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Volume 46 Issue 1
Feb.  2021
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Effect of nerve growth factor on the proliferation and differentiation of rabbit dental pulp stem cells in vitro

  • Received Date: 2019-12-26
    Accepted Date: 2020-12-01
  • ObjectiveTo investigate the effects of nerve growth factor(NGF) on the proliferation and differentiation of rabbit dental pulp stem cells(DPSCs) in vitro, and provide a new clue for the repair of dental pulp tissue.MethodsThe DPSCs from healthy New Zealand rabbits were isolated and cultured by tissue enzymedigestion method, and the morphology and growth changes of cells were observed under light microscope.The third passage DPSCs were divided into the blank group(only DPSCs), experimental group(DPSCs mixed with 100 g/L NGF) and control group(DPSCs mixed with mineralization solution).The cell morphology and alkaline phosphatase(ALP) activity in three groups were investigated after 7 days and 14 days of culture, and the mRNA expression levels of COL-1, Runx-2 and OCN were detected using RT-qPCR.ResultsThe DPSCs cultured in vitro grew well, adhered to the wall, and most of them were polygonal or spindle shaped with nest or colony growth.The ALP activity value in three groups after 14 d of culture were higher than that on the 7th day of culture, and the differences of which among three groups after 7 and 14 d of culture were significant(P < 0.05).The results of RT-qPCR showed that the expression levels of COL-1, Runx-2 and OCN mRNA in experimental group after 14 d of culture were higher than those in blank group(P < 0.05).ConclusionsThe NGF can promote the proliferation and osteogenic differentiation of rabbit DPSCs, and the combined application effect of the two is better than that of DPSCs alone.
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  • [1] HWANG HI, LEE TH, KANG KJ, et al. Immunomic screening of cell surface molecules on undifferentiated human dental pulp stem cells[J]. Stem Cells Dev, 2015, 24(16): 1934. doi: 10.1089/scd.2014.0493
    [2] GALLER KM, WIDBILLER M, BUCHALLA W, et al. EDTA conditioning of dentine promotes adhesion, migration and differentiation of dental pulp stem cells[J]. Int End J, 2016, 49(6): 581. doi: 10.1111/iej.12492
    [3] YUSOF MFH, ZAHARI W, HASHIM SNM, et al. Angiogenic and osteogenic potentials of dental stem cells in bone tissue engineering[J]. J Oral Biol Craniofac Res, 2018, 8(1): 48. doi: 10.1016/j.jobcr.2017.10.003
    [4] 庄友梅, 木合塔尔·霍加, 徐辉, 等. 转化生长因子-β3和牙髓干细胞在兔面神经损修复中的作用[J]. 中华实用诊断与治疗杂志, 2015, 29(7): 637.
    [5] STROJNY C, BOYLE M, BARTHOLOMEW A, et al. Interferon gamma-treated dental pulp stem cells promote human mesenchymal stem cell migration in vitro[J]. J Endod, 2015, 41(8): 1259. doi: 10.1016/j.joen.2015.02.018
    [6] DECROLI E, MANAF A, SYAHBUDDIN S, et al. The correlation between malondialdehyde and nerve growth factor serum level with diabetic peripheral neuropathy score[J]. Open Access Maced J Med Sci, 2019, 7(1): 103 doi: 10.3889/oamjms.2019.029
    [7] JIANG L, YE B, WANG Y, et al. Effect and mechanisms of sacral nerve stimulation on visceral hypersensitivity mediated by nerve growth factor[J]. J Cell Mol Med, 2019, 23(12): 8019. doi: 10.1111/jcmm.14660
    [8] 崔传举, 袁树华, 王玉香, 等. 鼠神经生长因子联合神经节苷脂治疗脑出血的效果观察[J]. 河南医学研究, 2018, 27(12): 2173. doi: 10.3969/j.issn.1004-437X.2018.12.024
    [9] HWANG HI, LEE TH, KANG KJ, et al. Immunomic screening of cell surface molecules on undifferentiated human dental pulp stem cells[J]. Stem Cells Dev, 2015, 24(16): 1934. doi: 10.1089/scd.2014.0493
    [10] GALLER KM, WIDBILLER M, BUCHALLA W, et al. EDTA conditioning of dentine promotes adhesion, migration and differentiation of dental pulp stem cells[J]. Int Endod J, 2016, 49(6): 581. doi: 10.1111/iej.12492
    [11] GRONTHOS S, ARTHUR A, BARTOLD PM, et al. A method to isolate and culture expand human dental pulp stem cells[J]. Methods Mol Bid, 2011, 698: 107.
    [12] HILKENS P, DRIESEN RB, WOLFS E, et al. Cryopreservation and banking of dental stem cells[J]. Adv Exp Med Biol, 2016, 951: 199.
    [13] TAKEBE Y, TATEHARA S, FUKUSHIMA T, et al. Cryopreservation method for the effective collection of dental pulp stem cells[J]. Tissue Eng Part C Methods, 2017, 23(5): 251. doi: 10.1089/ten.tec.2016.0519
    [14] CRISTALDI M, MAUCERI R, TOMASELLO L, et al. Dental pulp stem cellsfor bone tissue engineering: a review of the current literature and alook to the future[J]. Regen Med, 2018, 13(2): 207. doi: 10.2217/rme-2017-0112
    [15] 崔国胜, 曾剑玉, 张婧, 等. 神经生长因子对2型糖尿病小鼠骨髓基质细胞体外成骨能力的影响[J]. 中华口腔医学杂志, 2018, 53(2): 97. doi: 10.3760/cma.j.issn.1002-0098.2018.02.005
    [16] HUAI WJ, HAN XF, ZHU YH. Effect of pereutaneous local injection of nerve growth factor on fracture healing[J]. China Pharms, 2015, 24(9): 41.
    [17] LIU YT, HUANG CT, CHEN LF, et al. The research on rat nerve growth factor in the treatment of tibial fracture nonunion[J]. Chin J Clin Anat, 2016, 34(3): 326.
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Effect of nerve growth factor on the proliferation and differentiation of rabbit dental pulp stem cells in vitro

