外源性TGF-β1与PDGF-BB联合应用对正畸牙压力侧Pyk2蛋白和基因表达的影响

doi:10.19439/j.sjos.2019.05.005

摘要/Abstract

摘要： 目的探讨外源性生长因子-β1与外源性血小板衍生生长因子BB联合应用对牙周组织破骨细胞内Pyk2表达的影响。方法 160只SD大鼠随机分为2组,建立正畸牙移动模型。A组注射含rhTGF-β1与rhPDGF-BB的混合液 0.1 mL,B组注射相同容量的PBS。加力后1、4、7、10、14 d,分别处死每大组的1小组大鼠。测量牙移动距离变化,TRAP染色行破骨细胞计数,Pyk2蛋白和基因水平分别用免疫组织化学染色法和RT-PCR检测。采用SPSS19.0软件包对数据进行统计学分析。结果 2组牙移动距离在第4、7、10、14天均具有统计学差异（P<0.05）。A组压力侧破骨细胞计数显著高于B组,除第14天外,其余各时间点均具有显著差异（P<0.05）。A组Pyk2蛋白和基因表达均比B组高,2组均在第7天达到最高峰,除第1天外,其余时间点Pyk2蛋白表达均具有统计学差异（P<0.05）。第4、7天,2组Pyk2基因表达均具有统计学差异（P<0.05）。结论外源性TGF-β1与PDGF-BB联合应用可从蛋白和分子水平上调正畸牙牙周组织压力侧Pyk2的表达,这可能是加速正畸牙移动速率的原因之一。

关键词: rhTGF-β, 1, rhPDGF-BB, 正畸牙, 破骨细胞, Pyk2

Abstract: PURPOSE: To explore the combined effect of recombinant human transforming growth factor-β1 (rhTGF-β1) and recombinant human platelet derived growth factor-BB (rhPDGF-BB) on the expression of Pyk2 in the osteoclasts of the pressure side of orthodontic tooth in rats. METHODS: One hundred and sixty male Sprague-Dawley rats were randomly divided into 2 groups: experimental group(A) and control group(B) to establish orthodontic tooth movement model. Rats in the experimental group received combined injection of 5 ng rhTGF-β1 and 10 ng rhPDGF-BB in the buccal submucosal area of the left upper first molar every other day, while rats in the control group received equivalent volumes of PBS. Rats in each group were sacrificed at each of 5 time points(1,4,7,10 and 14 days) after appliance placement. The distance of the tooth movement was measured with stereomicroscope, the change of the amount of osteoclasts was detected with tartrate-resistant acid phosphatase histochemistry (TRAP), the protein and mRNA expression of Pyk2 was detected by immunohistochemistry and real time PCR(RT-PCR). SPSS19.0 software package was used for statistical analysis. RESULTS: The distance of teeth in group A moved more rapidly than in group B, except for the first day, significant differences were found (P<0.05) in all time points.The number of osteoclasts in group A was significantly higher than that in group B, except for the 14th day, compared with the control group(P<0.05). Pyk2 protein and gene expression of group A was significantly higher than group B, both groups reached a peak in 7 days and gradually decreased thereafter, except in the first day, the expression of protein Pyk2 was significantly different at other time points (P<0.05). CONCLUSIONS: rhTGF-β1 and rhPDGF-BB up-regulated the expression of Pyk2 protein and mRNA gene on the pressure side of orthodontic tooth in rats, which may be one of the reasons for accelerating the rate of orthodontic tooth movement.

Key words: rhPDGF-BB, rhTGF-β1, Orthodontic tooth, Osteoclasts, Pyk2

中图分类号:

R783.5

梅梅, 易杰, 张疆弢, 黄瑾, 江策, 丰雷. 外源性TGF-β1与PDGF-BB联合应用对正畸牙压力侧Pyk2蛋白和基因表达的影响[J]. 上海口腔医学, 2019, 28(5): 472-477.

MEI Mei, YI Jie, ZHANG Jiang-tao, HUANG Jin, JIANG Ce, FENG Lei. Combined effect of rhTGF-β1 and rhPDGF-BB on the expression of Pyk2 protein and mRNA gene during orthodontic tooth movement in SD rats[J]. Shanghai Journal of Stomatology, 2019, 28(5): 472-477.

