吉林大学学报(医学版)  2020, Vol. 46 Issue (01): 56-60     DOI: 10.13481/j.1671-587x.20200110

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李雪洋, 尹硕, 谢金芳, 李晶, 胡雪, 耿文韬, 张颖丽
LI Xueyang, YIN Shuo, XIE Jinfang, LI Jing, HU Xue, GENG Wentao, ZHANG Yingli
促红细胞生成素对大鼠即刻再植牙牙髓血运重建的促进作用
Promotion effect of erythropoietin on immediate revascularization of replanted teeth in rats
吉林大学学报(医学版), 2020, 46(01): 56-60
Journal of Jilin University (Medicine Edition), 2020, 46(01): 56-60
10.13481/j.1671-587x.20200110

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收稿日期: 2019-07-07
促红细胞生成素对大鼠即刻再植牙牙髓血运重建的促进作用
李雪洋1 , 尹硕2 , 谢金芳1 , 李晶1 , 胡雪1 , 耿文韬1 , 张颖丽1     
1. 吉林大学口腔医院牙体牙髓科, 吉林 长春 130021;
2. 吉林省长春市口腔医院修复科, 吉林 长春 130022
[摘要]: 目的 探讨血管内皮细胞生长因子(VEGF)在大鼠即刻再植牙牙髓愈合过程中的表达,阐明促红细胞生成素(EPO)对大鼠即刻再植牙牙髓血运重建的作用及机制。方法 80只4周龄健康雄性Wistar大鼠随机分为未拔牙组、阴性对照(生理盐水)组、阳性药对照(庆大霉素)组和EPO组,每组20只,各组大鼠拔除的牙齿在其相对应的溶液中浸泡4min后再植,于再植术后3、7、14、21和28 d分别处死4只大鼠,制作术区标本,行HE染色观察各组大鼠在不同时间段内再植牙牙髓血运重建的情况,免疫组织化学染色观察不同时间段各组大鼠再植牙牙体组织中VEGF蛋白表达水平。结果 HE染色,与未拔牙组比较,生理盐水组大鼠再植牙牙髓组织中炎症较重,牙根发育情况较差,修复性牙本质和牙骨质沉积较少,根尖孔较宽;庆大霉素组和EPO组大鼠再植牙牙髓组织中炎症较轻,牙根发育情况相对较好,修复性牙本质和牙骨质沉积较多,根尖孔缩窄。免疫组织化学染色,与未拔牙组比较,3、7和14 d时生理盐水组、庆大霉素组和EPO组大鼠再植牙牙体组织中VEGF呈强阳性表达,随着时间的延长,VEGF阳性表达强度逐渐减弱;VEGF阳性表达区的平均吸光度(AOD)值,EPO组 > 庆大霉素组 > 生理盐水组 > 未拔牙组。与未拔牙组比较,3、7、14和21 d时生理盐水组、庆大霉素组和EPO组大鼠牙体组织中VEGF蛋白表达水平明显升高(P < 0.05),而在28 d时各组大鼠牙体组织中VEGF蛋白表达水平差异无统计学意义(P > 0.05);与生理盐水组比较,3、7、14和21d时庆大霉素组和EPO组大鼠牙体组织中VEGF蛋白表达水平明显升高(P < 0.05),而在28 d时庆大霉素组和EPO组大鼠牙体组织中VEGF蛋白表达水平差异无统计学意义(P > 0.05);与庆大霉素组比较,各时间点EPO组大鼠牙体组织中VEGF蛋白表达水平差异均无统计学意义(P > 0.05)。结论 EPO通过上调VEGF表达,诱导牙髓组织中血管新生,为再植牙提供了丰富的血管床,从而发挥牙髓的防御修复潜能,促进了再植牙的牙髓愈合。
关键词: 促红细胞生成素    再植牙    牙髓血运重建    血管内皮细胞生长因子    
Promotion effect of erythropoietin on immediate revascularization of replanted teeth in rats
LI Xueyang1 , YIN Shuo2 , XIE Jinfang1 , LI Jing1 , HU Xue1 , GENG Wentao1 , ZHANG Yingli1     
1. Department of Endodontics, Stomatology Hospital, Jilin University, Changchun 130021, China;
2. Department of Prosthodontics, Changchun Stomatology Hospital, Jilin Province, Changchun 130022, China
[ABSTRACT]: Objective To investigate the expression of vascular endothelial cell growth factor (VEGF) in the immediate replantation of pulp healing in the rats, and to clarify the effect and its mechanism of erythropoietin (EPO) on immediate pulp reconstruction in the rats. Methods Eighty 4-week-old healthy male Wistar rats were randomly divided into non-tooth extraction group, negative control (normal saline) group, positive control (gentamicin) group and EPO group; there were twenty rats in each group.The teeth in each group were immersed in its corresponding solution for 4 min before replantation. Four rats were killed on the days 3, 7, 14, 21 and 28, respectively, and the specimens were made in the operation area. HE staining was used to observe the pulp revascularization in different time periods. Immunohistochemical staining was used to observe the protein expression levels of VEGF in odontal tissue of the rats in each group in different time periods. Results The HE staining results showed that compared with non-tooth extraction group, the pulp tissue of replanted teeth of the rats in normal saline group had more inflammation, less root development, less restorative dentin and cementum deposition, and wider apical pores; the inflammation of pulp tissue of replanted teeth of the rats in gentamicin group and EPO group was mild, and the root development was relatively good; there were more deposits of restorative dentin and cementum, and the apical pores were narrowed. The immunohistochemical results showed that compared with non-tooth extraction group, the positive expressions of VEGF in odontal tissue of the rats at the days 3, 7 and 14 in the other groups were strong. Afterwards, the positive expression levels of VEGF were decreased gradually with the prolongation of time. The average optical density (AOD) of VEGF positive area indicated that EPO group > gentamycin group > normal saline group > non-tooth extraction group. Compared with non-tooth extraction group, the protein expression levels of VEGF in odontal tissue of the rats in normal saline group, gentamicin group and EPO group at 3, 7, 14 and 21 d after operation were significanty increased (P < 0.05), but the protein expression levels of VEGF in odontal tissue of the rats at 28 d had no significant differences (P > 0.05). Compared with normal saline group, the protein expression levels of VEGF in odontal tissue of the rats in gentamicin group and EPO group at 3, 7, 14 and 21 d after operation were significantly increased (P < 0.05), but the protein expression levels of VEGF in odontal tissue of the rats at 28 d had no significant differences (P > 0.05).Compared with gentamicin group, the protein expression levels of VEGF in odontal tissue of the rats in EPO group at every time points had no significant differences(P > 0.05). Conclusion: EPO can increase the expression of VEGF, induce angiogenesis in pulp tissue, and provide the rich vascular bed for replantated teeth, so as to exert the potential of dental pulp defense and repair, and promote the healing of replanted teeth.
KEYWORDS: erythropoietin    replanted of teeth    pulp revascularization    vascular endothelial cell growth factor    

