加入无菌PBS,通过MTT比色法筛选对大鼠成骨细胞增殖具有促进作用的ODN。
实验2不同ODN对大鼠成骨细胞成熟活化的影响
取传至第3代的大鼠成骨细胞,以每孔5×103个细胞铺96孔细胞培养板,选用实验1中提到的11条不同序列的ODN,等体积无菌PBS液作为对照,分别与上述在96孔板中培养的大鼠成骨细胞共培养72 h后用ALP检测试剂盒检测各组ALP表达水平,同时确定ODN影响大鼠成骨细胞成熟活化的最佳工作浓度。
实验3 ODN对实验性大鼠牙移动动物模型骨改建过程的影响
通过体内实验探讨经实验1和实验2筛选出的特定序列的ODN对Wistar大鼠实验性牙移动模型的影响。采用形态学观察评价大鼠牙槽骨的组织形态学变化；测量正畸牙移动的距离；采用免疫组织化学染色方法,检测ODN体内作用对骨改建相关因子RANKL、OPG、Runx-2、Osterix和Collagen-Ⅰ表达水平的影响,初步探讨特定序列的ODN在实验性大鼠牙移动模型牙槽骨组织改建过程中的作用及机制。
实验4不同ODN对人源性成骨样细胞MG 63增殖活化的影响
采用人源性成骨样细胞MG 63作为研究对象,取传代培养至第3代的MG 63细胞,实验组细胞分别与11条不同序列的ODN共培养,对照组中加入等体积无菌PBS液,通过MTT比色法和ALP检测试剂盒筛选对MG 63细胞增殖和成熟活化有影响作用的ODN。
实验5 ODN促人源性成骨样细胞MG 63增殖活化作用的机制研究
应用流式细胞术检测经实验4筛选出的特定序列ODN作用于MG 63细胞后细胞周期的变化情况；采用Realtime-PCR和Western blot技术定量检测特定序列ODN体外刺激MG 63细胞骨改建相关因子Sp7、Runx-2、Collagen-Ⅰ、RANKL和OPG等基因转录和蛋白表达水平的变化。探讨特定序列ODN促进人源性成骨样细胞MG 63增殖与成熟活化的作用机制。
通过上述5部分实验,得出以下结果：
实验111条ODNs作用于大鼠成骨细胞24 h后,编号第2、3、6、9、10条ODN可以促大鼠成骨细胞增殖(P<0.05),作用48 h后,第1、6、9、10条ODN可以促大鼠成骨细胞增殖(P<0.05)；作用72 h后,第1、6、9、10条ODN可以促大鼠成骨细胞增殖(P<0.05)；作用96 h后,第1、2、8、9条ODN可以促大鼠成骨细胞增殖(P<0.05)；其中第9条ODN在整个作用过程中均表现出显著的促增殖作用。
实验2与PBS对照组ALP表达量相比,初步筛选出5条ODN(第2,3,8,9,10条)可不同程度刺激大鼠成骨细胞ALP表达水平的上调(P<0.05)。将初步筛选出的第2,3,8,9,10条ODN,以终浓度4.0、2.0、1.0和0.5 mg.L-1分别与大鼠成骨细胞共培养72 h后,进行ALP活性检测,结果发现：ODN工作浓度为4.0和0.5 mg.L-1时,实验组与对照组无统计学差异(P>0.05),工作浓度为2.0和1.0 mg.L-1时,第9,10条ODN可促进大鼠成骨细胞表达ALP水平显著升高(P<0.05)。
实验3局部注射ODN MT01的实验组大鼠第一磨牙牙移动距离均小于对照组(P<0.05)；HE染色光镜下观察可见实验组压力侧牙槽骨吸收破坏的程度较对照组为轻；免疫组织化学染色法检测ODN MT01体内刺激牙槽骨改建相关因子RANKL、OPG、Runx-2、Osterix和Collagen-Ⅰ的表达变化,结果显示实验组OPG、Runx-2、Osterix和Collagen-Ⅰ的表达水平均较对照组增强,而RANKL的表达则有所减弱。
实验4 11条ODNs作用于MG 63细胞24 h后,编号第2、3、5、9条ODN可以促MG 63细胞增殖(P<0.05)；作用48 h后,第3、5、6、9、10条ODN可以促MG63细胞增殖(P<0.05)；作用72 h后,第3、5、9、10条ODN可以促MG 63细胞增殖(P<0.05)；作用96 h后,第6、8、9条ODN可以促MG 63细胞增殖(P<0.05)。取第3、5、6、9、10条ODN与MG 63细胞共培养,进行ALP活性检测,发现第9条ODN (ODN MT01)可以促MG 63细胞ALP表达水平显著升高(P<0.05)。
实验5采用流式细胞术检测结果显示：与对照组相比,在各个作用时间点(24 h、48 h和72 h)ODN MT01处理组MG 63细胞中处于G1期的细胞比例均明显较空白对照组减少,处于S期、G2期的细胞比例增加；应用实时定量PCR方法,检测ODN MT01作用于MG 63细胞后细胞内成骨相关基因mRNA的表达变化,发现实验组Runx-2、Sp7和collagen-Ⅰ的mRNA表达水平均出现不同程度的增高；应用Western blot技术,检测ODN MT01作用于MG 63细胞后细胞内骨改建相关因子蛋白水平的表达变化,发现与对照组相比,实验组Runx-2、Sp7、collagen-Ⅰ和OPG的表达均出现不同程度的增高,而RANKL的表达出现了明显的降低。
通过以上实验结果,可以得出以下结论：
结论1所选11条ODNs中有3条特定序列的ODN,第6、9、10条(ODN BW006, ODN MT01, ODN YW001),可以促进体外培养的大鼠成骨细胞的增殖；其中第9条ODN (ODN MTO1)在整个作用过程中均表现出显著的促增殖作用。
结论2工作浓度为2.0和1.0 mg.L-1时,第9,10条ODN (ODN MT01, ODNYW001)可促进体外培养的大鼠成骨细胞的成熟活化。
结论3ODN MT01能够在体内调控大鼠实验性牙移动模型牙槽骨的改建,抑制大鼠实验性牙移动,有效保护牙槽骨结构的完整性,防止破坏性骨吸收的发生。
结论4ODN MT01对体外培养的人源性成骨样细胞MG 63同样具有显著的促增殖及促成熟活化的作用。