  • 1. Department of Stomatology, The First Affiliated Hospital of Bengbu Medical College, Bengbu Anhui 233004
  • 2. Department of Stomatology Bengbu Medical College, Bengbu Anhui 233030, China

Abstract: ObjectiveTo investigate the effects of nerve growth factor(NGF) on the proliferation and differentiation of rabbit dental pulp stem cells(DPSCs) in vitro, and provide a new clue for the repair of dental pulp tissue.MethodsThe DPSCs from healthy New Zealand rabbits were isolated and cultured by tissue enzymedigestion method, and the morphology and growth changes of cells were observed under light microscope.The third passage DPSCs were divided into the blank group(only DPSCs), experimental group(DPSCs mixed with 100 g/L NGF) and control group(DPSCs mixed with mineralization solution).The cell morphology and alkaline phosphatase(ALP) activity in three groups were investigated after 7 days and 14 days of culture, and the mRNA expression levels of COL-1, Runx-2 and OCN were detected using RT-qPCR.ResultsThe DPSCs cultured in vitro grew well, adhered to the wall, and most of them were polygonal or spindle shaped with nest or colony growth.The ALP activity value in three groups after 14 d of culture were higher than that on the 7th day of culture, and the differences of which among three groups after 7 and 14 d of culture were significant(P < 0.05).The results of RT-qPCR showed that the expression levels of COL-1, Runx-2 and OCN mRNA in experimental group after 14 d of culture were higher than those in blank group(P < 0.05).ConclusionsThe NGF can promote the proliferation and osteogenic differentiation of rabbit DPSCs, and the combined application effect of the two is better than that of DPSCs alone.

  • 牙髓干细胞(dental pulp stem cells,DPSCs)是外胚层来源的干细胞,具有较高的细胞活性及多向分化潜能,因其取材丰富、易于培养等优点一直受到研究者的青睐。DPSCs可参与牙髓的修复与重建[1-2],具有高增殖、自我更新的能力,在适当的诱导下可以分化为脂肪组织、软骨组织和神经组织等,因其高增殖率、高克隆潜力、高矿化潜力和较低的免疫原性,目前被认为是口腔医学中骨组织工程的种子细胞[3-5]

    神经生长因子(nerve growth factor, NGF)是目前医学上应用较为广泛的外源性调节因子,具有营养神经和修复神经损伤等方面的作用[6-7],在伤害性感受器的功能调节中起关键作用。外源性NGF还可通过注射的方式补充内源性NGF的含量,避免其凋亡,保护受损的神经元[8]。本研究在前期的实