牙外伤作为临床上十分普遍的牙急性损伤,多发生于年轻恒牙[1], 而牙外伤中以牙脱位较为常见,此时青少年正处于生长发育期,根尖孔尚未完全闭合。牙脱位会带来美学、功能和心理上的负担,鉴于生物学和心理学上的支持[2],国际牙外伤协会(IADT)指南建议即刻再植是治疗牙脱位的最佳方法,同时该指南指出为了获得牙髓血运重建和牙根的持续发育,对根尖孔开放的全脱位牙来说,除非有临床或影像学证据显示牙髓坏死,否则应避免进行根管治疗[3]。MELO等[4]研究表明:在理想的情况下,牙髓的血运重建和牙根的继续发育是可能发生的,因此再植牙的牙髓治疗并不适用于牙根尚未发育完全的牙齿。随着研究的深入,人们逐渐认识到牙髓细胞具有再生潜能,细胞迁移和血管新生在牙髓修复再生中均具有重要的作用[5-6]。因此,如何诱导牙髓组织内血管新生以发挥牙髓的防御修复潜能,是牙髓修复再生和临床活髓保存研究的重点。研究[7-10]显示:促红细胞生成素(erythropoietin,EPO)可通过募集基质细胞至受损区域促进组织的再生修复,体内外实验也已证实EPO在脑缺血再灌注的保护、烧伤皮肤的愈合和股骨头坏死修复中均发挥了促进血管新生的作用。目前对于再植牙的研究则大多集中于储存介质和牙周组织的愈合[11],而EPO在再植牙牙髓修复方面的研究较少。因此本研究旨在探讨EPO对再植牙牙髓血运重建的影响,为研究牙髓损伤的修复再生提供理论依据。

