牙根不同发育阶段牙髓组织中的MMP-8及Ⅰ型胶原的免疫组化研究
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摘要
前言
细胞外基质(extracellular matrix，ECM)是细胞之间的物质，构成细胞生活的微环境，具有支持、连结、营养、防御和保护等作用。基质金属蛋白酶(matrix metalloproteinstes，MMPs)是一种内肽酶家族，通过降解细胞外基质参与多种生理病理过程。基质金属蛋白酶-8(MMP-8)是MMPs中的一种，其作用底物为Ⅰ、Ⅱ、Ⅲ、Ⅶ、Ⅹ型胶原。胶原与机体组织细胞的分化、成熟和损伤修复等密切相关。MMP-8主要功能是降解胶原，且降解Ⅰ型胶原的能力最强。年轻恒牙处于牙齿发育的重要阶段，因此有必要对该时期牙髓中MMP-8及胶原的表达及相关关系进行研究。本实验研究目的是研究MMP-8及Ⅰ型胶原在牙齿发育中的表达和作用。
材料和方法
牙髓标本取自健康牙齿。所有研究对象均无全身系统性疾病，近三个月未曾服用免疫制剂和抗菌药物。按牙根发育阶段将牙髓分为3组，即牙根开始发育组、牙根发育中组及根尖闭合组。牙齿拔除后立即将牙齿纵向劈开，完整取出牙髓组织，放入4％的多聚甲醛液中，4℃下固定48小时。梯度酒精脱水，二甲苯透明，石蜡包埋。制备冠根纵向连续石蜡切片，厚5μm。进行Ⅰ型胶原和MMP-8免疫组化染色(兔抗人Ⅰ型胶原、MMP-8多克隆抗体，SABC法)，空白实验以0.01MPBS代替一抗，其余染色步骤相同。利用coolsnapfx显微照相系统(Roper，Japan)及MetaMorph分析软件(UIC，US)对免疫组化染色标本进行图像分析。
实验结果
1型胶原免疫组织化学染色表明其存在于牙髓中的成牙本质
细胞、成纤维细胞、血管内皮细胞胞浆，血管壁周围及细胞外基质
中。在三组之中，牙根刚开始发育组染色最强，根尖孔闭合组染色
最弱，根尖孔闭合前后差异有统计学意义汀＜o．01人牙髓外周
与牙髓中心区的表达强弱无统计学差异（P＞O．05人＊＊P－8的
免疫组化染色表明MMP－8存在于牙髓中的成牙本质细胞胞浆。
成纤维细胞胞浆、血管内皮细胞胞浆、前期牙本质、血管壁周围及
细胞外基质中。在三组之中，牙根刚开始发育组染色最强，根尖孔
闭合组染色最弱，但在根尖孔闭合之前两组间差异无统计学意义
汀＞0·05广它们与根尖孔闭合组差异有统计学意义｛ P＜0·05人
1型胶原与＊＊P－8的表达呈正相关关系卜＝O．6乃，0＜r＜1人
讨 论
在本实验中各组成牙本质细胞均有1型胶原的阳性染色，随
着牙根的发育，染色变弱，提示成牙本质细胞具有分泌1型胶原的
能力，随着牙根的发育，成牙本质细胞分泌二型胶原的能力减弱，
I型胶原合成减少。本实验发现在年轻恒牙牙髓细胞外基质中1
型胶原的表达均呈阳性，它们呈网状或条索状分布。虽然镜下可
见牙髓外周有明显阳性染色带，但是牙髓外周与中心部二型胶原
表达无统计学差异，可能与牙髓中心区血管较多，而且血管壁可见
1型胶原阳性包绕有关。越是牙根发育早期，I型胶原表达越强
烈，根尖孔闭合时其表达明显减弱，这可能是与牙根闭合以前牙髓
细胞丰富、产生胶原的能力强有关。牙髓中除了成牙本质细胞，部
蛐纤维细胞胞浆染色阳性。推测成纤维细胞是牙髓细胞外互型
胶原来源的主要途径。
本实验首次采用免疫组化的方法对人的不同发育阶段牙髓中
·二·
MMP－8的表达进行研究。发现在成牙本质细胞的胞浆、大部分
的血管壁周围及部分成纤维细胞中MMP－8呈阳性染色，这一结
果为MMP－8存在于中性白细胞以外的其他细胞提供了又一直
接证据。本实验显示前期牙本质中MMP－8呈阳性表达，而在牙
本质中表达呈阴性。MMP－8可能参与牙本质矿化前某些组分降
解、清除这一过程。我们还发现牙髓细胞外基质中有MMP－8阳
性染色。部分成纤维细胞胞浆中有MMP－8表达，考虑成纤维细
胞可能是牙髓细胞外基质中MMP－8的主要来源。推测成纤维
细胞既合成胶原也降解胶原。年轻恒牙牙髓中MMP－8的表达
强于根尖孔闭合组，可见越是胶原分泌旺盛时期它的表达也越强。
推测它可能通过对胶原的降解来维持正常牙髓中胶原量的稳定。
本实验研究表明MMP－8与牙髓中1型胶原的表达呈正相
关关系，可见MMP－8是牙齿发育各个时期 1型胶原表达的调解
因素。但是MMP－8与牙髓中其它类型胶原的关系、牙齿发育中
MMP—8是否对调节各发育时期胶原内部构成比起一定作用还有
待研究。
结 论
1牙本质和前期牙本质中1型胶原主要是由成牙本质细胞分
泌的，且随着牙齿发育，成牙本质细胞分泌胶原能力减弱。
2．MMP－8参与牙齿成熟过程。
3．MMP－8参与牙齿发育中1型胶原的降解，维持牙髓中1
型胶原的稳定。
Preface
Extracellular matrix is an intercellular matter that forms a micro - environment for cells to live in and has the effects of support, join, nutrition, defense and protection. . The matrix metalloproteinstes (MMPs) belongs to the endopeptidase family which participates in multiple physiological and pathological processes by degrading extracellular matrix . The matrix metalloproteinste - 8 ( MMP - 8 ) is one of MMPs, whose active substrates are Type I , II , III, VII and X col-lagens. Collagen is closely related with the differentiation, maturity and injury repair of cells in the body tissue. The main function of MMP - 8 is to degrade collagen. The immature permanent tooth is an important stage of development. Therefore it is necessary to make a study of the expression of MMP - 8 and collagen in the dental pulp in the period. But study on the expression and distribution of MMP - 8 in pulps of immature perment teeth has not been reported. The purpose of this experimental research is to study the expression and