结论5ODN MT01能够影响MG 63细胞的细胞周期,通过促使细胞进入S期使MG 63细胞的增殖活性增强；ODN MT01可能通过增加MG 63细胞中Runx-2、Sp7和Collagen-Ⅰ等成骨相关因子的表达水平进而促进MG 63细胞的增殖和成熟活化；ODNMT01可能通过上调OPG同时抑制RANKL的表达水平,增加OPG/RANKL的比值,经OPG-RANK-RANKL信号通路调控了成骨细胞的增殖与成熟活化的过程。
Orthodontic tooth movement is closely related to the remodeling of alveolar bone. In physiological conditions, bone remodeling process is controlled by a complex network of endocrine hormones and local cytokines. Generally, the regeneration of alveolar bone processes are as follows:growth factor-inducing bone marrow mesenchymal stem cells or periodontal ligament stem cells differentiate into osteoblast, which then synthesizes the mineralized bone matrix, and ultimately induces the formation of bone in a complex biological route. In the process of alveolar bone repair and regeneration, the proliferation and differentiation of osteoblast are influenced by various factors in vivo and in vitro. In order to satisfy the application requirement of clinical medicine and look for safety and steady, high-efficiency, low toxic and low cost new medicament, the regulation of the alveolar bone repair is increasingly becoming a hot spot in oral medicine research field. Since osteoblast is directly associated with the synthesis of mineralized bone matrix, the proliferation and activation of osteoblast using growth factors and other agents will be an attractive issue in the alveolar bone repair and regeneration.
ODN (oligodeoxynucleotides, ODN) refers to the multi-nucleotide chains containing 50 or less nucleotide monomers. ODN is absorbed into cells through the form of receptor-mediated, and then perform a function in cells. Thio-modified phosphate could enhance the capability of ingesting ODN and resisting the digestion of nuclease, which is good for keeping stable in vivo and increasing the intracellular uptake rate. Previous studies have shown that ODN can use the classical mode of action to regulate the balance of bone formation and resorption, which demonstrated that ODN could play an important role in the process of osteoblast differentiation. In the previous study of our group,12 different sequences ODN were screened in and 2 ODNs could affect the bone marrow mesenchymal stem cell proliferation and osteogenic differentiation. Early preliminary results suggested that specific ODN sequences could promote bone marrow mesenchymal stem cell to differentiate into osteoblast, which provided a theoretical basis and experimental basis for further studies of ODNs in the regeneration of alveolar bone tissue.