1 材料与方法 1.1 实验动物、主要试剂及仪器

80只3周龄雄性Wistar大鼠购自吉林大学实验动物中心,动物许可证号:SCXF(吉)2015-0001,标准饲粮、随机加水喂养,使其适应环境1周,术前体质量约100 g。EPO(沈阳三生制药有限责任公司),庆大霉素(上海现代哈森药业有限公司),生理盐水(辽宁民康药业有限公司),血管内皮细胞生长因子(vascular endothelial cell growth factor,VEGF)抗体和SABC免疫组织化学染色试剂盒(武汉博士生物工程有限公司),DAB显色试剂盒(北京中山金桥生物技术有限公司)。双目光学显微镜(吉林大学口腔医院病理教研室提供,配有日本QLYMPUS照相机)。ImageProPlus6.0(For windows)专业图像分析软件包,SPSS24.0统计软件。

1.2 实验动物分组和给药

80只4周龄雄性Wistar大鼠随机分为未拔牙组、阴性对照(生理盐水)组、阳性药对照(庆大霉素)组和EPO组,再根据观察时间随机分为3、7、14、21和28 d组。除外未拔牙组,其他3组大鼠固定后用30 g ·L-1水合氯醛(30 mL·kg-1体质量)腹腔注射麻醉,显效后用自制拔牙钳完整拔出上颌第一磨牙,将拔出的牙齿置于无菌一次性器械盘中,分别在生理盐水、庆大霉素和EPO溶液中浸泡4 min,再轻柔地植回牙槽窝内,实现牙齿在5 min内再植。牙再植术后严密观察大鼠复苏情况,自由饮水和摄食,每100 g饲料中混入阿莫西林0.5g,疗程为1周。

1.3 标本采集和处理

分别在再植术后3、7、14、21和28 d取各组大鼠上颌第一磨牙及其周围组织,用4%多聚甲醛溶液4℃下固定24~48h,于10%EDTA溶液中脱钙12周,然后将标本放入梯度乙醇中脱水,浸蜡,包埋,通过牙齿颊舌面沿牙齿长轴过根尖孔做厚度为3μm的连续切片。

1.4 免疫组织化学染色和HE染色

将石蜡切片放入烤箱中2 h,常规脱蜡水化,PBS冲洗后用3%双氧水灭活10 min,PBS洗3次;再放入EDTA抗原修复液中进行高压修复2 min,待冷却至室温时,PBS洗3次;随后滴加5%BSA封闭液封闭30 min,滴加VEGF抗体过夜;隔天PBS洗3次后滴加二抗30 min,PBS冲洗后滴加SABC 30 min,PBS冲洗后滴加DAB显示剂,显微镜下观察反应,终止后复染、分色、返蓝和透明,中性树脂封片,400倍显微镜下观察拍照,观察VEGF阳性表达情况,用ImageProPlus6.0专业图像分析软件包测定上述切片中VEGF平均吸光度(AOD)值,代表VEGF蛋白表达水平。经HE染色,中性树脂封片,200倍显微镜下观察再植牙的牙根发育情况。

1.5 统计学分析

采用SPSS24.0统计软件进行统计学分析。大鼠再植牙牙体组织中VEGF蛋白表达水平以x±s表示,组间比较采用SNK-q检验。以P < 0.05为差异有统计学意义。

