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中华口腔医学研究杂志(电子版) ›› 2014, Vol. 8 ›› Issue (02) : 104 -111. doi: 10.3877/cma.j.issn.1674-1366.2014.02.004

基础研究

人非ATP 酶依赖性调节颗粒13对人牙周膜细胞的调节作用
赵曦1, 巢永烈2,(), 陈丕修2, 卿列华1   
  1. 1.618000 德阳市人民医院口腔科
    2.四川大学华西口腔医学院修复科
  • 收稿日期:2013-12-16 出版日期:2014-04-01
  • 通信作者: 巢永烈

The effect of hRpn13 (a subunit of 26S proteasome) regulating human periodontal ligament cells

Xi Zhao1, Yonglie Chao1,(), Pixiu Chen1, Liehua Qin1   

  1. 1.Department of Stomatology, People′s Hospital of Deyang City, Sichuan Province, Deyang 618000, China
  • Received:2013-12-16 Published:2014-04-01
  • Corresponding author: Yonglie Chao
引用本文:

赵曦, 巢永烈, 陈丕修, 卿列华. 人非ATP 酶依赖性调节颗粒13对人牙周膜细胞的调节作用[J/OL]. 中华口腔医学研究杂志(电子版), 2014, 8(02): 104-111.

Xi Zhao, Yonglie Chao, Pixiu Chen, Liehua Qin. The effect of hRpn13 (a subunit of 26S proteasome) regulating human periodontal ligament cells[J/OL]. Chinese Journal of Stomatological Research(Electronic Edition), 2014, 8(02): 104-111.

目的

探讨人非ATP 酶依赖性调节颗粒13(hRpn13)通过泛素-蛋白酶体通路(UPP)途径对人牙周膜细胞(PDLC)的作用,从而揭示其对PDLC 增殖、分化和功能的调节作用。

方法

通过转染敲除来改变PDLC 中hRpn13 的表达后,用MTT 法观测细胞形态,4′,6-二脒基-2-苯基吲哚(DAPI)来检测细胞增殖情况,通过检测碱性磷酸酶(ALP)和Ⅰ型胶原纤维的分泌情况来检测细胞成骨分化情况,最后通过检测NF-κB 受体活化子配体(RANKL)、骨保护素(OPG)、泛素化蛋白的改变来研究hRpn13 对PDLC 功能的调节作用及其可能机制。

结果

hRpn13 对成纤维细胞的增殖和ALP 的活性,Ⅰ型胶原蛋白以及RANKL、OPG 的表达起到了负性调节作用,同时hRpn13 使泛素化蛋白聚集减少。

结论

hRpn13 可以通过影响UPP 通路来调节成纤维细胞的增殖、分化和功能。

Objective

To investigate the role of human regulatory particle non-ATPase 13(hRpn13) on the ubiquitin-26S proteasome pathway in human periodontal ligament cells (PDLCs) and the effects of hRpn13 on cell proliferation, differentiation, and function of human PDLCs.

Methods

The effects of altered hRpn13 gene expression on cell proliferation, apoptosis and differentiation of PDLCs were examined by MTT assay, DAPI staining, ALP ELISA Kit and elevation of collagenⅠ. And κappa B ligand/osteoprotegerin (RANKL/OPG) and ubiquitin gene expression was detectd by western blot.

Results

hRpn13 played a negative role in the proliferation, ALP activity and expression of collagen Ⅰ, RANKL and OPG. Moreover, the expression of ubiquitined protein decreased with over expression of hRpn13.

Conclusion

HRpn13 can influence cell proliferation, differentiation, and function of human PDLCs by the ubiquitin-26S proteasome pathway.