effect of MMP - 8 in the tooth development.
For the first time, by use of immunohistochemical and image a-nalysis techniques, we presented the expression and distribution pat-
tern of type I collagen and MMP - 8 in pulps of permanent teeth at different root development stage.
Materials and Methods
Dental pulps were obtained from healthy caries - free permanent teeth in need of extraction. All subjects had no systematic disease and didnt take any immune - inhibited drugs within three months. Based on the root development status, the pulp tissue was classified into three groups-, root just starting development, root being in development and root finishing development. After extracted, the dental pulp was rapidly removed, then fixed in 0.4% paraformaladehyde (PH7. 4) at 4^ for 48 hours. Tissue blocks were dehydrated in ascending series of ethanol, cleared in xylene and embedded in paraffin. Serial sections were cut 5jjtm thick. Anti - human polyclone antibody was used for staining. The sampies were observed under light microscope. Meta Morph/coolsnapfx/AX70 image analysis software was used to detect the gray value of type I collagen and MMP - 8 positive stained in dental pulp. The data were statistically analyzed by use of SPSS.
Results
Staining of immunohistochemistry for type I collagen in dental pulps revealed that there were positive reactions in the cytoplasm of fi-beroblast, odontoblast, and epithelial cells in blood vessel wall in immature and mature permanent teeth, especially at the stage of root just starting development.
Image analysis of type I collagen indicated the with the development of root formation, the gray value of the positive reaction in three groups were higher and higher and were significantly different when
root finishing development (P < 0.01). For all groups, there were no staisticaldifference between gray values in different parts.
Staining of immunohistochemistry for MMP - 8 in dental pulps revealed that there were positive reactions in the cytoplasm of fiber-oblast, odontoblast, and epithelial cells in blood .vessel wall and immature permanent teeth, especially at the stage of root just starting development.
Image analysis of MMP - 8 indicated the with the development of root formation, the gray value of the positive reaction in three groups were higher and were significantly different when root finishing development (P<0.05).
MMP - 8 is positively related with the expression of Type I collagen in the pulp. ( 0 < r < 1)
Disscussion
In this experiment each group of odontoblast is positive for Type I collagen by immunohistochemistry stain. With the development of the root of tooth, the stain is weakened, signifying the odontoblast is capable to secrete Type I collagen. With the development of the root of tooth, the ability of the odontoblast to secrete Type I collagen is weakened and the synthesis of Type I collagen is reduced.