2 结果 2.1 免疫组织化学染色检测各组大鼠牙体组织中VEGF蛋白表达

各组大鼠再植牙牙体组织中,成牙本质细胞、前期牙本质、血管内皮细胞和牙髓细胞中VEGF蛋白呈阳性表达;与固有髓核比较,各组大鼠牙体组织中VEGF蛋白在成牙本质细胞层阳性表达出现的时间较早且较强。与未拔牙组比较,其他3组大鼠再植牙牙体组织在3、7和14 d时VEGF蛋白呈强阳性表达,随着时间的推移,VEGF蛋白阳性表达强度逐渐减弱。与未拔牙组和生理盐水组比较,庆大霉素组和EPO组大鼠再植牙牙体组织中VEGF蛋白的表达均较强。见图 1(插页四)。

图 1 术后不同时间各组大鼠再植牙牙体组织中VEGF蛋白表达情况(免疫组织化学,×400) Fig. 1 Expressions of VEGF protein in tooth tissue of replanted teeth of rats in various groups at different time after operation(Immunohistochemistry, ×400)
2.2 各组大鼠再植牙牙体组织中VEGF蛋白表达水平

未拔牙组、生理盐水组、庆大霉素组和EPO组大鼠再植牙牙体组织的AOD值从高到低依次为:EPO组>庆大霉素组>生理盐水组>未拔牙组。与未拔牙组比较,3、7、14和21 d时生理盐水组、庆大霉素组和EPO组大鼠再植牙牙体组织中VEGF蛋白表达水平明显升高(P < 0.05),而28 d时各组大鼠再植牙牙体组织中VEGF蛋白表达水平差异无统计学意义(P > 0.05);与生理盐水组比较,3、7、14和21 d时庆大霉素组和EPO组大鼠再植牙牙体组织VEGF蛋白表达水平明显升高(P < 0.05),而在28d时VEGF蛋白表达水平差异无统计学意义(P > 0.05);与庆大霉素组比较,术后各时间点EPO组大鼠再植牙牙体组织中VEGF蛋白表达水平差异均无统计学意义(P > 0.05)。见表 1

表 1 不同时间点各组大鼠再植牙牙体组织中VEGF蛋白表达水平 Tab. 1 Expression levels of VEGF protein in odontal tissue of replanted teeth of rats in various groups at different time points
(n=4, x±s)
Group Expression level of VEGF protein
(t/d) 3 7 14 21 28
Non-tooth extraction 0.123±0.012 0.112±0.014 0.116±0.016 0.091±0.015 0.095±0.024
Normal saline 0.171±0.032* 0.171±0.024* 0.156±0.019* 0.108±0.014* 0.093±0.021
Gentamycin 0.237±0.019*△ 0.246±0.038*△ 0.233±0.041*△ 0.153±0.051*△ 0.104±0.020
EPO 0.260±0.048*△ 0.263±0.085*△ 0.239±0.029*△ 0.158±0.027*△ 0.091±0.010
* P < 0.05 vs non-tooth extraction group; P < 0.05 vs normal saline group.
2.3 HE染色观察各组大鼠再植牙牙髓血运重建情况

再植术后3d,未拔牙组大鼠再植牙牙根持续发育,血运丰富;生理盐水组大鼠再植牙可见炎症浸润灶;庆大霉素组大鼠再植牙纤维结缔组织长入;与未拔牙组和生理盐水组比较,EPO组大鼠再植牙血管内皮细胞和血管腔数目明显增多。再植术后7d,未拔牙组大鼠再植牙牙本质增厚;生理盐水组大鼠再植牙可见疏松结缔组织长入;庆大霉素组大鼠再植牙血管腔增多;与未拔牙组比较,EPO组大鼠再植牙可见钙化组织和修复性牙本质;与生理盐水组比较,庆大霉素组和EPO组大鼠再植牙的根尖孔均缩小。再植术后14 d,生理盐水组大鼠再植牙血管充血扩张;庆大霉素组大鼠再植牙开始出现钙化;与未拔牙组比较,EPO组大鼠再植牙髓腔内出现类骨样组织,牙本质小管排列紊乱。再植术后21 d,未拔牙组和EPO组大鼠再植牙根管壁持续增厚;生理盐水组大鼠再植牙出现牙髓坏死;庆大霉素组大鼠再植牙可见牙骨质和修复性牙本质沉积,根尖孔缩小。再植术后28 d,未拔牙组和EPO组大鼠再植牙牙根逐渐发育成熟;生理盐水组大鼠再植牙牙骨质处可见骨吸收陷窝和破骨细胞,出现牙根吸收;庆大霉素组大鼠再植牙髓腔内出现类骨样组织。见图 2(插页四)。