图1 改变hRpn13 表达后的MTT 分析(以对照组为100 来比较) 注:图1A 为敲除组;图1B 为转染组;siRNA scramble:转入不敲除任何基因的序列,即对照组;hRpn13 siRNA:转入敲除hRpn13的序列;Empty vector:转入空载体,即对照组;hRpn13 vector:转入载有hRpn13 质粒的载体
图2 改变hRpn13 表达后细胞形态和DAPI 染色图片 注:A 为倒置显微镜下的细胞形态(×40);B 为荧光显微镜(×100)下DAPI 染色后的细胞形态图;H:未做任何处理的PDLC 正常细胞; hRpn13 siRNA:转入hRpn13 siRNA 干扰PDLC 细胞hRpn13 的基因表达; hRpn13 vector:转入hRpn13 vector 增加PDLC 细胞hRpn13 的基因表达
图3 改变hRpn13 表达后的碱性磷酸酶活性检测 注:图3A 为敲除组;图3B 为转染组;siRNA scramble:转入不敲除任何基因的序列,即对照组;hRpn13 siRNA:转入干扰hRpn13的序列;Empty vector:转入空载体,即对照组;hRpn13 vector:转入载有hRpn13 质粒的载体
表1 hRpn13 表达改变后Ⅰ型胶原纤维的分泌情况(μg/ml,±s)
图4 hRpn13 表达改变后OPG、RANKL 的表达变化 注:图4A 为干扰hRpn13 基因表达后(hRpn13 siRNA)RANKL、OPG mRNA 相对于对照组(siRNA scramble)的变化;图4B 为hRpn13 基因过表达后(hRpn13 vector)RANKL、OPG mRNA 表达相对于对照组(Empty vector)的变化
图5 hRpn13 表达改变后泛素化蛋白的表达变化 干扰hRpn13 基因表达后(hRpn13 siRNA)泛素化蛋白聚集相对于对照组(siRNA scramble)增加;hRpn13 基因过表达后(hRpn13 vector)泛素化蛋白相对于对照组(Empty vector)减少
[1]
Fretz JA, Shevde NK, Singh S, et al. Receptor activator of nuclear factor-kappaB ligand-induced nuclear factor of activated T Cells(C1) autoregulates its own expression in osteoclasts and mediates the up-regulation of tartrate-resistant acid phosphatase[J]. Mol Endocrinol, 2008,22(3):737-750.
[2]
Qiu XB, Quyang SY, Li CJ, et al. hRpn13/ADRM1/GP110 is a novel proteosome subunie that binds the deubiquitinating enzyme, UCH37[J]. EMBO J, 2006,25(24):5742-5753.
[3]
Husnjak K, Elsasser S, Zhang N, et al. Proteasome subunit Rpn13 is a novel ubiquitin receptor [J]. Nature, 2008,453(7194):481-488.
[4]
Nojima N, Kobayashi M, Shionome M, et al. Fibroblastic cells derived from bovine periodontal ligaments have the phenotypes osteoblast[J]. J Periodontal Res, 1990,25(3):179-185.
[5]
Higuchi Y, Kabasawa Y, Sato M, et al. Effect of recombinant human fibroblast growth factor-2 on bone formation in rabbit mandibular distraction models using beta-tricalcium phosphate[J]. Congenit Anom(Kyoto), 2010,50(2):95-104.
[6]
饶寒敏,徐荣辉,朱雅萍,等. 成纤维细胞成骨潜能的体外培养研究[J]. 中华骨科杂志, 1995,15(12):845-848.
[7]
邓廉夫,柴本甫,齐进. 损伤性和骨关节炎性滑膜细胞成骨作用的体外研究[J]. 中华骨科杂志, 1997,17(11):693-695.
[8]
Jørgensen JP, Lauridsen AM, Krisensen P, et al. Adrm1, a putative cell adhension regulating protein, is a novel proteasomeassociated factor[J]. J Mol Biol, 2006,28,360(5):1043-1052.
[9]
Kawano M, Ariyoshi W, Iwanaqa K, et al. Mechanism involved in enhancement of osteoblast differentiation by hyaluronic acid[J]. Biochem Biophys Res Commun, 2011,405(4):575-580.
[10]
Hong YJ, Chun JS, Lee WK. Association of collagen with calcium phosphate promoted osteogenic responses of osteoblastlike MG63 cells [J]. Colloids SurfB Biointerfaces, 2011,83(2):245-253.
[11]
Chien HH, Lin WL, Cho MI. Expression of TGF-beta isoforms and their receptors during minerized nodule formation by rat periodontal ligament cells in vitro[J]. J Periodontal Res, 1999,34(6):301-309.
