摘要
牙周疾病是以牙周支持组织破坏为特征,最终导致牙齿丧失的严重危害口腔健康的疾病,牙周疾病造成的牙周附着丧失和牙槽骨缺损是临床急需解决的重要问题。针对宿主免疫反应在牙周组织破坏中的作用,结合牙周组织修复再生的调控机制,基于寡核苷酸(ODN)在抗感染免疫及其调控破骨细胞分化的作用机制,本课题通过体内和体外实验,研究ODN对骨髓间充质干细胞(BMSCs)向成骨细胞分化的影响,并将其应用于实验性大鼠牙周炎动物模型,探讨ODN对牙周骨组织改建的影响和作用机制。
首先,选用吉林大学基础医学院分子生物学教研室设计的12条ODN,通过大量体外筛选实验观察ODN对BMSCs生物学特性的影响,从人工合成的12条不同序列的寡核苷酸中,筛选出2条对骨髓间充质干细胞增殖及成骨分化等生物学特性均有影响作用的ODN。
根据筛选结果,选取可促进BMSCs向成骨分化的ODN,进一步通过实时定量PCR等分子生物学手段研究其成骨分化机制,结果发现:特定序列的ODN可显著上调经成骨诱导的骨髓间充质干细胞内RANKL,OPG,Runx2和Osterix等成骨相关因子mRNA的表达。
在体外研究的基础上,将ODN应用于大鼠牙周炎动物模型,采用实验动物学、组织病理学、免疫组织化学以及影像学技术,体内研究ODN对实验性动物牙周炎牙槽骨吸收量、组织病理学改变以及RANKL、OPG、Osterix、Runx2和Ⅰ型胶原等蛋白表达水平的变化,结果显示:特定序列的ODN对牙周骨组织改建具有一定的影响作用。
本实验将免疫学领域的研究热点ODN引入口腔牙周骨组织改建,经大量反复的筛选实验,开发出对骨髓间充质干细胞向成骨细胞分化具有调节作用的ODN,采用实时定量PCR等分子生物学手段对其作用机制进行了初步探讨;首次体内研究ODN对牙周骨组织改建的影响,为牙周组织再生性修复提供新的治疗途径并奠定实验基础。
Periodontal disease is one of the two main types of oral diseases. The late 20th century, more and more clinical and research data indicate that periodontal disease has a two-way close relationship with the systemic health and systemic disease. The defect of the periodontal tissue is the most common signs of periodontal disease which is caused by various kinds of reasons.
Regeneration of periodontal tissue defects has been the nodu and hot spot in clinical and basic research. Finding effective biological or chemical treatment method to promote the regeneration of periodontal tissue defects, can effectively reduce the periodontal tissue defects due to periodontal disease and some systemic diseases like the cardiovascular which is caused by periodontal disease.
The main manifestations of periodontal disease include the destruction and reconstruction of periodontal soft and hard tissue. The rebuilding of periodontal tissue assumes cyclicity, including the cycle of destruction and reconstruction. Both of the two modality determine the character and processes of the disease. The regeneration of bone occurrs in the healing of alveolar bone, its mechanism is that the immune system could prevent the formation of osteoclasts and increase the death of osteoclasts, by this way the activity of osteoclasts would been significantly reduced. The other mechanism is activating osteoblasts to induce reproducibility bone healing. Understanding the healing process will help us to develop the healing of periodontal tissue into the direction of regeneration during periodontal treatment.
The capacity of periodontal tissue regeneration is limited. In recent years, a considerable number of studies dedicated to the development of technology and materials which can increase the capacity of periodontal tissue regeneration. The treatment of periodontal tissue defects caused by periodontal disease includes that the application of guided tissue regeneration , tissue engineering methods to obtain new attachment of pericementum and repairing the natural anatomy of periodontal tissue.
At present, many studies focus on the activation of osteoblasts, periodontal ligament cells and bone marrow-derived mesenchymal stem cells, as a way of promoting regeneration healing. Because the extraction and culture of osteoblasts and periodontal ligament cells are difficult, more researches select the bone marrow-derived mesenchymal stem cells. The bone marrow derived mesenchymal stem cells has the characteristics of mesenchymal stem cell, shows strong proliferative ability and variety of cell differentiation potential. Under certain inducing conditions, the bone marrow derived mesenchymal stem cells can differentiate to osteoblasts. Bone marrow derived mesenchymal stem cells can be easily abstracted and cultured, it is one of the most ideal seed cells for tissue engineering research.
ODN (oligodeoxynucleotides, ODN) refers to the multi-nucleotide chains containing 50 or less nucleotide monomers. ODN exists in some inferior organism’s genomic DNA, It can simulate the immune stimulating effect of bacterial DNA to activate the various types of immune cells to produce proinflammatory cytokines and other inflammatory chemical mediator, to participate the anti-infection immunity in the body. DNA or RNA degradation products frequently contain ODN. ODN can be absorbed into cells through the form of receptor-mediated without the need of constructing a vector, thio-modified phosphate may enhance their cellular uptake capability and may resist nuclease digestion in vivo, these modifications of ODN are good for keeping stable in vivo and increasing the intracellular uptake rate. In addition, ODN also has the advantages of high efficiency, low toxicity, easy-synthesis and stability. Therefore, ODN becomes the hot spot in basic medical field over the past decade. ODN has been applied to the current pre-clinical studies to evaluate the clinical results of local application to the treatment of mastitis, allergic rhinitis and conjunctivitis. Some researches show that specific ODN sequences can regulate osteoclast differentiation through different signaling pathways, which provide a theoretical basis for the application of ODN to regulate the rebuilding of periodontal bone.
Present studies about ODN focus on the CG-rich motif ODN (CpG ODN). In recent studies showed that CpG ODN could regulate osteoclast formation and differentiation in TLR9 dependent individual through Toll-like receptor 9 (Toll like receptor 9, TLR9)-mediated molecular pathway. CpG ODN can inhibit osteoclast differentiation by upregulating the expression of IL-12 to inhibited RANKL, which suggests the possibility of its use of the inhibition to prevent the possibility of pathological bone resorption. CpG ODN via TLR9 produces effective TNF-α, which mediated the expression of RANKL reduced in co-culture of bone marrow stromal cells and osteoblasts, which was specificity inhibited by chloroquine. The results suggested that CpG ODN regulated osteoblast-osteoclast balance through its classical mode of action. Jae-Ho Chang’s research shows that the CpG-KSK13 inhibites the activation of osteoclasts via TLR9 is mediated by the down expression of TREM-2. In addition, a specific sequence of CpG ODN can induce dephosphorylation reaction to hinder the need of sustained phosphorylation of extracellular signal kinase (ERK) in order to maintain a high level expression of c-fos, as an essential condition in the procedure of osteoclast differentiation. The above studies have shown that a specific sequence of ODN can be effected osteoclast differentiation and regulation of osteoblast - osteoclast balance through different signaling pathways, which provided a possibility for the further research of ODN.