In this study, based on preliminary studies, we presented a scientific hypothesis that a specific sequence of ODN plays a regulatory role in the proliferation, maturation and activation of osteoblasts. The research was performed as follows:obtaining rat osteoblast cells and human osteoblast cells by cell culture in vitro; screening the ODNs with the ability to promote osteoblastic proliferation and activation by by methyl thiazolyl tetrazolium (MTT) and alkaline phosphatase (ALP) assays; investigating the effect of ODNs on the expression level of Sp7, runx-2, collagen-1 and RANKL at the transcription and translation levels by real time PCR and Western blotting analysis; observing the changes of histological structure of alveolar bone, bone mass and osteoclast count; elucidating the effect and mechanism of the sequence-specific oligonucleotides to the proliferation and activation of the osteoblast cells, which supplied the theoretical foundation for the research of regulation of ODN in the process of remodeling of periodontal tissue of the orthodontic tooth movement. In this study, the 11 ODNs were designed and synthesized by College of Molecular Biology in Jilin University. According to the characteristics of immune function, these ODNs were divided into three types:(1) immunostimulant, including BW001, FC001, BW006, FC002, YW001, FC004 and YW002; (2) immunosuppressant, including SAT05f, MT01 and FC003; (3) immunologic inertia, such as MS 19.
The content of this research is divided into five parts:
Experiment 1:The influence of various ODNs on rats osteoblast proliferation activity.
Wistar rats were used as research subjects. Rat osteoblasts were cultured and identified by alizarin red staining. After determining the best concentration,11 different sequences of ODNs were added to the cells in the experimental groups, meanwhile PBS was added to the control groups. The ODNs, which had the effect on proliferation of osteoblastic cells, were selected by MTT colorimetry.
Experiment2:The influence of various ODNs on rats osteoblast muturation activity.
The third generation of the rat osteoblast cells were used, and cultivated in 96-well plates with 5 x 103 cells per well. The 11 ODNs mentioned in Experiment 1 were co-cultivated respectively with the rat osteoblast cells in 96-well plates for 72 hours, with equal volume of PBS solution as control. After that, the expression level of ALP of each group was detected by ALP assay kit. And then the best concentration of the effect on rats osteoblast muturation activity was determined.
Experiment 3:The influence of ODN on bone remodeling of rat tooth movement model.
The influence of ODN on periodontal tissue of tooth movement model of wistar rats was discussed through studies in vivo. The changes of alveolar bone in rats were evaluated morphologically. Then the distance of orthodontic tooth movement was measured. Meanwhile, the expression of osteoblasts/osteoclast-related factors (RANKL, OPG, Runx-2, Osterix and Collagen-Ⅰ) under the stimulation of ODN was detected by immunohistochemical staining. Furthermore, the mechanism for the effect of ODN on periodontal remodeling of experimental rat tooth movement model was explored.
Experiment 4:The influence of ODN on proliferation and activation of human osteoblast-like MG 63 cells.
The human osteoblast-like cell line MG 63 cells were used as research objects, and the three generations of MG 63 cells were taken. The 11 ODNs were co-cultivated respectively with the cells of experimental groups. Meanwhile, equal volume of PBS solution was added to the control group. The ODNs, which had the effect on proliferation and activation of human osteoblast-like MG 63 cells, were selected by MTT colorimetry and ALP assay kit.
Experiment 5:The study of mechanism for promotion of ODN on proliferation and activation of bone cells.
The changes of cell cycle of MG 63 cells, which was induced by ODN MT01, were detected by flow cytometry. Then the difference of transcription and protein expression level of related factors (Sp7, Runx-2, Collagen-Ⅰ, RANKL and OPG) of MG 63 cells, which were induced by ODN in vitro, was tested quantitatively by Real-time PCR and Western blot respectively at the transcription and translation levels. At last, the mechanism for promotion of ODN on proliferation and activation of bone cells was discussed.
Through the above 5 experiments, the results were as follows:
Experiment 1
After 24 h, NO.2,3,6,9 and 10 ODNs could promote the proliferation of osteoblasts (P <0.05). After 48 h, NO.1,6,9 and 10 ODNs could promote the proliferation of osteoblasts (P <0.05). After 72 h, NO.1,6,9 and 10 ODNs could promote the proliferation of osteoblasts (P O.05). After 96 h, NO.1,2,8 and 9 ODNs could promote the proliferation of osteoblasts (P <0.05), of which the effect of NO.9 ODN (ODN MT01) in the whole process had a significant difference (P<0.01).