In this experiment it is found that in the pulp of immature and mature permanent teeth, the expression of Type
细胞外基质(extracellular matrix，ECM)是细胞之间的物质，构成细胞生活的微环境，具有支持、连结、营养、防御和保护等作用。基质金属蛋白酶(matrix metalloproteinstes，MMPs)是一种内肽酶家族，通过降解细胞外基质参与多种生理病理过程。基质金属蛋白酶-8(MMP-8)是MMPs中的一种，其作用底物为Ⅰ、Ⅱ、Ⅲ、Ⅶ、Ⅹ型胶原。胶原与机体组织细胞的分化、成熟和损伤修复等密切相关。MMP-8主要功能是降解胶原，且降解Ⅰ型胶原的能力最强。年轻恒牙处于牙齿发育的重要阶段，因此有必要对该时期牙髓中MMP-8及胶原的表达及相关关系进行研究。本实验研究目的是研究MMP-8及Ⅰ型胶原在牙齿发育中的表达和作用。
材料和方法
牙髓标本取自健康牙齿。所有研究对象均无全身系统性疾病，近三个月未曾服用免疫制剂和抗菌药物。按牙根发育阶段将牙髓分为3组，即牙根开始发育组、牙根发育中组及根尖闭合组。牙齿拔除后立即将牙齿纵向劈开，完整取出牙髓组织，放入4％的多聚甲醛液中，4℃下固定48小时。梯度酒精脱水，二甲苯透明，石蜡包埋。制备冠根纵向连续石蜡切片，厚5μm。进行Ⅰ型胶原和MMP-8免疫组化染色(兔抗人Ⅰ型胶原、MMP-8多克隆抗体，SABC法)，空白实验以0.01MPBS代替一抗，其余染色步骤相同。利用coolsnapfx显微照相系统(Roper，Japan)及MetaMorph分析软件(UIC，US)对免疫组化染色标本进行图像分析。
实验结果
1型胶原免疫组织化学染色表明其存在于牙髓中的成牙本质
细胞、成纤维细胞、血管内皮细胞胞浆，血管壁周围及细胞外基质
中。在三组之中，牙根刚开始发育组染色最强，根尖孔闭合组染色
最弱，根尖孔闭合前后差异有统计学意义汀＜o．01人牙髓外周
与牙髓中心区的表达强弱无统计学差异（P＞O．05人＊＊P－8的
免疫组化染色表明MMP－8存在于牙髓中的成牙本质细胞胞浆。
成纤维细胞胞浆、血管内皮细胞胞浆、前期牙本质、血管壁周围及
细胞外基质中。在三组之中，牙根刚开始发育组染色最强，根尖孔
闭合组染色最弱，但在根尖孔闭合之前两组间差异无统计学意义
汀＞0·05广它们与根尖孔闭合组差异有统计学意义｛ P＜0·05人
1型胶原与＊＊P－8的表达呈正相关关系卜＝O．6乃，0＜r＜1人
讨 论
在本实验中各组成牙本质细胞均有1型胶原的阳性染色，随
着牙根的发育，染色变弱，提示成牙本质细胞具有分泌1型胶原的
能力，随着牙根的发育，成牙本质细胞分泌二型胶原的能力减弱，
I型胶原合成减少。本实验发现在年轻恒牙牙髓细胞外基质中1
型胶原的表达均呈阳性，它们呈网状或条索状分布。虽然镜下可
见牙髓外周有明显阳性染色带，但是牙髓外周与中心部二型胶原
表达无统计学差异，可能与牙髓中心区血管较多，而且血管壁可见
1型胶原阳性包绕有关。越是牙根发育早期，I型胶原表达越强
烈，根尖孔闭合时其表达明显减弱，这可能是与牙根闭合以前牙髓
细胞丰富、产生胶原的能力强有关。牙髓中除了成牙本质细胞，部
蛐纤维细胞胞浆染色阳性。推测成纤维细胞是牙髓细胞外互型
胶原来源的主要途径。
本实验首次采用免疫组化的方法对人的不同发育阶段牙髓中
·二·
MMP－8的表达进行研究。发现在成牙本质细胞的胞浆、大部分
的血管壁周围及部分成纤维细胞中MMP－8呈阳性染色，这一结
果为MMP－8存在于中性白细胞以外的其他细胞提供了又一直
接证据。本实验显示前期牙本质中MMP－8呈阳性表达，而在牙
本质中表达呈阴性。MMP－8可能参与牙本质矿化前某些组分降
解、清除这一过程。我们还发现牙髓细胞外基质中有MMP－8阳
性染色。部分成纤维细胞胞浆中有MMP－8表达，考虑成纤维细
胞可能是牙髓细胞外基质中MMP－8的主要来源。推测成纤维
细胞既合成胶原也降解胶原。年轻恒牙牙髓中MMP－8的表达
强于根尖孔闭合组，可见越是胶原分泌旺盛时期它的表达也越强。
推测它可能通过对胶原的降解来维持正常牙髓中胶原量的稳定。
本实验研究表明MMP－8与牙髓中1型胶原的表达呈正相
关关系，可见MMP－8是牙齿发育各个时期 1型胶原表达的调解
因素。但是MMP－8与牙髓中其它类型胶原的关系、牙齿发育中
MMP—8是否对调节各发育时期胶原内部构成比起一定作用还有
待研究。
结 论
1牙本质和前期牙本质中1型胶原主要是由成牙本质细胞分
泌的，且随着牙齿发育，成牙本质细胞分泌胶原能力减弱。
2．MMP－8参与牙齿成熟过程。
3．MMP－8参与牙齿发育中1型胶原的降解，维持牙髓中1
型胶原的稳定。
Preface
Extracellular matrix is an intercellular matter that forms a micro - environment for cells to live in and has the effects of support, join, nutrition, defense and protection. . The matrix metalloproteinstes (MMPs) belongs to the endopeptidase family which participates in multiple physiological and pathological processes by degrading extracellular matrix . The matrix metalloproteinste - 8 ( MMP - 8 ) is one of MMPs, whose active substrates are Type I , II , III, VII and X col-lagens. Collagen is closely related with the differentiation, maturity and injury repair of cells in the body tissue. The main function of MMP - 8 is to degrade collagen. The immature permanent tooth is an important stage of development. Therefore it is necessary to make a study of the expression of MMP - 8 and collagen in the dental pulp in the period. But study on the expression and distribution of MMP - 8 in pulps of immature perment teeth has not been reported. The purpose of this experimental research is to study the expression and