In view of the above studies, aiming at how to regulate periodontal tissue repair and regeneration in periodontal disease, according to the specific sequence of ODN could regulate the osteoblast-osteoclast balance and the research of bone remodeling and bone marrow-derived mesenchymal stem cell, we propose the hypothesis that the specific sequence of ODN has some influence on the process of bone marrow-derived mesenchymal stem cells differentiate to osteoblasts. Around this assumption, we set the following experiment:
Research 1: Filtration experiments in vitro. There has not yet seen the research on the effects of ODN to the bone marrow-derived mesenchymal stem cells, so we carried out a large number of initial screening in the beginning of this experiment. First of all, we choose 12 different sequences of ODN (in which partial sequence has applied for National Science and invention patents) which were designed and built by Jilin University, college of Basic Medical, department of Molecular Biology. According to the biological characteristics of ODN that could be absorbed into cells through the form of receptor-mediated without the need of constructing a vector, we took ODN and bone marrow-derived mesenchymal stem cells co-culture. By MTT colorimetric assay different ODN effects to the proliferation of BMSCs, and mapping dose-response curves and time curves about ODN impact on proliferation of different sources of rat BMSCs at different time points. The result showed that there are four different sequences ODN which can significantly stimulate the proliferation of BMSCs, initially confirmed that ODN has a certain effect on the biological characteristics of BMSCs. Second, different sequences of ODN were added in the BMSCs which were induced to osteoblast at the same time, the alkaline phosphatase kit was applicated to detect the expression of alkaline phosphatase in BMSCs in which with different concentrations of ODN at different time, the results show that there were two ODN can significantly enhance the osteoblastic differentiation of BMSCs, we successfully screened out the ODN which can promote the osteoblastic differentiation of BMSCs, the ODN has an impact in the process of osteoblastic differentiation of BMSCs was further confirmed.
Research 2, the mechanism research of the specific ODN sequences has an impact in the process of osteoblastic differentiation of BMSCs. The previous experiments have confirmed that a specific sequence of ODN can promote the osteoblastic differentiation of BMSCs. And then, the cells with ODN added in whether have abnormal expression of the relevant factors in the process of osteoblastic differentiation of BMSCs? We undertook the following experiment. First, rat BMSCs were isolated and cultured, the third generation of BMSCs were taken for osteoblastic induction, while adding the specific sequence of ODN co-cultured for 1d, 3d and 5d. Real-time quantitative PCR were used to analysis the mRNA expression of bone-related factors OPG, RANKL, Osterix, Runx2 and typeⅠcollagen in experimental group and control group BMSCs. The results showed that in the early time(3d and 5d), the ODN can significantly increase the mRNA expression of Osterix-related factors such as RANKL, OPG, Runx2 and the RANKL / OPG ratio had an evident declining trend while the action time of ODN increased. The result suggested that the mechanism that the ODN could promote the osteoblastic differentiation of BMSCs may be related to the abnormal expression of osteoblast-related factors.
Research 3, the purpose of this study is to find the active substance which can promote the osteoblastic differentiation of BMSCs and have the characteristics like high-performance, low-cost, easy synthesis and stable.That would provide an experimental basis for the future application in the field of periodontal tissue remodeling. Therefore, we used the ODN which was screened out from the vitro experiment for the studies in vivo to confirm whether the ODN has an impact in tissue repairing and regeneration of alveolar bone in vivo. First of all, the animal models of experimental periodontitis in rats was established by using the silk ligation which was currently accepted, after that, the ODN was injected into the local periodontal tissue of rats. In this process, experimental zoology, histopathology, immunohistochemistry chemistry and imaging technology were used in vivo studies to research the absorbed dose of alveolar bone, the histopathological changes and the expression of RANKL, OPG, Osterix, Runx2 and typeⅠcollagen in protein level. The results showed that the resorption of alveolar bone had no significant difference between the experimental group and the control group when 2 weeks after injection, however, when 4 weeks after injection, the resorption of alveolar bone in the experimental group was reduced than the control group (p<0.05), especially the resorption of palatal bone reduced significantly. We also detected that some protein expression of bone-associated factor appeared different expression in the experimental group and control group, the expression of osteoblast associated protein RANKL, OPG, etc. had increased.
Integrated the experimental results, we believe that the specific sequence of ODN has significant influence on the biological characteristics of bone marrow-derived mesenchymal stem cells, especially it plays an important role in the process of osteoblast differentiation. In the process, bone remodeling regulatory factors were abnormally expressed; in vivo, ODN promoted the bone rebuilding, we can infer that ODN should have a certain effect on the periodontal bone remodeling. Therefore, the results of this study show that: ODN may has the influence on periodontal alveolar bone remodeling caused by periodontal infection. The results may provide new treatment ideas to block absorption of alveolar bone caused by periodontal infection, and to promote the periodontal tissue repairing and regeneration.
ODN is a prominent achievement in the field of immunization scientific research in recent years. The innovative point of this study is to apply ODN to the field of oral medicine research, successfully screened and exploited the specific ODN which have a role in the process of bone marrow-derived mesenchymal stem cells differentiated to osteoblast. We made a preliminary exploration of its effect, mechanism and expected to provide a new treatment ideas on reparation and regeneration of periodontal tissue.
引文
[1]Tokunaga T, Yamamoto H, Shimada S, Abe H, Fukuda T, Fujisawa Y, et al. Antitumor activity of deoxyribonucleic acid fraction from Mycobacterium bovis BCG. I. Isolation, physicochemical characterization, and antitumor activity. [J] J Natl Cancer Inst. 1984 Apr;72(4):955-962.
[2]Walther W, Stein U. Therapeutic genes for cancer gene therapy. [J] Mol Biotechnol. 1999 Nov;13(1):21-28.
[3]Tokunaga T, Yano O, Kuramoto E, Kimura Y, Yamamoto T, Kataoka T, et al. Synthetic oligonucleotides with particular base sequences from the cDNA encoding proteins of Mycobacterium bovis BCG induce interferons and activate natural killer cells. [J] Microbiol Immunol. 1992;36(1):55-66.
[4]Tokunaga T, Yamamoto T, Yamamoto S. How BCG led to the discovery of immunostimulatory DNA. [J] Jpn J Infect Dis. 1999 Feb;52(1):1-11.
[5]Yamamoto S. Commemorative lecture of receiving Imamura Memorial Prize. II. Mode of action of oligonucleotide fraction extracted from Mycobacterium bovis BCG. [J] Kekkaku. 1994 Sep;69(9):571-574.
[6]Scheule RK. The role of CpG motifs in immunostimulation and gene therapy. [J] Adv Drug Deliv Rev. 2000 Nov 15;44(2-3):119-134.