    验研究中发现NGF可以在体外将骨髓间充质干细胞定向分化为软骨并促进其向成骨方向分化,但NGF对DPSCs的影响机制尚不明确, 且NGF联合DPSCs用于种植体周围是否能够促进种植体早期骨结合也尚未可知。因此,本研究旨在探讨NGF对兔DPSCs的增殖活性及成骨分化的影响,分析NGF和DPSCs的联合应用对牙髓损伤的修复,以期为探索NGF和DPSCs促进骨生成提供一定的依据和思路。

1.   资料与方法
  • 选取健康的新西兰兔3只,3~4周龄,体质量1.5~2.0 kg,雌雄不限(由东方集团提供)。饲养环境:温度(25±2)℃、湿度(60±5)%,动物生产许可证号SCXK(苏)2015-0005。新西兰兔于蚌埠医学院科研中心适应性喂养1周后用于实验。主要试剂:胎牛血清(FBS)(美国GIBCO公司)、DMEM培养液(美国Hyclone公司)、0.25% EDTA胰蛋白酶消化液(北京Solarbio公司)、青霉素-链霉素混合液(美国Hyclone公司)、Ⅰ型胶原酶(北京Solarbio公司)。仪器:细胞培养箱(德国Eppendorf公司)、离心机(上海卢湘仪)、倒置显微镜(上海蔡康光学)、超净台(苏州德汇)、移液枪(北京康林)。

  • 在局麻环境下拔除兔前牙及前磨牙,放入三倍双抗液中浸泡30 min,在超净台中用0.1 mmol/L的PBS溶液冲洗3次,用无菌拔髓针拔出牙髓组织,剪去距根尖1mm左右组织,余下牙髓组织剪成1 mm×1 mm×1 mm大小块状,置于15 mL离心管中。在离心管中加入3 g/L Collagenase-1和4 g/L分解酶各1 mL,混匀后置入37 ℃水浴中消化50 min,观察细胞的消化情况。通过细胞筛网(孔径200目)筛拣出未通过的组织块,用0.1 mmol/L的PBS溶液冲洗3次,加入培养液后,置入CO2培养箱中,待细胞贴壁后,3 d换液一次。

  • 显微镜下观察细胞形态的变化,瓶底的细胞铺满80%左右时,去除原培养液,用PBS溶液冲洗3次。加适量的胰蛋白酶,置于37 ℃的细胞培养箱中孵育3 min。后离心加入培养基,放入培养箱中培养,每隔3 d换一次液,在倒置显微镜下观察细胞的生长,2~5代的细胞用于后续实验。

  • 将生长良好的第3代DPSCs设置为3组:空白组为纯DPSCs组,含10%FBS,1%双抗的DMEM高糖培养液;实验组为DPSCs加NGF组,10%FBS,1%双抗的DMEM高糖培养液加入100 μg/L的NGF;对照组为DPSCs加矿化液组,10%FBS,1%双抗的DMEM高糖培养液加入10 mmol/L β-甘油磷酸钠、100 nmol/L地塞米松、50 mg/L抗坏血酸。分别在7、14 d后收集细胞进行后续实验。

  • 第3代DPSCs以5×104个/孔细胞接种于24孔板中,随机分为3组,在培养7、14 d时各组取一个24孔板,用0.1 mmol/L PBS冲洗3次,490 nm处酶标仪测吸光度值计算裂解液中ALP水平。

  • 取第三代细胞1×106个/孔接种于6孔板中分组培养,分别于培养7、14 d时提取细胞RNA,第一链cDNA模板通过逆转录试剂盒合成,以β-actin为内参对照,设置无模板阴性对照。PCR反应条件: 50 ℃ 2 min,95 ℃ 10 min,95 ℃ 30 s,60 ℃ 30 s,40循环(引物序列见表 1)。

    基因 正向引物
    反向引物
    产物大小
    COL-1 5′- TGC TTG AAG ACC CGA GTG G-3′ 5′-GGT TGC TGG CAG GAC AAT G-3′ 151 bps
    Runx-2 5′- ATG GGA CTG TGG TTA CTG-3′ 5′- GGG AGG ATT TGT GAA GAC-3′ 176 bps
    OCN 5′- TCA GCA AGC AGG AGT ATG-3′ 5′- CCA ATC TCG TCT CGT TTC-3′ 133 bps
  • 采用t检验、方差分析和q检验。

