MTA对胎鼠颅骨细胞表达4种标志性蛋白mRNA 与细胞培养环境的关联研究

摘要/Abstract

摘要： 目的： 探讨MTA对胎鼠颅骨细胞标志性蛋白Cbfa1、ALP、Col-Ⅰ和 BGPmRNA 表达及细胞培养环境中钙、磷元素变化的关联及影响。方法： 采用2种酶混合多次消化，获得原代胎鼠颅骨细胞，将细胞与MTA共培养。采用RT-PCR技术检测Cbfα1 、ALP、Col-Ⅰ和 BGPmRNA在第4、7、14、21天时的表达变化，使用原子吸收分光光度法每2 d检测细胞外钙、磷元素的含量。采用SPSS 10.0软件包对数据进行统计学分析。结果： 第4天时，P3-含量显著降低（P<0.05），此时，MTA组ALPmRNA的增幅最大，峰值为对照组的40倍；第14~18天，Ca2+、P3-含量再次显著降低时（P<0.05），MTA组先是BGPmRNA的增幅最大，约为对照组的7.71倍；后是CbfαlmRNA增幅最大，约为对照组的7.38倍。各时间点Col-ⅠmRNA的增幅最低。结论： P3-含量的变化可能是MTA促进胎鼠颅骨细胞体外矿化的启动因素，当Ca2+积累到一定程度后，将大幅度加速矿化进程；同时调节ALPmRNA、BGPmRNA、Col-ⅠmRNA与CbfαlmRNA的表达，这可能是MTA利于骨质形成的关键。

关键词: 无机三氧化物聚合物, 成骨细胞, mRNA, 培养环境

Abstract: PURPOSE: To discuss the influence of MTA on mRNA expression of Cbfa1, ALP, Col-Ⅰand BGP which are 4 kinds of iconic protein in cells of fetal rats skull, and explore its influence on cell culture environment and association of changes of calcium, phosphorus. METHODS： Cells were obtained by 2 kinds of mixed enzymatic digestion for 3 steps from gestation fetal rat calvarial bone. The expression of Cbfa1mRNA, ALPmRNA, Col-1mRNA, BGP mRNA and extracellular calcium were detected. Phosphorus (P) and calcium concentration of fetal rat skull cells co-cultured with MTA for 3 weeks at different stages of cell differentiation was assessed atomic absorption spectrophotometry. The data was statistically analyzed using SPSS10.0 software package. RESULTS: At the 4th day, P3- content decreased significantly (P<0.05), while ALPmRNA in MTA group increased most greatly and was 40 times of the control group. At the 14th to 18th day, the Ca2+ and P3- content reduced significantly (P < 0.05), and then the BGPmRNA in MTA group rised most greatly which was about 7.71 times of the control group. Then Cbfalpha l mRNA in MTA group increased most strongly later which was about 7.38 times of the control group. Col Ⅰ mRNA increased minimally in all time points. CONCLUSIONS: The change of P3- content may be the initiating factor when MTA promoted differentiation of fetal rats skull cells in vitro, and Ca2+ could greatly accelerate the process of mineralization when accumulated to a certain extent. At the same time, the expression of ALPmRNA, BGPmRNA, Col Ⅰ mRNA and Cbfalpha lmRNA were regulated accordingly, which is the key to explain osteogenetic mechanism of MTA.

Key words: Mineral trioxide aggregate, Osteoblasts, mRNA, Culture environment

中图分类号:

R781.4

郑钧元，何俐，胡图强. MTA对胎鼠颅骨细胞表达4种标志性蛋白mRNA 与细胞培养环境的关联研究[J]. 上海口腔医学, 2016, 25(1): 16-21.

ZHENG Jun-Yuan, HE Li, HU Tu-qiang.. The relationship between the effects of MTA on mRNA expression of four iconic proteins in cells of fetal rat skull and cell culture environment[J]. Shanghai Journal of Stomatology, 2016, 25(1): 16-21.