Experiment 2
Compared to the PBS vehicle control group expression, initially selected five ODN (2, 3,8,9 and 10) could increase the expression of osteoblast ALP at different degrees (P<0.05). These ODNs were co-cultivated with rat osteoblasts for 72h as the concentration of 4.0,2.0, 1.0 and 0.5 mg.L-1 respectively, and then the ALP activity was detected. The results showed that the experimental groups and control group had no significant difference (P> 0.05) with the concentration of 4.0 and 0.5 mg.L-1, and NO.9,10 ODNs could promote the expression levels of osteoblast ALP significantly (P<0.05) with the concentration of 2.0 and 1.0 mg.L-1.
Experiment 3
The moving distance of first molar teeth of experimental groups was less than that of the control group (P<0.05); specific sequences ODN could influence the balance of bone formation and resorption during the remodeling of rat alveolar bone, and inhibit the movement of tooth. The expression of osteoblasts/osteoclast-related factors (RANKL, OPG, Runx-2, Osterix and Collagen-Ⅰ) under the stimulation of ODN was detected by immunohistochemical staining. The results showed that the expression of OPG, Runx-2, Osterix and Collagen-Ⅰof experimental groups was increased compared with control group, while the expression of RANKL was weakened.
Experiment 4
After 24 h, NO.2,3,5,9 ODNs could promote the proliferation of MG 63 cell (P <0.05); after 48h, NO.3,5,6,9,10 ODNs could promote the proliferation of MG 63 cell (P <0.05); after 72 h, NO.3,5,9,10 ODNs could promote the proliferation of MG 63 cell (P <0.05); after 96h, NO.6,8,9 ODNs could promote the proliferation of MG 63 cell (P<0.05). NO.3,5,6,9,10 ODNs were selected to co-cultivated with MG 63 cells, and alkaline phosphatase activity was detected. The results displayed that NO.9 ODN could increase the expression of ALP of MG 63 cells significantly (P<0.05)
Experiment 5
The results of flow cytometry assay showed that more cells at S phase were observed in group treated with ODN MT01 than control group. It demonstrated that ODN MT01 might affect the cell cycle of MG 63 cells, and promote the proliferation of MG 63 cells by increasing the proportion of cells at DNA synthesis phase. The mRNA expression levels of osteoblasts-related genes of MG 63 cells treated with ODN was determined by real time quantitative PCR. The results showed that the mRNA expressions of Runx-2, Sp7 and collagen-Ⅰof experimental groups were higher than control group at different degrees. The protein expression level of osteoblasts-related factors of MG 63 cells treated with ODN was determined by western blot. The results displayed that compared with control group, the expression of Runx-2, Sp7, collagen-Ⅰand OPG were increased at different degrees, while the expression of RANKL had a marked decrease. This suggested that a specific sequence ODN might increase OPG/RANKL ratio by upregulating the expression of OPG and downregulating the expression of RANKL at the same time, and promote the maturation and activation of osteoblasts through the OPG-RANK-RANKL pathway.