[7]Weigel BJ, Rodeberg DA, Krieg AM, Blazar BR. CpG oligodeoxynucleotides potentiate the antitumor effects of chemotherapy or tumor resection in an orthotopic murine model of rhabdomyosarcoma. [J] Clin Cancer Res. 2003 Aug 1;9(8):3105-3114.
[8]Wilson HL, Dar A, Napper SK, Marianela Lopez A, Babiuk LA, Mutwiri GK. Immune mechanisms and therapeutic potential of CpG oligodeoxynucleotides. [J] Int Rev Immunol. 2006 May-Aug;25(3-4):183-213.
[9]Krieg AM, Yi AK, Matson S, Waldschmidt TJ, Bishop GA, Teasdale R, et al. CpG motifs in bacterial DNA trigger direct B-cell activation. [J] Nature. 1995 Apr 6;374(6522):546-549.
[10]Cho YJ, Ahn BY, Song ES, Park SA, Lee DH, Kim DS, et al. Bacterial DNA containing methylated CpG motifs retains immunostimulatory activity in synergy with modified lipopolysaccharides. [J] Microbiol Immunol. 2007;51(2):211-222.
[11]Wilson A, Pitt B, Li S. Complex roles of CpG in liposomal delivery of DNA and oligonucleotides. [J] Biosci Rep. 2002 Apr;22(2):309-322.
[12]Bird AP. CpG-rich islands and the function of DNA methylation. [J] Nature. 1986May 15-21;321(6067):209-213.
[13]Klinman DM, Barnhart KM, Conover J. CpG motifs as immune adjuvants. [J] Vaccine. 1999 Jan;17(1):19-25.
[14]Bird AP, Taggart MH, Nicholls RD, Higgs DR. Non-methylated CpG-rich islands at the human alpha-globin locus: implications for evolution of the alpha-globin pseudogene. [J] EMBO J. 1987 Apr;6(4):999-1004.
[15]Kandimalla ER, Bhagat L, Cong YP, Pandey RK, Yu D, Zhao Q, et al. Secondary structures in CpG oligonucleotides affect immunostimulatory activity. [J] Biochem Biophys Res Commun. 2003 Jul 11;306(4):948-953.
[16]Sester DP, Naik S, Beasley SJ, Hume DA, Stacey KJ. Phosphorothioate backbone modification modulates macrophage activation by CpG DNA. [J] J Immunol. 2000 Oct 15;165(8):4165-4173.
[17]Yu D, Kandimalla ER, Zhao Q, Bhagat L, Cong Y, Agrawal S. Requirement of nucleobase proximal to CpG dinucleotide for immunostimulatory activity of synthetic CpG DNA. [J] Bioorg Med Chem. 2003 Feb 6;11(3):459-464.
[18]Krieg AM, Davis HL. Enhancing vaccines with immune stimulatory CpG DNA. [J] Curr Opin Mol Ther. 2001 Feb;3(1):15-24.
[19]Krieg AM. The role of CpG motifs in innate immunity. [J] Curr Opin Immunol. 2000 Feb;12(1):35-43.
[20]Krieg AM. Therapeutic potential of Toll-like receptor 9 activation. [J] Nat Rev Drug Discov. 2006 Jun;5(6):471-484.
[21]Krieg A, Ruckstuhl T, Seeger S. Towards single-molecule DNA sequencing: assays with low nonspecific adsorption. [J] Anal Biochem. 2006 Feb 15;349(2):181-185.
[22]Marshall JD, Fearon K, Abbate C, Subramanian S, Yee P, Gregorio J, et al. Identification of a novel CpG DNA class and motif that optimally stimulate B cell and plasmacytoid dendritic cell functions. [J] J Leukoc Biol. 2003 Jun;73(6):781-792.
[23]Hartmann G, Weeratna RD, Ballas ZK, Payette P, Blackwell S, Suparto I, et al. Delineation of a CpG phosphorothioate oligodeoxynucleotide for activating primate immune responses in vitro and in vivo. [J] J Immunol. 2000 Feb 1;164(3):1617-1624.
[24]Verthelyi D, Ishii KJ, Gursel M, Takeshita F, Klinman DM. Human peripheral blood cells differentially recognize and respond to two distinct CPG motifs. [J] J Immunol. 2001 Feb 15;166(4):2372-2377.
[25]Lackner TE, R GH, J JH, Davey C, Guay DR. Pneumococcal polysaccharide revaccination: immunoglobulin g seroconversion, persistence, and safety in frail, chronically illolder subjects. [J] J Am Geriatr Soc. 2003 Feb;51(2):240-245.
[26]Vollmer J, Weeratna RD, Jurk M, Samulowitz U, McCluskie MJ, Payette P, et al. Oligodeoxynucleotides lacking CpG dinucleotides mediate Toll-like receptor 9 dependent T helper type 2 biased immune stimulation. [J] Immunology. 2004 Oct;113(2):212-223.
[27]Vollmer J, Weeratna R, Payette P, Jurk M, Schetter C, Laucht M, et al. Characterization of three CpG oligodeoxynucleotide classes with distinct immunostimulatory activities. [J] Eur J Immunol. 2004 Jan;34(1):251-262.
[28]Hartmann G, Battiany J, Poeck H, Wagner M, Kerkmann M, Lubenow N, et al. Rational design of new CpG oligonucleotides that combine B cell activation with high IFN-alpha induction in plasmacytoid dendritic cells. [J] Eur J Immunol. 2003 Jun;33(6):1633-1641.
[29]Rothenfusser S, Hornung V, Ayyoub M, Britsch S, Towarowski A, Krug A, et al. CpG-A and CpG-B oligonucleotides differentially enhance human peptide-specific primary and memory CD8+ T-cell responses in vitro. [J] Blood. 2004 Mar 15;103(6):2162-2169.
[30]Krug A, Rothenfusser S, Selinger S, Bock C, Kerkmann M, Battiany J, et al. CpG-A oligonucleotides induce a monocyte-derived dendritic cell-like phenotype that preferentially activates CD8 T cells. [J] J Immunol. 2003 Apr 1;170(7):3468-3477.
[31]Kadowaki N, Antonenko S, Liu YJ. Distinct CpG DNA and polyinosinic- polycytidylic acid double-stranded RNA, respectively, stimulate CD11c- type 2 dendritic cell precursors and CD11c+ dendritic cells to produce type I IFN. [J] J Immunol. 2001 Feb 15;166(4):2291-2295.
[32]Blackwell SE, Krieg AM. CpG-A-induced monocyte IFN-gamma-inducible protein- 10 production is regulated by plasmacytoid dendritic cell-derived IFN-alpha. [J] J Immunol. 2003 Apr 15;170(8):4061-4068.