参考文献

[1] Brandl A, Angele P, Christina R, et al.Influence of the growth factors PDGF-BB, TGF-β1 and bFGF on the replicative aging of human articular chondrocytes during in vitro expansion[J]. J Orthop Res, 2010, 28(3): 354-360.
[2] Qi YX, Jiang J, Jiang XH, et al.PDGF-BB and TGF-β1 on cross-talk between endothelia-l and smooth muscle cells in vascular remodeling induced by low shear stress[J]. Proc Natl Acad Sci USA, 2011, 108(5): 1908-1913.
[3] 刘亚, 王小琴, 马艳宁. 血小板衍生生长因子BB和转化生长因子-β1联合应用对大鼠正畸牙移动的影响[J].中国药物与临床, 2013, 13(6): 719-721.
[4] Cao J, Chen Y, Fu J, et al.High expression of proline-rich tyrosine kinase 2 is associated with poor survival of hepatocellular carcinoma via regulating phosphatidylinositol 3-Kinase/AKT pathway[J]. Ann Surg Oncol, 2013, 20(Suppl 3): 312-323.
[5] Kacena MA, Eleniste PP, Cheng YH, et al.Mega karyocytes regulate expression of Pyk2 isoforms and caspase-mediated cleavage of actin in osteoblasts[J]. J Biol Chem, 2012, 287(21): 17257-17268.
[6] 张疆弢, 梅梅, 刘建国, 等. rhPDGF-BB与rhTGF-β1联合应用对大鼠正畸牙压力侧破骨细胞FAK mRNA表达的影响[J].实用口腔医学杂志, 2014, 24(5): 619-623.
[7] 张疆弢, 梅梅, 江策, 等. rhPDGF-BB与rhTGF-β1联合应用对大鼠正畸牙破骨细胞FAK蛋白表达的影响[J]. 上海口腔医学, 2015, 24(4): 423-427.
[8] Belal MH, Watanabe H, Ichinose S, et al.Effect of PDGF-BB combined with EDTA gel on adhesion and proliferation to the root surface[J]. Odontology, 2012, 100(2): 206-214.
[9] Li X, McFarland DC, Velleman SG. Transforming growth factor-β1-induced satellite cell apoptosis in chickens is associated with β1 integrin-mediated focal adhesion kinase activation[J]. Poult Sci, 2009, 88(8): 1725-1734.
[10] Cooke JW, Sarment DP, Whitesman LA, et al.Effect of rhPDGF-BB delivery on mediators of periodontal wound repair[J]. Tissue Eng, 2006, 12(6): 1441-1450.
[11] 黄瑾, 刘建国, 宋琦, 等. 重组人血小板衍生生长因子BB与转化生长因子β1 联合应用对大鼠正畸牙牙周组织改建的影响[J].中华口腔医学杂志, 2013, 48(s1): 72-76.
[12] Tabatabaei FS, Torshabi M.Effects of non-collagenous proteins,TGF-β1,and PDG F-BB on viability and proliferation of dental pulp stem cells[J]. J Oral Maxillofac Res, 2016, 7(1): e4.
[13] Mills RD, Mita M, Walsh MP.A role for the Ca²+-dependent tyrosine kinase Pyk2 in tonic depolarization-induced vascular smooth muscle contraction[J]. J Muscle Res Cell Motil, 2015, 36(6): 479-489.
[14] Torigoe G, Nagao M, Tanabe N, et al.PYK2 mediates BzATP-induced extracellular matrix proteins synthesis[J]. Biochem Biophys Res Commun, 2017, 494(3-4): 663-667.
[15] Canobbio I, Cipolla L, Guidetti GF, et al.The focal adhesion kinase Pyk2 links Ca²+ signalling to Src family kinase activation and protein tyrosine phosphorylation in thrombin stimulated platelets[J]. Biochem J, 2015, 469(2): 199-210.
[16] Zhu X, Bao Y, Guo Y, et al.Proline-rich protein tyrosine kinase 2 in inflammation and cancer[J]. Cancers(Basel), 2018, 10(5): E139.
[17] 武茜茗, 寇育荣. Pyk2参与细胞信号转导通路的研究进展[J]. 生物学杂志, 2018, 35(2): 73-76.
[18] Chien PT, Lin CC, Hsiao LD, et al.c-Src/Pyk2/EGFR/PI3K/Akt/CREB-activated pathway contributes to human cardiomyocyte hypertrophy: role of COX-2 induction[J]. Mol Cell Endocrinol, 2015, 409: 59-72.