According to the results above, we can draw the following conclusions:a specific sequence of the ODN (ODN MT01) could promote the proliferation, maturation and activation of osteoblast in vitro, and be able to regulate the remodeling of alveolar bone of rat tooth movement model in vivo. This effect could be achieved through the regulation of OPG-RANK-RANKL pathway, which increased the expression of OPG and inhibited the expression of RANKL.
寡核苷酸(oligodeoxynucleotides, ODN)是指含有50个以下核苷酸单体的多核苷酸链。以往的研究表明,ODN可通过受体介导的形式被细胞摄取,内吞进入胞内发挥作用；经磷酸硫代化修饰可增强ODN被细胞摄取与抵抗体内核酸酶消化的能力,增加胞内摄取率并有利于其在体内的稳定。此外,ODN可通过TLR9介导的分子通路调控成骨-破骨平衡,提示ODN可能对成骨细胞分化过程有重要作用。本研究组在前期的研究中已从人工合成的12条不同序列的ODN中成功筛选出2条对骨髓间充质干细胞的增殖及向成骨分化均有作用的ODN。前期研究初步证实特定序列的ODN可以促骨髓间充质干细胞向成骨细胞分化,为进一步研究ODN对成骨细胞增殖与成熟活化的影响提供了重要的理论依据并奠定了实验基础。
本研究以前期研究工作为基础,提出了特定序列的ODN能够对成骨细胞增殖和成熟活化发挥调控作用这一科学假设。本研究中所采用的11条ODN均由吉林大学基础医学院分子生物学教研室设计并合成,按其免疫学作用特点可分成三类：(1)具有免疫刺激性的ODN,包括BW001、FC001、BW006、FC002、YW002、FC004和YW001；(2)具有免疫抑制性的ODN,包括SAT05f、MT01和FC003；(3)免疫惰性ODN,比如MS19。本研究通过一系列的体内外生物学实验展开探讨：首先应用体外细胞培养技术获取鼠源性及人源性成骨细胞；采用MTT法和碱性磷酸酶检测等细胞生物学技术,筛选促成骨细胞增殖及活化的寡核苷酸；观察大鼠实验性牙移动模型局部应用特定序列ODN后牙槽骨组织学结构的变化,测量比较实验组和对照组正畸牙移动的距离,采用免疫组织化学染色的方法观察比较骨改建相关蛋白表达的情况：采用流式细胞仪检测特定序列ODN作用于人源性成骨样细胞MG 63后对细胞周期的影响；进一步通过Real Time-PCR、Western blot等分子生物学技术,从转录和翻译水平研究特定序列ODN对体外培养的MG 63细胞骨改建相关因子表达的影响,阐明特定序列的ODN对成骨细胞增殖及成熟活化的影响与作用机制,为ODN用于调控正畸牙移动牙槽骨重建的研究奠定理论基础。
本研究具体内容分五部分实验展开：
实验1不同ODN对大鼠成骨细胞增殖活性的影响
采用Wistar大鼠作为研究对象,原代培养大鼠成骨细胞,茜素红染色鉴定细胞来源。确定细胞最佳铺板浓度后,在实验组细胞中分别加入11条不同序列的ODN,对照组细胞中加入无菌PBS,通过MTT比色法筛选对大鼠成骨细胞增殖具有促进作用的ODN。
实验2不同ODN对大鼠成骨细胞成熟活化的影响
取传至第3代的大鼠成骨细胞,以每孔5×103个细胞铺96孔细胞培养板,选用实验1中提到的11条不同序列的ODN,等体积无菌PBS液作为对照,分别与上述在96孔板中培养的大鼠成骨细胞共培养72 h后用ALP检测试剂盒检测各组ALP表达水平,同时确定ODN影响大鼠成骨细胞成熟活化的最佳工作浓度。
实验3 ODN对实验性大鼠牙移动动物模型骨改建过程的影响
通过体内实验探讨经实验1和实验2筛选出的特定序列的ODN对Wistar大鼠实验性牙移动模型的影响。采用形态学观察评价大鼠牙槽骨的组织形态学变化；测量正畸牙移动的距离；采用免疫组织化学染色方法,检测ODN体内作用对骨改建相关因子RANKL、OPG、Runx-2、Osterix和Collagen-Ⅰ表达水平的影响,初步探讨特定序列的ODN在实验性大鼠牙移动模型牙槽骨组织改建过程中的作用及机制。
实验4不同ODN对人源性成骨样细胞MG 63增殖活化的影响
采用人源性成骨样细胞MG 63作为研究对象,取传代培养至第3代的MG 63细胞,实验组细胞分别与11条不同序列的ODN共培养,对照组中加入等体积无菌PBS液,通过MTT比色法和ALP检测试剂盒筛选对MG 63细胞增殖和成熟活化有影响作用的ODN。
实验5 ODN促人源性成骨样细胞MG 63增殖活化作用的机制研究
应用流式细胞术检测经实验4筛选出的特定序列ODN作用于MG 63细胞后细胞周期的变化情况；采用Realtime-PCR和Western blot技术定量检测特定序列ODN体外刺激MG 63细胞骨改建相关因子Sp7、Runx-2、Collagen-Ⅰ、RANKL和OPG等基因转录和蛋白表达水平的变化。探讨特定序列ODN促进人源性成骨样细胞MG 63增殖与成熟活化的作用机制。
通过上述5部分实验,得出以下结果：
实验111条ODNs作用于大鼠成骨细胞24 h后,编号第2、3、6、9、10条ODN可以促大鼠成骨细胞增殖(P<0.05),作用48 h后,第1、6、9、10条ODN可以促大鼠成骨细胞增殖(P<0.05)；作用72 h后,第1、6、9、10条ODN可以促大鼠成骨细胞增殖(P<0.05)；作用96 h后,第1、2、8、9条ODN可以促大鼠成骨细胞增殖(P<0.05)；其中第9条ODN在整个作用过程中均表现出显著的促增殖作用。