[33]Kerkmann M, Rothenfusser S, Hornung V, Towarowski A, Wagner M, Sarris A, et al. Activation with CpG-A and CpG-B oligonucleotides reveals two distinct regulatory pathways of type I IFN synthesis in human plasmacytoid dendritic cells. [J]J Immunol. 2003 May 1;170(9):4465-4474.
[34]Yi AK, Krieg AM. CpG DNA rescue from anti-IgM-induced WEHI-231 B lymphoma apoptosis via modulation of I kappa B alpha and I kappa B beta and sustained activation of nuclear factor-kappa B/c-Rel. [J] J Immunol. 1998 Feb 1;160(3):1240-1245.
[35]McCluskie MJ, Davis HL. CpG DNA is a potent enhancer of systemic and mucosal immune responses against hepatitis B surface antigen with intranasal administration to mice. [J] J Immunol. 1998 Nov 1;161(9):4463-4466.
[36]Verthelyi D, Zeuner RA. Differential signaling by CpG DNA in DCs and B cells: not just TLR9. [J] Trends Immunol. 2003 Oct;24(10):519-522.
[37]Lee S, Hong J, Choi SY, Oh SB, Park K, Kim JS, et al. CpG oligodeoxynucleotides induce expression of proinflammatory cytokines and chemokines in astrocytes: the role of c-Jun N-terminal kinase in CpG ODN-mediated NF-kappaB activation. [J] J Neuroimmunol. 2004 Aug;153(1-2):50-63.
[38]Kuo CC, Lin WT, Liang CM, Liang SM. Class I and III phosphatidylinositol 3'-kinase play distinct roles in TLR signaling pathway. [J] J Immunol. 2006 May 15;176(10):5943-5949.
[39]丛中一,包木胜,万敏,王莉,李大鹏,王丽颖,等.新型CpG ODN增强乙肝疫苗诱导IgG2a类抗体产生的实验研究. [J]中国免疫学杂志; 2006. 11(3): 987-991.
[40]Klinman DM. Immunotherapeutic uses of CpG oligodeoxynucleotides. [J] Nat Rev Immunol. 2004 Apr;4(4):249-258.
[41]Krieg AM. CpG motifs: the active ingredient in bacterial extracts? [J] Nat Med. 2003 Jul;9(7):831-835.
[42]Lubaroff DM, Karan D, Andrews MP, Acosta A, Abouassaly C, Sharma M, et al. Decreased cytotoxic T cell activity generated by co-administration of PSA vaccine and CpG ODN is associated with increased tumor protection in a mouse model of prostate cancer. [J] Vaccine. 2006 Aug 28;24(35-36):6155-6162.
[43]Tanimizu N, Tsujimura T, Takahide K, Kodama T, Nakamura K, Miyajima A. Expression of Dlk/Pref-1 defines a subpopulation in the oval cell compartment of rat liver. [J] Gene Expr Patterns. 2004 Dec;5(2):209-218.
[44]Hemmi H, Takeuchi O, Kawai T, Kaisho T, Sato S, Sanjo H, et al. A Toll-like receptor recognizes bacterial DNA. [J] Nature. 2000 Dec 7;408(6813):740-745.
[45]Ashkar AA, Rosenthal KL. Toll-like receptor 9, CpG DNA and innate immunity. [J] Curr Mol Med. 2002 Sep;2(6):545-556.
[46]Bialecki E, Paget C, Fontaine J, Capron M, Trottein F, Faveeuw C. Role of marginal zone B lymphocytes in invariant NKT cell activation. [J]J Immunol. 2009 May 15;182(10):6105-6113.
[47]Mutwiri G, Pontarollo R, Babiuk S, Griebel P, van Drunen Littel-van den Hurk S, Mena A, et al. Biological activity of immunostimulatory CpG DNA motifs in domestic animals. [J] Vet Immunol Immunopathol. 2003 Jan 30;91(2):89-103.
[48]Nichani AK, Kaushik RS, Mena A, Popowych Y, Dent D, Townsend HG, et al. CpG oligodeoxynucleotide induction of antiviral effector molecules in sheep. [J] Cell Immunol.2004 Jan;227(1):24-37.
[49]Kamstrup S, Verthelyi D, Klinman DM. Response of porcine peripheral blood mononuclear cells to CpG-containing oligodeoxynucleotides. [J] Vet Microbiol. 2001 Feb 26;78(4):353-362.
[50]Leifer CA, Verthelyi D, Klinman DM. Heterogeneity in the human response to immunostimulatory CpG oligodeoxynucleotides. [J] J Immunother. 2003 Jul-Aug;26(4):313-9.
[51]Krieg AM. CpG motifs in bacterial DNA and their immune effects. [J] Annu Rev Immunol. 2002;20:709-60.
[52]Verthelyi D, Klinman DM. Immunoregulatory activity of CpG oligonucleotides in humans and nonhuman primates. [J] Clin Immunol. 2003 Oct;109(1):64-71.
[53]Halperin SA, Van Nest G, Smith B, Abtahi S, Whiley H, Eiden JJ. A phase I study of the safety and immunogenicity of recombinant hepatitis B surface antigen co-administered with an immunostimulatory phosphorothioate oligonucleotide adjuvant. [J] Vaccine. 2003 Jun 2;21(19-20):2461-2467.
[54]Heikenwalder M, Polymenidou M, Junt T, Sigurdson C, Wagner H, Akira S, et al. Lymphoid follicle destruction and immunosuppression after repeated CpG oligodeoxynucleotide administration. [J] Nat Med. 2004 Feb;10(2):187-192.
[55]Svelander L, Erlandsson Harris H, Lorentzen JC, Trollmo C, Klareskog L, Bucht A. Oligodeoxynucleotides containing CpG motifs can induce T cell-dependent arthritis in rats. [J] Arthritis Rheum. 2004 Jan;50(1):297-304.
[56]Weeratna RD, McCluskie MJ, Xu Y, Davis HL. CpG DNA induces stronger immune responses with less toxicity than other adjuvants. [J] Vaccine. 2000 Mar 6;18(17):1755-1762.
[57]Cooper CL, Davis HL, Morris ML, Efler SM, Krieg AM, Li Y, et al. Safety and immunogenicity of CPG 7909 injection as an adjuvant to Fluarix influenza vaccine. [J] Vaccine. 2004 Aug 13;22(23-24):3136-3143.
[58]van Rijnsoever M, Grieu F, Elsaleh H, Joseph D, Iacopetta B. Characterisation of colorectal cancers showing hypermethylation at multiple CpG islands. [J] Gut. 2002 Dec;51(6):797-802.
[59]Gramzinski RA, Doolan DL, Sedegah M, Davis HL, Krieg AM, Hoffman SL. Interleukin-12- and gamma interferon-dependent protection against malaria conferred by CpG oligodeoxynucleotide in mice. [J] Infect Immun. 2001 Mar;69(3):1643-1649.