2.   结果
  • 酶解组织法顺利培养出兔DPSCs,2 d即可见周围组织块有细胞游出(见图 1),细胞呈集落生长,胞质均匀,形状呈圆形。5~6 d后可汇合达80%,此时可进行再次传代。实验7、14 d时3组细胞形态见图 2。实验7 d时,与空白组和对照组比较,实验组细胞呈聚集性生长,梭形细胞逐渐向多角形分化;14 d时,实验组细胞间隙增多,多角形改变更加明显。

  • 实验14 d时的空白组、实验组和对照组的ALP活性值均高于7 d时的活性值(P < 0.01);实验7 d和14 d时3组间ALP活性值差异均有统计学意义(P < 0.01),实验组高于空白组(P < 0.05),对照组高于实验组(P < 0.05)(见表 2)。

    分组 n 7 d 14 d t P
    空白组 6 0.21±0.01 0.25±0.01 6.04 < 0.01
    实验组 6 0.30±0.01* 0.39±0.02* 8.46 < 0.01
    对照组 6 0.39±0.02*# 0.45±0.01*# 7.75 < 0.01
    F 206.95 325.12
    P < 0.01 < 0.01
    MS组内 0.000 0.000
    q检验:与空白组比较*P < 0.05;与实验组比较#P < 0.05
  • 各组培养在7 d时实验组中COL-1和OCN基因的表达值均高于空白组(P < 0.05);培养14 d时实验组中COL-1、Runx-2和OCN基因的表达值均高于空白组(P < 0.05)(见表 3~5)。

    分组 n 7 d 14 d t P
    空白组 6 4.71±0.89 4.46±1.02 0.46 >0.05
    实验组 6 9.82±1.48* 9.07±1.78* 0.80 >0.05
    对照组 6 15.33±3.85*# 12.65±2.97*# 1.34 >0.05
    F 28.44 23.28
    P < 0.01 < 0.01
    MS组内 5.943 4.351
    q检验:与空白组比较*P < 0.05;与实验组比较#P < 0.05
    分组 n 7 d 14 d t P
    空白组 6 0.72±0.31 5.44±2.07 5.51 0.01
    实验组 6 1.22±0.41 10.84±3.97* 2.04 >0.05
    对照组 6 2.17±0.83*# 11.24±4.77 4.59 < 0.01
    F 10.20 4.42
    P < 0.01 >0.05
    MS组内 0.319 31.527
    q检验:与空白组比较*P < 0.05;与实验组比较#P < 0.05
    分组 n 7 d 14 d t P
    空白组 6 0.48±0.14 4.61±1.74 5.79 < 0.01
    实验组 6 1.27±0.46* 10.53±3.78* 5.95 < 0.01
    对照组 6 1.33±0.49* 12.47±4.56* 5.95 < 0.01
    F 8.67 7.92
    P < 0.01 < 0.01
    MS组内 0.157 12.709
    q检验:与空白组比较*P < 0.05
3.   讨论
  • DPSCs具有成骨分化能力,而且其是存在于牙髓组织中的一类成体干细胞,具有高度自我更新能力和多向分化潜能,在口腔颅颌面部的缺损修复中具有一定的同源优越性。自GRONTHOS等[11]成功从成人牙髓源性细胞中分离出间充质干细胞以来,DPSCs就开始在再生医学的应用中发挥着重要的作用。

    NGF是多种组织再生中的理想细胞因子,由于其潜在的成骨诱导作用,也让其成为近期的研究热点[12-13]。NGF是在1952年发现的第一个神经营养因子。早期研究证实了NGF是重要的神经营养蛋白,可以促进神经的再生、营养和发育[14]。相关研究[15-16]显示NGF有很好的促进骨修复的作用,可以促进兔胫骨骨折部位的愈合,也能促进人体四肢骨的骨折愈合,使其愈合时间缩短。近期研究[17]显示,NGF可在小鼠肋骨的骨折部位检测到,2 d达到高峰值,说明NGF与骨的形成有关。

    NGF可加速干细胞向成骨方向分化,因此,本研究拟在前期基础上,通过ALP检测、RT-qPCR等技术明确DPSCs体外在NGF作用下的成骨影响,研究结果, 发现实验组ALP水平明显高于空白组,RT-qPCR结果显示14 d时实验组中COL-1、Runx-2和OCN基因的表达量均高于空白组,具有一定加速成骨向分化的作用。

    综上所述,本研究证实NGF体外能够促进兔DPSCs向成骨向分化,缩短临床种植骨结合修复的时间,为其运用到颌面部及种植体周围骨缺损的修复提供新的视角和思路。

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