实验2与PBS对照组ALP表达量相比,初步筛选出5条ODN(第2,3,8,9,10条)可不同程度刺激大鼠成骨细胞ALP表达水平的上调(P<0.05)。将初步筛选出的第2,3,8,9,10条ODN,以终浓度4.0、2.0、1.0和0.5 mg.L-1分别与大鼠成骨细胞共培养72 h后,进行ALP活性检测,结果发现：ODN工作浓度为4.0和0.5 mg.L-1时,实验组与对照组无统计学差异(P>0.05),工作浓度为2.0和1.0 mg.L-1时,第9,10条ODN可促进大鼠成骨细胞表达ALP水平显著升高(P<0.05)。
实验3局部注射ODN MT01的实验组大鼠第一磨牙牙移动距离均小于对照组(P<0.05)；HE染色光镜下观察可见实验组压力侧牙槽骨吸收破坏的程度较对照组为轻；免疫组织化学染色法检测ODN MT01体内刺激牙槽骨改建相关因子RANKL、OPG、Runx-2、Osterix和Collagen-Ⅰ的表达变化,结果显示实验组OPG、Runx-2、Osterix和Collagen-Ⅰ的表达水平均较对照组增强,而RANKL的表达则有所减弱。
实验4 11条ODNs作用于MG 63细胞24 h后,编号第2、3、5、9条ODN可以促MG 63细胞增殖(P<0.05)；作用48 h后,第3、5、6、9、10条ODN可以促MG63细胞增殖(P<0.05)；作用72 h后,第3、5、9、10条ODN可以促MG 63细胞增殖(P<0.05)；作用96 h后,第6、8、9条ODN可以促MG 63细胞增殖(P<0.05)。取第3、5、6、9、10条ODN与MG 63细胞共培养,进行ALP活性检测,发现第9条ODN (ODN MT01)可以促MG 63细胞ALP表达水平显著升高(P<0.05)。
实验5采用流式细胞术检测结果显示：与对照组相比,在各个作用时间点(24 h、48 h和72 h)ODN MT01处理组MG 63细胞中处于G1期的细胞比例均明显较空白对照组减少,处于S期、G2期的细胞比例增加；应用实时定量PCR方法,检测ODN MT01作用于MG 63细胞后细胞内成骨相关基因mRNA的表达变化,发现实验组Runx-2、Sp7和collagen-Ⅰ的mRNA表达水平均出现不同程度的增高；应用Western blot技术,检测ODN MT01作用于MG 63细胞后细胞内骨改建相关因子蛋白水平的表达变化,发现与对照组相比,实验组Runx-2、Sp7、collagen-Ⅰ和OPG的表达均出现不同程度的增高,而RANKL的表达出现了明显的降低。
通过以上实验结果,可以得出以下结论：
结论1所选11条ODNs中有3条特定序列的ODN,第6、9、10条(ODN BW006, ODN MT01, ODN YW001),可以促进体外培养的大鼠成骨细胞的增殖；其中第9条ODN (ODN MTO1)在整个作用过程中均表现出显著的促增殖作用。
结论2工作浓度为2.0和1.0 mg.L-1时,第9,10条ODN (ODN MT01, ODNYW001)可促进体外培养的大鼠成骨细胞的成熟活化。
结论3ODN MT01能够在体内调控大鼠实验性牙移动模型牙槽骨的改建,抑制大鼠实验性牙移动,有效保护牙槽骨结构的完整性,防止破坏性骨吸收的发生。
结论4ODN MT01对体外培养的人源性成骨样细胞MG 63同样具有显著的促增殖及促成熟活化的作用。
结论5ODN MT01能够影响MG 63细胞的细胞周期,通过促使细胞进入S期使MG 63细胞的增殖活性增强；ODN MT01可能通过增加MG 63细胞中Runx-2、Sp7和Collagen-Ⅰ等成骨相关因子的表达水平进而促进MG 63细胞的增殖和成熟活化；ODNMT01可能通过上调OPG同时抑制RANKL的表达水平,增加OPG/RANKL的比值,经OPG-RANK-RANKL信号通路调控了成骨细胞的增殖与成熟活化的过程。
Orthodontic tooth movement is closely related to the remodeling of alveolar bone. In physiological conditions, bone remodeling process is controlled by a complex network of endocrine hormones and local cytokines. Generally, the regeneration of alveolar bone processes are as follows:growth factor-inducing bone marrow mesenchymal stem cells or periodontal ligament stem cells differentiate into osteoblast, which then synthesizes the mineralized bone matrix, and ultimately induces the formation of bone in a complex biological route. In the process of alveolar bone repair and regeneration, the proliferation and differentiation of osteoblast are influenced by various factors in vivo and in vitro. In order to satisfy the application requirement of clinical medicine and look for safety and steady, high-efficiency, low toxic and low cost new medicament, the regulation of the alveolar bone repair is increasingly becoming a hot spot in oral medicine research field. Since osteoblast is directly associated with the synthesis of mineralized bone matrix, the proliferation and activation of osteoblast using growth factors and other agents will be an attractive issue in the alveolar bone repair and regeneration.