[60]Amcheslavsky A, Zou W, Bar-Shavit Z. Toll-like receptor 9 regulates tumor necrosis factor-alpha expression by different mechanisms. Implications for osteoclastogenesis. [J] J Biol Chem. 2004 Dec 24;279(52):54039-54045.
[61]Zou W, Amcheslavsky A, Bar-Shavit Z. CpG oligodeoxynucleotides modulate the osteoclastogenic activity of osteoblasts via Toll-like receptor 9. [J] J Biol Chem. 2003 May 9;278(19):16732-16740.
[62]Chang JH, Chang EJ, Kim HH, Kim SK. Enhanced inhibitory effects of a novel CpG motif on osteoclast differentiation via TREM-2 down-regulation. [J] Biochem Biophys Res Commun. 2009 Nov 6;389(1):28-33.
[63]Amcheslavsky A, Bar-Shavit Z. Toll-like receptor 9 ligand blocks osteoclast differentiation through induction of phosphatase. [J] J Bone Miner Res. 2007 Aug;22(8):1301-1310.
[64]Kileng O, Albuquerque A, Robertsen B. Induction of interferon system genes in Atlantic salmon by the imidazoquinoline S-27609, a ligand for Toll-like receptor 7. [J] Fish Shellfish Immunol. 2008 May;24(5):514-522.
[65]Weiss ML, Medicetty S, Bledsoe AR, Rachakatla RS, Choi M, Merchav S, et al. Human umbilical cord matrix stem cells: preliminary characterization and effect of transplantation in a rodent model of Parkinson's disease. [J] Stem Cells. 2006 Mar;24(3):781-792.
[66]舒胜文,李盛林,马绪臣.大白鼠骨髓间充质干细胞体外培养体系的建立及分化研究. [J]现代口腔医学杂志2002;16(2):97-99.
[67]Sun X, Gan Y, Tang T, Zhang X, Dai K. In vitro proliferation and differentiation of human mesenchymal stem cells cultured in autologous plasma derived from bone marrow. [J] Tissue Eng Part A. 2008 Mar;14(3):391-400.
[68]裴国献,金丹.骨组织工程学种子细胞研究进展. [J]中华创伤骨科杂志. 2003;5(1):4-7.
[69]Wohrer S, Rabitsch W, Shehata M, Kondo R, Esterbauer H, Streubel B, et al. Mesenchymal stem cells in patients with chronic myelogenous leukaemia or bi-phenotypic Ph+ acute leukaemia are not related to the leukaemic clone. [J] Anticancer Res. 2007 Nov-Dec;27(6B):3837-3841.
[70]顾祖超,李起鸿,赵玉峰.兔骨髓基质细胞在诱导条件下的成骨特性[J]第三军医大学学报. 2002;24(5):502-506.
[71]胡炜,俞兴,徐林.骨髓基质细胞诱导分化成骨方法及相关研究进展. [J]中国组织工程研究与临床康复. 2009;13(1):169-172.
[72]Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, et al. Multilineage potential of adult human mesenchymal stem cells. [J] Science. 1999 Apr2;284(5411):143-147.
[73]崔鹏程,陈文弦,张志培.诱导的人骨髓间充质干细胞在裸鼠体内生成软骨的实验研究. [J]第一军医大学学报. 2003, ;23(8):766-769.
[74]王骏骅,杨惠林,陈亮,等.大鼠脂肪基质干细胞的培养及其向成骨细胞分化的研究[J]中国脊柱脊髓杂志, 2005;15(9):553-555.
[75]张科强,刘一,王宝刚,罗扬.体外冲击波诱导骨髓间充质干细胞向成骨细胞转化过程中c-fos、c-jun的表达[J]山东医药,2008;48(4):63-.65
[76]Thiel M. Application of shock waves in medicine. [J] Clin Orthop Relat Res. 2001 Jun(387):18-21.
[77]Orciani M, Trubiani O, Vignini A, Mattioli-Belmonte M, Di Primio R, Salvolini E. Nitric oxide production during the osteogenic differentiation of human periodontal ligament mesenchymal stem cells. [J] Acta Histochem. 2009;111(1):15-24.
[78]Diniz P, Shomura K, Soejima K, Ito G. Effects of pulsed electromagnetic field (PEMF) stimulation on bone tissue like formation are dependent on the maturation stages of the osteoblasts. [J] Bioelectromagnetics. 2002 Jul;23(5):398-405.
[79]宋晋刚,许建中,周强,罗飞,李洪鹏.不同频率脉冲电磁场诱导人骨髓间充质干细胞成骨分化的研究[J]中华物理医学与康复杂志. 2005;3(3):134-137.
[80]Z T, Q z, P G, al e. Research on promoting periodontal regeneration with human basic fibroblast growth factor-modified bone marrow mesenchymal stromal cell gene therapy. [J] Cytotherapy. 2009;11(3):317-325.
[81]梅红,李一雷,王心蕊,等.人骨髓间充质干细胞与成骨细胞共培养诱导破骨细胞方法的建立和鉴定. [J]吉林大学学报(医学版). 2007;33(5):824-826.
[82]von Knoch F, Jaquiery C, Kowalsky M, Schaeren S, Alabre C, Martin I, et al. Effects of bisphosphonates on proliferation and osteoblast differentiation of human bone marrow stromal cells. [J] Biomaterials. 2005 Dec;26(34):6941-6949.
[83]Domon T, Yamazaki Y, Fukui A, Ohnishi Y, Takahashi S, Yamamoto T, et al. Ultrastructural study of cell-cell interaction between osteoclasts and osteoblasts/stroma cells in vitro. [J] Ann Anat. 2002 May;184(3):221-227.
[84]Diefenderfer DL, Osyczka AM, Reilly GC, Leboy PS. BMP responsiveness in human mesenchymal stem cells. [J] Connect Tissue Res. 2003;44 Suppl 1:305-311.
[85]Urist MR. Bone: formation by autoinduction. Science. 1965 Nov 12;150 (698): 893-899.
[86]Guan CC, Yan M, Jiang XQ, Zhang P, Zhang XL, Li J, et al. Sonic hedgehog alleviates the inhibitory effects of high glucose on the osteoblastic differentiation of bone marrow stromal cells. [J] Bone. 2009 Dec;45(6):1146-1152.
[87]Wang L, Huang Y, Pan K, Jiang X, Liu C. Osteogenic responses to different concentrations/ratios of BMP-2 and bFGF in bone formation. [J] Ann Biomed Eng. Jan;38(1):77-87.
[88]Langer HF, Stellos K, Steingen C, Froihofer A, Schonberger T, Kramer B, et al. Platelet derived bFGF mediates vascular integrative mechanisms of mesenchymal stem cells in vitro. [J] J Mol Cell Cardiol. 2009 Aug;47(2):315-325.