参考文献

[1] Caron G, Azérad J, Faure MO, et al. Use of a new retrograde filling material (Biodentine) for endodontic surgery: two case reports [J]. Int J Oral Sci, 2014, 6(4): 250-253.
[2] Huang SC, Wu BC, Kao CT, et al. Role of the p38 pathway in mineral trioxide aggregate-induced cell viability and angiogenesis-related proteins of dental pulp cell in vitro[J]. Int Endod J, 2015, 48(3): 236-245.
[3] Yan M, Wu J, Yu Y, et al. Mineral trioxide aggregate promotes the odonto/osteogenic differentiation and dentinogenesis of stem cells from apical papilla via nuclear factor kappa B signaling pathway [J]. J Endod, 2014, 40(5): 640-647.
[4] Shirvani A, Hassanizadeh R, Asgary S. Mineral trioxide aggregate vs. calcium hydroxide in primary molar pulpotomy: a systematic review [J]. Iran Endod J, 2014, 9(2): 83-88.
[5] Torabinejad M, Hong CU, Lee SJ, et al. Investigation of mineral trioxide aggregate for root-end filling in dogs [J]. J Endod, 1995, 21(12): 603-608.
[6] Duan X, Xu H, Wang Y, et al. Expression of core-binding factor alpha1 and osteocalcin in fluoride-treated fibroblasts and osteoblasts [J]. J Trace Elem Med Biol, 2014, 28(3): 278-283.
[7] Lynch MP, Stein JL, Stein GS, et al. The influence of type I collagen on the development and maintenance of the osteoblast phenotype in primary and passaged rat calvarial osteoblasts: modification of expression of genes supporting cell growth, adhesion, and extracellular matrix mineralization[J]. Exp Cell Res, 1995, 216(1): 35-45.
[8] Stein GS, Lian JB, Owen TA. Bone cell differentiation: a functionally coupled relationship between expression of cell-growth- and tissue-specific genes [J]. Curr Opin Cell Biol, 1990, 2(6): 1018-1027.
[9] Fensky F, Reichert JC, Traube A, et al. Chondrogenic predifferentiation of human mesenchymal stem cells in collagen type I hydrogels [J]. Biomed Tech (Berl), 2014, 59(5): 375-383.
[10] Pérez AL, Spears R, Gutmann JL, et al. Osteoblasts and MG-63 osteosarcoma cells behave differently when in contact with ProRoot MTA and White MTA[J]. Int Endod J, 2003, 36(8): 564-570.
[11] 李晓霞, 唐明, 邓旭亮, 等. 国产无机三氧化物聚合物的生物相容性研究 [J]. 第四军医大学学报, 2009, 30(20): 2157-2159.
[12] Opperman LA, Passarelli RW, Morgan EP, et al. Cranial sutures require tissue interactions with dura mater to resist osseous obliteration in vitro [J]. J Bone Miner Res, 1995, 10(12): 1978-1987.
[13] Gordin DM, Gloriant T, Chane-Pane V, et al. Surface characterization and biocompatibility of titanium alloys implanted with nitrogen by Hardion+ technology [J]. J Mater Sci Mater Med, 2012, 23(12): 2953-2966.
[14] 傅德皓,杨述华,马德彰,等.鼠胚成骨细胞的原代培养与鉴定 [J]. 创伤外科杂志, 2006, 8(2): 157-160.
[15] 周国君. 原子吸收法测定血清中的无机磷和钙 [J]. 分析测试通报, 1989, 8(4): 36-39.
[16] Zhao X, He W, Song Z, et al. Mineral trioxide aggregate promotes odontoblastic differentiation via mitogen-activated protein kinase pathway in human dental pulp stem cells[J]. Mol Biol Rep, 2012, 39(1): 215-220.
[17] 杨琳. ERK/p38信号通路在MTA诱导的人根尖乳头细胞分化过程中的作用[D]. 济南: 山东大学, 2014.
[18] Montgomery SR, Nargizyan T, Meliton V, et al. A novel osteogenic oxysterol compound for therapeutic development to promote bone growth: activation of hedgehog signaling and osteogenesis through smoothened binding[J]. J Bone Miner Res, 2014, 29(8): 1872-1885.
[19] Shapiro R, Heaney RP. Co-dependence of calcium and phosphorus for growth and bone development under conditions of varying deficiency [J]. Bone, 2003, 32(5): 532-540.
[20] Caverzasio J, Bonjour JP. Characteristics and regulation of Pi transport in osteogenic cells for bone metabolism[J]. Kidney Int, 1996, 49(4): 975-980.
[21] Ploumis A, Donovan JM, Olurinde MO, et al. Association between alendronate, serum alkaline phosphatase level, and heterotopic ossification in individuals with spinal cord injury[J]. J Spinal Cord Med, 2015, 38(2): 193-198.
[22] Laager R, Ninnis R, Keller U. Comparison of the effects of recombinant human insulin-like growth factor-I and insulin on glucose and leucine kinetics in humans[J]. J Clin Invest, 1993, 92(4): 1903-1909.
[23] 张伟国. 成骨细胞表型分化及基因调控研究进展 [J]. 上海口腔医学, 1998, 7(3): 57-59.
[24] Wu M, Li C, Zhu G, et al. Deletion of core-binding factor beta (Cbfbeta) in mesenchymal progenitor cells provides new insights into Cbfbeta/Runxs complex function in cartilage and bone development [J]. Bone, 2014, 65(1): 49-59.
[25] Chen Y, Yan Y, Li X, et al. Application of ultrasound on monitoring the evolution of the collagen fiber reinforced nHAC/CS composites in vivo [J]. Biomed Res Int, 2014, 2014: 418302.
[26] 于明香, 金慰芳, 顾淑珠, 等. 体外培养人成骨细胞骨相关基因表达 [J]. 中国骨质疏松杂志, 2002, 8(3): 195-198.
[27] Zhou W, Liu Z, Yao J, et al. The effects of exenatide microsphere on serum BGP and ALP levels in ZDF rats after implantation [J]. Clin Implant Dent Relat Res, 2015,17(4):765-770.
[28] 李云飞, 祝传宝, 何文, 等. 钙体内研究进展[J]. 中南药学, 2006, 4(1): 46-50.
[29] Masquelier D, Herbert B, Hauser N, et al. Morphologic characterization of osteoblast-like cell cultures isolated from newborn rat calvaria [J]. Calcif Tissue Int, 1990, 47(2): 92-104.