ODN (oligodeoxynucleotides, ODN) refers to the multi-nucleotide chains containing 50 or less nucleotide monomers. ODN is absorbed into cells through the form of receptor-mediated, and then perform a function in cells. Thio-modified phosphate could enhance the capability of ingesting ODN and resisting the digestion of nuclease, which is good for keeping stable in vivo and increasing the intracellular uptake rate. Previous studies have shown that ODN can use the classical mode of action to regulate the balance of bone formation and resorption, which demonstrated that ODN could play an important role in the process of osteoblast differentiation. In the previous study of our group,12 different sequences ODN were screened in and 2 ODNs could affect the bone marrow mesenchymal stem cell proliferation and osteogenic differentiation. Early preliminary results suggested that specific ODN sequences could promote bone marrow mesenchymal stem cell to differentiate into osteoblast, which provided a theoretical basis and experimental basis for further studies of ODNs in the regeneration of alveolar bone tissue.
In this study, based on preliminary studies, we presented a scientific hypothesis that a specific sequence of ODN plays a regulatory role in the proliferation, maturation and activation of osteoblasts. The research was performed as follows:obtaining rat osteoblast cells and human osteoblast cells by cell culture in vitro; screening the ODNs with the ability to promote osteoblastic proliferation and activation by by methyl thiazolyl tetrazolium (MTT) and alkaline phosphatase (ALP) assays; investigating the effect of ODNs on the expression level of Sp7, runx-2, collagen-1 and RANKL at the transcription and translation levels by real time PCR and Western blotting analysis; observing the changes of histological structure of alveolar bone, bone mass and osteoclast count; elucidating the effect and mechanism of the sequence-specific oligonucleotides to the proliferation and activation of the osteoblast cells, which supplied the theoretical foundation for the research of regulation of ODN in the process of remodeling of periodontal tissue of the orthodontic tooth movement. In this study, the 11 ODNs were designed and synthesized by College of Molecular Biology in Jilin University. According to the characteristics of immune function, these ODNs were divided into three types:(1) immunostimulant, including BW001, FC001, BW006, FC002, YW001, FC004 and YW002; (2) immunosuppressant, including SAT05f, MT01 and FC003; (3) immunologic inertia, such as MS 19.
The content of this research is divided into five parts:
Experiment 1:The influence of various ODNs on rats osteoblast proliferation activity.
Wistar rats were used as research subjects. Rat osteoblasts were cultured and identified by alizarin red staining. After determining the best concentration,11 different sequences of ODNs were added to the cells in the experimental groups, meanwhile PBS was added to the control groups. The ODNs, which had the effect on proliferation of osteoblastic cells, were selected by MTT colorimetry.
Experiment2:The influence of various ODNs on rats osteoblast muturation activity.
The third generation of the rat osteoblast cells were used, and cultivated in 96-well plates with 5 x 103 cells per well. The 11 ODNs mentioned in Experiment 1 were co-cultivated respectively with the rat osteoblast cells in 96-well plates for 72 hours, with equal volume of PBS solution as control. After that, the expression level of ALP of each group was detected by ALP assay kit. And then the best concentration of the effect on rats osteoblast muturation activity was determined.
Experiment 3:The influence of ODN on bone remodeling of rat tooth movement model.
The influence of ODN on periodontal tissue of tooth movement model of wistar rats was discussed through studies in vivo. The changes of alveolar bone in rats were evaluated morphologically. Then the distance of orthodontic tooth movement was measured. Meanwhile, the expression of osteoblasts/osteoclast-related factors (RANKL, OPG, Runx-2, Osterix and Collagen-Ⅰ) under the stimulation of ODN was detected by immunohistochemical staining. Furthermore, the mechanism for the effect of ODN on periodontal remodeling of experimental rat tooth movement model was explored.
Experiment 4:The influence of ODN on proliferation and activation of human osteoblast-like MG 63 cells.
The human osteoblast-like cell line MG 63 cells were used as research objects, and the three generations of MG 63 cells were taken. The 11 ODNs were co-cultivated respectively with the cells of experimental groups. Meanwhile, equal volume of PBS solution was added to the control group. The ODNs, which had the effect on proliferation and activation of human osteoblast-like MG 63 cells, were selected by MTT colorimetry and ALP assay kit.