[89]Xia L, Zhao M, Xu M. Experimental investigation of pEGFP-bFGF gene transfer to human limbal stem cells. [J] Sheng Wu Yi Xue Gong Cheng Xue Za Zhi. 2009 Feb;26(1):148-152.
[90]Huang W, Carlsen B, Wulur I, Rudkin G, Ishida K, Wu B, et al. BMP-2 exerts differential effects on differentiation of rabbit bone marrow stromal cells grown in two-dimensional and three-dimensional systems and is required for in vitro bone formation in a PLGA scaffold. [J] Exp Cell Res. 2004 Oct 1;299(2):325-334.
[91]黎晖,周健洪,陈东风,等.龟板对大鼠骨髓间充质干细胞向成骨分化的影响. [J]中药新药与临床病理. 2005;16(3):159-161.
[92]赛音其木格,娜仁图娅,杨健,等.大黄素诱导人骨髓间充质干细胞向成骨细胞分化的研究. [J]中国中西医结合外科杂志. 2006;12(5):448-452.
[93]吴云刚,张志平.右归饮含药血清对人骨髓基质干细胞诱导为成骨细胞的影响. [J]江西中医药. 2006;37(7):57-58.
[94]Tang TT, Lu B, Yue B, Xie XH, Xie YZ, Dai KR, et al. Treatment of osteonecrosis of the femoral head with hBMP-2-gene-modified tissue-engineered bone in goats. [J] J Bone Joint Surg Br. 2007 Jan;89(1):127-129.
[95]Gugala Z, Davis AR, Fouletier-Dilling CM, Gannon FH, Lindsey RW, Olmsted-Davis EA. Adenovirus BMP2-induced osteogenesis in combination with collagen carriers. [J] Biomaterials. 2007 Oct;28(30):4469-4479.
[96]Kimelman N, Pelled G, Gazit Z, Gazit D. Applications of gene therapy and adult stem cells in bone bioengineering. [J] Regen Med. 2006 Jul;1(4):549-561.
[97]刘宏伟,欧龙,庞劲凡.自体骨髓基质细胞用于牙周组织缺损的动物实验研究. [J]牙体牙髓牙周病学杂志. 2000;10(3):117-119.
[98]Reynolds MA, Aichelmann-Reidy ME, Branch-Mays GL, Gunsolley JC. The efficacyof bone replacement grafts in the treatment of periodontal osseous defects. [J] A systematic review. Ann Periodontol. 2003 Dec;8(1):227-265.
[99]Tan Z, Zhao Q, Gong P, Wu Y, Wei N, Yuan Q, et al. Research on promoting periodontal regeneration with human basic fibroblast growth factor-modified bone marrow mesenchymal stromal cell gene therapy. [J] Cytotherapy. 2009;11(3):317-325.
[100]Kamei N, Tanaka N, Oishi Y, Ishikawa M, Hamasaki T, Nishida K, et al. Bone marrow stromal cells promoting corticospinal axon growth through the release of humoral factors in organotypic cocultures in neonatal rats. [J] J Neurosurg Spine. 2007 May;6(5):412-419.
[101]曹采方,孟焕新,沙月琴,等. [M ]临床牙周病学.北京:北京大学医学出版社. 2006.
[102]Nonnenmacher C, Dalpke A, Zimmermann S, Flores-De-Jacoby L, Mutters R, Heeg K. DNA from periodontopathogenic bacteria is immunostimulatory for mouse and human immune cells. [J] Infect Immun. 2003 Feb;71(2):850-856.
[103]Azuma M. Fundamental mechanisms of host immune responses to infection. [J] J Periodontal Res. 2006 Oct;41(5):361-373.
[104]Wang PL, Ohura K, Fujii T, Oido-Mori M, Kowashi Y, Kikuchi M, et al. DNA microarray analysis of human gingival fibroblasts from healthy and inflammatory gingival tissues. [J] Biochem Biophys Res Commun. 2003 Jun 13;305(4):970-973.
[105]Franceschi RT. Biological approaches to bone regeneration by gene therapy. [J] J Dent Res. 2005 Dec;84(12):1093-1103.
[106]Shimono M, Ishikawa T, Ishikawa H, Matsuzaki H, Hashimoto S, Muramatsu T, et al. Regulatory mechanisms of periodontal regeneration. [J] Microsc Res Tech. 2003 Apr 1;60(5):491-502.
[107]Anusaksathien O, Giannobile WV. Growth factor delivery to re-engineer periodontal tissues. [J] Curr Pharm Biotechnol. 2002 Jun;3(2):129-139.
[108]Song AM, Shu R, Xie YF, Song ZC, Li HY, Liu XF, et al. A study of enamel matrix proteins on differentiation of porcine bone marrow stromal cells into cementoblasts. [J] Cell Prolif. 2007 Jun;40(3):381-396.
[109]宋忠臣,束荣,谢玉峰,等.釉基质蛋白联合骨髓基质细胞促进牙周再生. [J]上海交通大学学报(医学版). 2007;27(6):634-637.
[110]Zhu W, Boachie-Adjei O, Rawlins BA, Frenkel B, Boskey AL, Ivashkiv LB, et al. A novel regulatory role for stromal-derived factor-1 signaling in bone morphogenic protein-2osteogenic differentiation of mesenchymal C2C12 cells. [J] J Biol Chem. 2007 Jun 29;282(26):18676-18685.
[111]Kasten P, Vogel J, Beyen I, Weiss S, Niemeyer P, Leo A, et al. Effect of platelet-rich plasma on the in vitro proliferation and osteogenic differentiation of human mesenchymal stem cells on distinct calcium phosphate scaffolds: the specific surface area makes a difference. [J] J Biomater Appl. 2008 Sep;23(2):169-188.
[112]Komori T. Regulation of bone development and maintenance by Runx2. [J] Front Biosci. 2008;13:898-903.
[113]Komori T. Runx2, a multifunctional transcription factor in skeletal development. [J] J Cell Biochem. 2002;87(1):1-8.
[114]Okazaki K, Sandell LJ. Extracellular matrix gene regulation. [J] Clin Orthop Relat Res. 2004 Oct(427 Suppl):S123-128.
[115]Saito T, Ogawa M, Hata Y, Bessho K. Acceleration effect of human recombinant bone morphogenetic protein-2 on differentiation of human pulp cells into odontoblasts. [J] J Endod. 2004 Apr;30(4):205-208.
[116]Takeda S, Bonnamy JP, Owen MJ, Ducy P, Karsenty G. Continuous expression of Cbfa1 in nonhypertrophic chondrocytes uncovers its ability to induce hypertrophic chondrocyte differentiation and partially rescues Cbfa1-deficient mice. [J] Genes Dev. 2001 Feb 15;15(4):467-481.
[117]Zhang Y, Deng X, Scheller EL, Kwon TG, Lahann J, Franceschi RT, et al. The effects of Runx2 immobilization on poly (epsilon-caprolactone) on osteoblast differentiation of bone marrow stromal cells in vitro. [J] Biomaterials. Apr;31(12):3231-3236.