A-D:3 d; E-H:7 d; I-L:14 d; M-P:21 d; Q-T:28 d; A, E, I, M, Q:Non-tooth extraction group; B, F, J, N, R:Saline group; C, G, K, O, S:Gentamycin group; D, H, L, P, T:EPO group. 图 2 术后不同时间各组大鼠再植牙牙体组织形态表现(HE,×200) Fig. 2 Morphology of tooth tissue of replanted teeth of rats in various groups at different time after operation(HE, ×200)
3 讨论

对于牙根未发育成熟的脱位牙,实现即刻再植或再植前储存在合适的介质中,牙髓血运重建是有可能发生的[3]。研究[12-13]表明:大鼠的上颌第一磨牙在15 d时牙根开始发育,25~30 d时牙根形成1/2~2/3,大鼠的切牙末端存在被称为“apical bud”的特殊上皮结构,使切牙得以终生不断萌出。此外,有文献[14-15]指出:牙髓血运重建通常在再植后30 d左右建立,且牙周膜的修复在28 d完成。因此本实验选择4周龄大鼠的第一磨牙进行了为期28 d的观察,排除了个体自身发育的影响。

近年来研究者[16]发现了VEGF在人牙髓成纤维细胞中的表达。本研究结果显示:无论是对照组还是实验组,大鼠牙体组织中VEGF均呈阳性表达。还有研究[17]显示:牙本质基质中含有VEGF,其在损伤后从牙本质基质中释放,从而起到修复牙髓-牙本质复合体的作用。同时,成牙本质细胞的体外培养研究[18]表明:成牙本质细胞可以上调VEGF的表达。在本研究中,与固有髓核比较,成牙本质细胞层阳性表达出现的时间较早且阳性表达较强。

随着对VEGF研究的深入,有学者[19]发现:EPO通过蛋白质酪氨酸激酶2/信号传导转录激活因子3(JAK2/ STAT3)信号通路上调VEGF的表达。本研究结果显示:在再植术后3、7和14 d时EPO组和庆大霉素组大鼠再植牙牙体组织中VEGF均呈强阳性表达,可能与该时间内成牙本质细胞变性、炎症、牙髓间充质干细胞迁移和分化、牙髓血运重建和修复性牙本质形成活跃有关[5, 15, 20-21];EPO组大鼠再植牙牙体组织中VEGF表达水平略强于庆大霉素组,但差异无统计学意义,推测可能是因为在本实验中实现了即刻再植,与现实条件比较,操作均在相对无菌的条件下进行,细菌污染程度相对较轻。体外研究[22]显示:牙髓炎时,牙髓组织中EPO及其受体呈强阳性表达,表明EPO在炎症牙髓中发挥着一定的作用。并且EPO除了抗炎作用外,还具有促进血管再生、神经保护和促进成骨[7-10]的作用。最近的研究[23]表明:EPO通过促进Runt相关转录因子2(Runx2)、碱性磷酸酶(ALP)和骨钙素的表达以及促分裂素原活化蛋白激酶(mitogen-activated protein kinases, MAPK)途径上调人牙周膜间充质干细胞和牙周炎间充质干细胞的成骨能力。但本研究选择的是VEGF作为观察指标,而相关的研究[16]表明:VEGF的表达与牙髓炎中血管化的增加相一致,因此并不能说明EPO在增加再植牙成功率方面优于庆大霉素,还需要长期的观察和增加样本量以及检测细胞因子进行验证,但根据庆大霉素组和EPO组大鼠再植牙牙体组织中VEGF蛋白表达水平均强于生理盐水组和未拔牙组的结果推测,将VEGF和抗生素结合的微球作用于再植牙,可能会提高再植牙的远期成活率,这将成为本课题组未来的研究方向。

综上所述,本研究结果表明:EPO对再植牙的牙髓血运重建起到了一定的促进作用。

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