Experiment 5:The study of mechanism for promotion of ODN on proliferation and activation of bone cells.
The changes of cell cycle of MG 63 cells, which was induced by ODN MT01, were detected by flow cytometry. Then the difference of transcription and protein expression level of related factors (Sp7, Runx-2, Collagen-Ⅰ, RANKL and OPG) of MG 63 cells, which were induced by ODN in vitro, was tested quantitatively by Real-time PCR and Western blot respectively at the transcription and translation levels. At last, the mechanism for promotion of ODN on proliferation and activation of bone cells was discussed.
Through the above 5 experiments, the results were as follows:
Experiment 1
After 24 h, NO.2,3,6,9 and 10 ODNs could promote the proliferation of osteoblasts (P <0.05). After 48 h, NO.1,6,9 and 10 ODNs could promote the proliferation of osteoblasts (P <0.05). After 72 h, NO.1,6,9 and 10 ODNs could promote the proliferation of osteoblasts (P O.05). After 96 h, NO.1,2,8 and 9 ODNs could promote the proliferation of osteoblasts (P <0.05), of which the effect of NO.9 ODN (ODN MT01) in the whole process had a significant difference (P<0.01).
Experiment 2
Compared to the PBS vehicle control group expression, initially selected five ODN (2, 3,8,9 and 10) could increase the expression of osteoblast ALP at different degrees (P<0.05). These ODNs were co-cultivated with rat osteoblasts for 72h as the concentration of 4.0,2.0, 1.0 and 0.5 mg.L-1 respectively, and then the ALP activity was detected. The results showed that the experimental groups and control group had no significant difference (P> 0.05) with the concentration of 4.0 and 0.5 mg.L-1, and NO.9,10 ODNs could promote the expression levels of osteoblast ALP significantly (P<0.05) with the concentration of 2.0 and 1.0 mg.L-1.
Experiment 3
The moving distance of first molar teeth of experimental groups was less than that of the control group (P<0.05); specific sequences ODN could influence the balance of bone formation and resorption during the remodeling of rat alveolar bone, and inhibit the movement of tooth. The expression of osteoblasts/osteoclast-related factors (RANKL, OPG, Runx-2, Osterix and Collagen-Ⅰ) under the stimulation of ODN was detected by immunohistochemical staining. The results showed that the expression of OPG, Runx-2, Osterix and Collagen-Ⅰof experimental groups was increased compared with control group, while the expression of RANKL was weakened.
Experiment 4
After 24 h, NO.2,3,5,9 ODNs could promote the proliferation of MG 63 cell (P <0.05); after 48h, NO.3,5,6,9,10 ODNs could promote the proliferation of MG 63 cell (P <0.05); after 72 h, NO.3,5,9,10 ODNs could promote the proliferation of MG 63 cell (P <0.05); after 96h, NO.6,8,9 ODNs could promote the proliferation of MG 63 cell (P<0.05). NO.3,5,6,9,10 ODNs were selected to co-cultivated with MG 63 cells, and alkaline phosphatase activity was detected. The results displayed that NO.9 ODN could increase the expression of ALP of MG 63 cells significantly (P<0.05)
Experiment 5
The results of flow cytometry assay showed that more cells at S phase were observed in group treated with ODN MT01 than control group. It demonstrated that ODN MT01 might affect the cell cycle of MG 63 cells, and promote the proliferation of MG 63 cells by increasing the proportion of cells at DNA synthesis phase. The mRNA expression levels of osteoblasts-related genes of MG 63 cells treated with ODN was determined by real time quantitative PCR. The results showed that the mRNA expressions of Runx-2, Sp7 and collagen-Ⅰof experimental groups were higher than control group at different degrees. The protein expression level of osteoblasts-related factors of MG 63 cells treated with ODN was determined by western blot. The results displayed that compared with control group, the expression of Runx-2, Sp7, collagen-Ⅰand OPG were increased at different degrees, while the expression of RANKL had a marked decrease. This suggested that a specific sequence ODN might increase OPG/RANKL ratio by upregulating the expression of OPG and downregulating the expression of RANKL at the same time, and promote the maturation and activation of osteoblasts through the OPG-RANK-RANKL pathway.
According to the results above, we can draw the following conclusions:a specific sequence of the ODN (ODN MT01) could promote the proliferation, maturation and activation of osteoblast in vitro, and be able to regulate the remodeling of alveolar bone of rat tooth movement model in vivo. This effect could be achieved through the regulation of OPG-RANK-RANKL pathway, which increased the expression of OPG and inhibited the expression of RANKL.
引文
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