[118]Zhang X, Aubin JE, Inman RD. Molecular and cellular biology of new bone formation: insights into the ankylosis of ankylosing spondylitis. [J] Curr Opin Rheumatol. 2003 Jul;15(4):387-393.
[119]Nakashima K, Zhou X, Kunkel G, Zhang Z, Deng JM, Behringer RR, et al. The novel zinc finger-containing transcription factor osterix is required for osteoblast differentiation and bone formation. [J] Cell. 2002 Jan 11;108(1):17-29.
[120]Heymann MF, Riet A, Le Goff B, Battaglia S, Paineau J, Heymann D. OPG, RANK and RANK ligand expression in thyroid lesions. [J] Regul Pept. 2008 Jun 5;148(1-3):46-53.
[121]Ling L, Nurcombe V, Cool SM. Wnt signaling controls the fate of mesenchymal stem cells. [J] Gene. 2009 Mar 15;433(1-2):1-7.
[122]Giraud Guille MM, Mosser G, Helary C, Eglin D. Bone matrix like assemblies of collagen: from liquid crystals to gels and biomimetic materials.[J]Micron. 2005; 36(7-8):602-608.
[123]Tzaphlidou M. The role of collagen in bone structure: an image processing approach. [J] Micron. 2005;36(7-8):593-601.
[124]Nienhuijs ME, Meijer GJ, Merkx MA, Walboomers XF, Jansen JA. Bone substitutes, growth factors and distraction osteogenesis.[J] Ned Tijdschr Tandheelkd. 2008 Jun;115(6):297-304.
[125]于丽,刘霞,申玉芹,等.腺病毒介导TWEAK对大鼠骨髓间充质干细胞影响的实验研究. [J]口腔医学研究. 2009;25(01):12-15.
[126]Vollmer J. CpG motifs to modulate innate and adaptive immune responses. [J] Int Rev Immunol. 2006 May-Aug;25(3-4):125-134.
[127]Amcheslavsky A, Hemmi H, Akira S, Bar-Shavit Z. Differential contribution of osteoclast- and osteoblast-lineage cells to CpG-oligodeoxynucleotide (CpG-ODN) modulation of osteoclastogenesis. [J] J Bone Miner Res. 2005 Sep;20(9):1692-1699.
[128]Lin D. Comparison of three non-radioactive assays for measurement of cell-mediated immunity. [J] Bio Formosa. 2006;41(2):59-66.
[129]Amcheslavsky A, Bar-Shavit Z. Interleukin (IL)-12 mediates the anti- osteoclas- togenic activity of CpG-oligodeoxynucleotides. [J] J Cell Physiol. 2006 Apr;207(1):244-250.
[130]Arpornmaeklong P, Pripatnanont P, Suwatwirote N. Properties of chitosan-collagen sponges and osteogenic differentiation of rat-bone-marrow stromal cells. [J] Int J Oral Maxillofac Surg. 2008 Apr;37(4):357-366.
[131]申玉芹,孙新华,于丽,等.不同CpG ODN对大鼠骨髓间充质干细胞增殖作用的研究. [J]口腔医学研究. 2009;25(06):695-698.
[132]Sculean A, Nikolidakis D, Schwarz F. Regeneration of periodontal tissues: combinations of barrier membranes and grafting materials - biological foundation and preclinical evidence: a systematic review. [J] J Clin Periodontol. 2008 Sep;35(8 Suppl): 106-116.
[133]Shichinohe H, Kuroda S, Tsuji S, Yamaguchi S, Yano S, Lee JB, et al. Bone marrow stromal cells promote neurite extension in organotypic spinal cord slice: significance for cell transplantation therapy. [J] Neurorehabil Neural Repair. 2008 Sep-Oct;22(5):447-457.
[134]Zou W, Schwartz H, Endres S, Hartmann G, Bar-Shavit Z. CpG oligonucleotides: novel regulators of osteoclast differentiation. [J] FASEB J. 2002 Mar;16(3):274-282.
[135]Turnbull IR, Gilfillan S, Cella M, Aoshi T, Miller M, Piccio L, et al. Cutting edge: TREM-2 attenuates macrophage activation. [J] J Immunol. 2006 Sep 15;177(6):3520-3524.
[136]Gundberg CM. Biochemical markers of bone formation. [J] Clin Lab Med. 2000 Sep;20(3):489-501.
[137]Zhou GS, Su ZY, Cai YR, Liu YK, Dai LC, Tang RK, et al. Different effects of nanophase and conventional hydroxyapatite thin films on attachment, proliferation and osteogenic differentiation of bone marrow derived mesenchymal stem cells. [J] Biomed Mater Eng. 2007;17(6):387-395.
[138]Manzel L, Strekowski L, Ismail FM, Smith JC, Macfarlane DE. Antagonism of immunostimulatory CpG-oligodeoxynucleotides by 4-aminoquinolines and other weak bases: mechanistic studies. [J] J Pharmacol Exp Ther. 1999 Dec;291(3):1337-1347.
[139]Manzel L, Macfarlane DE. CpG-oligodeoxynucleotide-resistant variant of WEHI 231 cells. [J] J Leukoc Biol. 1999 Nov;66(5):817-821.
[140]Jeong JY, Choi JW, Jeon KI, Jue DM. Chloroquine decreases cell-surface expression of tumour necrosis factor receptors in human histiocytic U-937 cells. [J] Immunology. 2002 Jan;105(1):83-91.
[141]Weber SM, Levitz SM. Chloroquine antagonizes the proinflammatory cytokine response to opportunistic fungi by alkalizing the fungal phagolysosome. [J] J Infect Dis. 2001 Mar 15;183(6):935-942.
[142]Park J, Kwon D, Choi C, Oh JW, Benveniste EN. Chloroquine induces activation of nuclear factor-kappaB and subsequent expression of pro-inflammatory cytokines by human astroglial cells. [J] J Neurochem. 2003 Mar;84(6):1266-1274.
[143]Sanjuan MA, Rao N, Lai KT, Gu Y, Sun S, Fuchs A, et al. CpG-induced tyrosine phosphorylation occurs via a TLR9-independent mechanism and is required for cytokine secretion. [J] J Cell Biol. 2006 Mar 27;172(7):1057-1068.
[144]Di JM, Pang J, Pu XY, Zhang Y, Liu XP, Fang YQ, et al. Toll-like receptor 9 agonists promote IL-8 and TGF-beta1 production via activation of nuclear factor kappaB in PC-3 cells. [J] Cancer Genet Cytogenet. 2009 Jul 15;192(2):60-67.
[145]Yasuda H, Leelahavanichkul A, Tsunoda S, Dear JW, Takahashi Y, Ito S, et al. Chloroquine and inhibition of Toll-like receptor 9 protect from sepsis-induced acute kidney injury. [J] Am J Physiol Renal Physiol. 2008 May;294(5):F1050-1058.