[12]
Kostenuik PJ, Shalhoub V. Osteoprotegerin: a physiological and pharmacological inhibitor of bone resorption [J]. Curr Pharm Des, 2001,7(8):613-635.
[13]
Takahashi N,Maeda K,Ishihara A,et al. Regulatory mechanism of osteoclastogenesis by RANKL and Wnt signals [J]. Front Biosci(Landmark Ed), 2011(16):21-30.
[14]
Giuliani N,Bataille R,Mancini C, et al. Myeloma cells induce imbalance in the osteoprotegerin/osteoprotegerin ligand system in the human bone marrow enviroment [J]. Blood, 2001,98(13):3527-3533.
[15]
Anandarajah AP, Schwarz EM. Bone loss in the spondyloarthropathies:role of osteoclast,RANKL,RANK and OPG in the spondyloarthropathies[J]. Adv Exp Med Biol, 2009(649):85-99.
[16]
Boyce BF, Xing L. Functions of RANKL/RANK/OPG in bone modeling and remodeling [J]. Arch Biochem Biophys, 2008,473(2):139-146.
[17]
Belibasakis GN, Meier A, Guggenheim B, et al. The RANKLOPG system is differentially regulated by supragingival and subgingival biofilm supernatants [J]. Cytokine, 2011,55(1):98-103.
[18]
Borsje MA, Ren Y, de Haan-Visser HW, et al. Comparison of low-intensity pulsed ultrasound and pulsed electromagnetic field treatments on OPG and RANKL expression in human osteoblastlike cells[J]. Angle Orthod, 2010,80(3):498-503.
[19]
Kim T, Ha HI, Kim N, et al. Adrm1 interacts with Atp6v0d2 and regulates osteoclast differentiation [J]. Biochem Biophys Res Commun, 2009,390(3):585-590.
[20]
Glickrnan MH, Ciechanover A. The uhiquitin-proteasorne proLeolytic pathway: destruction for the sake of construction[J]. Physiol Rev, 2002,82(2):373-428.
[21]
Kessler BM, Edelmann MJ. PTMs in conversation: activity and function of deubiquitinating enzymes regulated via posttranslational modifications[J]. Cell Biochem Biophys, 2011,60(1-2):21-38.
[22]
Wrigley JD, Eckersley K, Hardern IM, et al. Enzymatic characterisation of USP7 deubiquitinating activity and inhibition[J]. Cell Biochem Biophys, 2011,60(1-2):99-111.
[23]
Hamazaki J, Iemura S, Natsume T, et al. A novel proteasome interacting protein recruits the deubipuitinating enzyme UCH37 to 26S proteasomes[J]. EMBO J, 2006,25(19):4524-4536.
[24]
Hölzl H, Kapelari B, Kellermann J, et al. The regulatory complex of Drosophlia melanogaster 26S proteasomes. Subunit composition and localization of a deubuquitylating enzyme[J]. J Cell Biol, 2000,150(1):119-130.
[25]
Uchiki T, Kim HT, Zhai B, et al. The ubiquitin-interacting motif protein, S5a, is ubiquitinated by all types of ubiquitin ligases by a mechanism different from typical substrate recognition[J]. J Biol Chem, 2009,284(19):12622-12632.
[26]
Elangovan M, Oh C, Sukumaran L, et al. Functional differences between two major ubiquitin receptors in the proteasome; S5a and hRpn13 [J]. Biochem Biophys Res Commun, 2010,396(2):425-428.
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