effect of MMP - 8 in the tooth development.
For the first time, by use of immunohistochemical and image a-nalysis techniques, we presented the expression and distribution pat-
tern of type I collagen and MMP - 8 in pulps of permanent teeth at different root development stage.
Materials and Methods
Dental pulps were obtained from healthy caries - free permanent teeth in need of extraction. All subjects had no systematic disease and didnt take any immune - inhibited drugs within three months. Based on the root development status, the pulp tissue was classified into three groups-, root just starting development, root being in development and root finishing development. After extracted, the dental pulp was rapidly removed, then fixed in 0.4% paraformaladehyde (PH7. 4) at 4^ for 48 hours. Tissue blocks were dehydrated in ascending series of ethanol, cleared in xylene and embedded in paraffin. Serial sections were cut 5jjtm thick. Anti - human polyclone antibody was used for staining. The sampies were observed under light microscope. Meta Morph/coolsnapfx/AX70 image analysis software was used to detect the gray value of type I collagen and MMP - 8 positive stained in dental pulp. The data were statistically analyzed by use of SPSS.
Results
Staining of immunohistochemistry for type I collagen in dental pulps revealed that there were positive reactions in the cytoplasm of fi-beroblast, odontoblast, and epithelial cells in blood vessel wall in immature and mature permanent teeth, especially at the stage of root just starting development.
Image analysis of type I collagen indicated the with the development of root formation, the gray value of the positive reaction in three groups were higher and higher and were significantly different when
root finishing development (P < 0.01). For all groups, there were no staisticaldifference between gray values in different parts.
Staining of immunohistochemistry for MMP - 8 in dental pulps revealed that there were positive reactions in the cytoplasm of fiber-oblast, odontoblast, and epithelial cells in blood .vessel wall and immature permanent teeth, especially at the stage of root just starting development.
Image analysis of MMP - 8 indicated the with the development of root formation, the gray value of the positive reaction in three groups were higher and were significantly different when root finishing development (P<0.05).
MMP - 8 is positively related with the expression of Type I collagen in the pulp. ( 0 < r < 1)
Disscussion
In this experiment each group of odontoblast is positive for Type I collagen by immunohistochemistry stain. With the development of the root of tooth, the stain is weakened, signifying the odontoblast is capable to secrete Type I collagen. With the development of the root of tooth, the ability of the odontoblast to secrete Type I collagen is weakened and the synthesis of Type I collagen is reduced.
In this experiment it is found that in the pulp of immature and mature permanent teeth, the expression of Type
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