[146]Zhu X, Ertel W, Ayala A, Morrison MH, Perrin MM, Chaudry IH. Chloroquine inhibits macrophage tumour necrosis factor-alpha mRNA transcription. [J] Immunology. 1993 Sep;80(1):122-126.
[147]Yeo SJ, Yoon JG, Yi AK. Myeloid differentiation factor 88-dependent post-transcriptional regulation of cyclooxygenase-2 expression by CpG DNA: tumor necrosisfactor-alpha receptor-associated factor 6, a diverging point in the Toll-like receptor 9-signaling. [J] J Biol Chem. 2003 Oct 17;278(42):40590-40600.
[148]吴翀,丁国富,周红,等.抗人TLR9抗体对CpG寡核苷酸介导的人外周血单核细胞肿瘤坏死因子-α释放的影响. [J]中国临床药理学与治疗学. 2006;11(3):324-327.
[149]Wang J, Huang WL, Liu RY. CpG-ODN enhances ingestion of apoptotic neutrophils by macrophages. [J] Clin Exp Med. 2009 Mar;9(1):37-43.
[150]Jang CH, Choi JH, Byun MS, Jue DM. Chloroquine inhibits production of TNF-alpha, IL-1beta and IL-6 from lipopolysaccharide-stimulated human monocytes/ macrophages by different modes. [J] Rheumatology (Oxford). 2006 Jun;45(6): 703- 710.
[151]Weber SM, Chen JM, Levitz SM. Inhibition of mitogen-activated protein kinase signaling by chloroquine. [J] J Immunol. 2002 May 15;168(10):5303-5309.
[152]Lim EJ, Lee SH, Lee JG, Kim JR, Yun SS, Baek SH, et al. Toll-like receptor 9 dependent activation of MAPK and NF-kB is required for the CpG ODN-induced matrix metalloproteinase-9 expression. [J] Exp Mol Med. 2007 Apr 30;39(2):239-245.
[153]Nakamura K, Miyazato A, Xiao G, Hatta M, Inden K, Aoyagi T, et al. Deoxynucleic acids from Cryptococcus neoformans activate myeloid dendritic cells via a TLR9-dependent pathway. [J] J Immunol. 2008 Mar 15;180(6):4067-4074.
[154]Rutz M, Metzger J, Gellert T, Luppa P, Lipford GB, Wagner H, et al. Toll-like receptor 9 binds single-stranded CpG-DNA in a sequence- and pH-dependent manner. [J] Eur J Immunol. 2004 Sep;34(9):2541-2550.
[155]El Kebir D, Jozsef L, Pan W, Wang L, Filep JG. Bacterial DNA activates endothelial cells and promotes neutrophil adherence through TLR9 signaling. [J] J Immunol. 2009 Apr 1;182(7):4386-4394.
[156]Tashiro K, Kondo A, Kawabata K, Sakurai H, Sakurai F, Yamanishi K, et al. Efficient osteoblast differentiation from mouse bone marrow stromal cells with polylysin-modified adenovirus vectors. [J] Biochem Biophys Res Commun. 2009 Jan 30;379(1):127-132.
[157]Al-kalaly A, Wu C, Wong R, Rabie AB. The assessment of cell cycle genes in the rat mandibular condyle. [J] Arch Oral Biol. 2009 May;54(5):470-478.
[158]Mendes Sdos S, Candi A, Vansteenbrugge M, Pignon MR, Bult H, Boudjeltia KZ, et al. Microarray analyses of the effects of NF-kappaB or PI3K pathway inhibitors on the LPS-induced gene expression profile in RAW264.7 cells: synergistic effects of rapamycin on LPS-induced MMP9-overexpression. [J] Cell Signal. 2009 Jul;21(7):1109-1122.
[159]Chen RS, Chen MH, Young TH. Induction of differentiation and mineralization in rat tooth germ cells on PVA through inhibition of ERK1/2. Biomaterials. 2009 Feb;30(4):541-547.
[160]Maehira F, Miyagi I, Eguchi Y. Effects of calcium sources and soluble silicate on bone metabolism and the related gene expression in mice. [J] Nutrition. 2009 May;25(5):581-589.
[161]Bu SY, Hunt TS, Smith BJ. Dried plum polyphenols attenuate the detrimental effects of TNF-alpha on osteoblast function coincident with up-regulation of Runx2, Osterix and IGF-I. [J] J Nutr Biochem. 2009 Jan;20(1):35-44.
[162]Jones CD, Yeung C, Zehnder JL. Comprehensive validation of a real-time quantitative bcr-abl assay for clinical laboratory use. [J] Am J Clin Pathol. 2003 Jul; 120(1):42-48.
[163]Schmittgen TD, Zakrajsek BA, Mills AG, Gorn V, Singer MJ, Reed MW. Quantitative reverse transcription-polymerase chain reaction to study mRNA decay: comparison of endpoint and real-time methods. [J] Anal Biochem. 2000 Oct 15; 285(2):194-204.
[164]Gori F, Hofbauer LC, Dunstan CR, Spelsberg TC, Khosla S, Riggs BL. The expression of osteoprotegerin and RANK ligand and the support of osteoclast formation by stromal-osteoblast lineage cells is developmentally regulated. [J] Endocrinology. 2000 Dec;141(12):4768-4776.
[165]Cai X, Li C, Du G, Cao Z. Protective effects of baicalin on ligature-induced periodontitis in rats. [J] J Periodontal Res. 2008 Feb;43(1):14-21.
[166]章魁华,于世凤,主编. [M]实验口腔病理学.北京:人民卫生出版社. 2002:36-37.
[167]Weinberg MA, Bral M. Laboratory animal models in periodontology. [J] J Clin Periodontol. 1999 Jun;26(6):335-340.
[168]Bezerra MM, de Lima V, Alencar VB, Vieira IB, Brito GA, Ribeiro RA, et al. Selective cyclooxygenase-2 inhibition prevents alveolar bone loss in experimental periodontitis in rats. [J] J Periodontol. 2000 Jun;71(6):1009-1014.
[169]Achong R, Nishimura I, Ramachandran H, Howell TH, Fiorellini JP, Karimbux NY. Membrane type (MT) 1-matrix metalloproteinase (MMP) and MMP-2 expression in ligature-induced periodontitis in the rat. [J] J Periodontol. 2003 Apr;74(4):494-500.
[170]Kuhr A, Popa-Wagner A, Schmoll H, Schwahn C, Kocher T. Observations on experimental marginal periodontitis in rats. [J] J Periodontal Res. 2004 Apr;39(2):101-106.
[171]于永利,杨光,王丽颖.一种具有免疫抑制功能的寡核苷酸. [p]中国,专利号: 200810135458; 2010-02-10.