β-catenin与APC在小鼠牙胚发育中的表达及相互关系的研究
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摘要
背景:牙胚的发生是胚胎时期口腔牙源性上皮与下方来源于颅神经嵴细胞的外胚间充质互相作用的结果,局部增生的口腔上皮发出信号作用于下方间充质,引起特异性生牙部位的间充质凝集并促进细胞分化,从而启动牙胚发育。目前牙胚发育的研究已从细胞水平深入到分子水平,研究发现,在牙胚形态发生和细胞分化过程中,有一系列信号分子调控,已知WNT经典通路参与了胚胎发育的过程,在其中扮演重要的角色。近年,WNT在牙胚发育中的作用是研究热点,其多个相关因子在牙胚中有表达。β-catenin(β连环蛋白)、APC(结肠腺瘤性息肉病基因)均为WNT经典通路的重要因子,近年关于β—catenin和APC的研究多在肿瘤发生方面,在牙胚发育方面的研究较少,国内未见报道。
目的:本实验欲通过观察小鼠牙胚发育过程中WNT经典通路因子β-catenin、APC的表达情况,了解其在牙胚发育过程中的调控作用;并对各因子的表达情况进行相关性分析,探讨WNT信号家族与牙胚发育的关系,为进一步了解牙胚发育过程中的分子调控机制打下基础。为体外培养牙胚,牙齿再生工程提供基础研究依据。
材料和方法:培养获得怀孕昆明小鼠,采用免疫组化SP法和DAB显色法检测不同时期胎鼠下颌第一磨牙胚内β-catenin和APC蛋白的表达情况,以细胞出现黄色颗粒为阳性表达。
结果:
1.β-catenin在蕾状期时在口腔及牙板上皮中均弱阳性表达,牙蕾末端突起阳性表达,邻近间充质细胞为弱阳性,着色部位主要在细胞膜、细胞质及细胞核;帽状期在口腔上皮、成釉器外釉上皮和星网状层表达弱阳性,内釉上皮阳性表达,尤其在内釉上皮中央处原发性釉结为强阳性,邻近牙乳头细胞中见弱阳性表达,着色在细胞膜、细胞质、细胞核;钟状期在外釉上皮表达阳性,内釉上皮强阳性表达,星网状层弱阳性表达;牙乳头内前成牙本质细胞阳性表达,着色在细胞膜、细胞质。
2. APC蕾状期时在口腔上皮,牙蕾上皮中均强阳性表达,着色在细胞膜、细胞质,在牙蕾周围间充质细胞的细胞膜可见弱阳性表达;帽状期时在口腔上皮和星网状层表达阳性,内釉上皮表达强阳性,外釉上皮为弱阳性,牙囊细胞中可见弱阳性表达,着色在细胞膜、细胞质。钟状期在内釉上皮阳性表达,在牙囊中的表达弱阳性,着色在细胞膜、细胞质;在牙乳头中各期均无表达。
3.β-catenin和APC的表达有统计学相关性。
结论:
1.β-catenin随着牙胚发育其表达逐渐增强,尤其以内釉上皮及釉结处的表达更为明显,而且早期位于细胞核及细胞质中这一事实推测其参与了牙胚早期的细胞增殖作用。
2. APC随着牙胚发育其表达逐渐减弱,而且在同一期内,内釉上皮及牙囊的表达较其它部位较强,这一现象提示其主要参与了牙胚早期细胞增殖,随着细胞增殖活性下降其作用减弱。
3.β-catenin与APC在牙胚发育过程的时空表达有重叠现象,表达结果分析有显著负相关性,这符合WNT经典信号通路中两者的作用关系,推测WNT经典信号通路参与了牙胚的发育,并主要作用在发育早期。
Background: Tooth morphogenesis is a process that is regulated by sequential and reciprocal interactions between the epithelial and mesenchymal tissues,during which the simple oral ectoderm thickens, buds, grows and folds to form the complex shape of the tooth crown. During tooth initiation the ectoderm thickens and forms a placode that buds to the underlying neural-crest-derived mesenchyme. The epithelium signals to the mesenchyme, which then condenses around the epithelial bud. During subsequent morphogenesis the epithelium folds and grows to surround the dental papilla mesenchyme (cap stage).It is known that there are a serious of signals including WNT which plays an essential roal during tooth development with the research level from cell to melocular. In recent years, more and more reaearchers have confirmed the function of WNT and much correlatation factor’s expression within the tooth germ. Beta-catenin and APC(adenomatous polyposis coli),two key factors in the WNT signal pathway have been frequently found in the tumorigenesis. But there are few study of them about tooth germ in domestic.
Objective: In this research, through examining the gene distributions of and APC in the tooth germ. The message of function of the WNT signal pathway in the tooth development is obtained, and the functional relationship between beta-catenin and APC is also discussed.
Method: Embryos are obtained by mating km male and female mouse.The age is determined by the day of apperance of the vaginal plug. Moalr tooth germ are dissected from day 13.5 to17.5. The distributions of beta-catenin and APC are examined in the paraffin-embedded sections by immunohistochemistry methods. Yellow staining in the cell are masculine.
Result:
1. beta-catenin: During the bud stages, weakly positive is found in the oral epithelium and the dental lamina, masculine expression are found in the top of the tooth bud .The staining were whole cell; During the cap stages, except the strong masculine staining in the middle of the inner dental epithelium (primary enamel knot),there are all weakly positive in other location. In the dental papilla,only adjacent cell have weakly positon. The staining were whole cell. In the bell stage, the outer enamal epithelium expresses the beta-catenin gene in the normal level, the inner dental epithelium is strong expression, and the stratum intermedium is weakly expression. No stainging in the dental papilla except for the cell that differented into odontoblast. The staining in the enmel organ were not found in cell nucleus.
2. APC: During the bud stages, strong positive is found in the oral epithelium and the dental lamina, and weakly expression is found in the cytomembrane of mesenchymal cell nearby; During the cap stages, there is strong masculine staining in the inner dental epithelium and masculine in the oral epithelium and the stratum intermedium. In the outer enamal epithelium and the cytomembrane of mesenchymal cell in the dental follicle, there is weakly staining. In the bell stage, the inner enamal epithelium expressed the APC gene in the normal level, the expression in the dental follicle were weakly. There is no stainging in the dental papilla in all the stages.
3. There is statistics sig correlation between beta-catenin and APC.
Conclusion:
1. Beta-catenin expression in the whole stages of tooth germ is in an up-regulation tendency.(P<0.01). In both the inner enamel epithelium and the enamel knot, the expression of beta-catenin is stronger than the other place. The staining in the mesenchymal cell is masculine .The result suggests its contribution in the early development of enamel organ and the proliferation of cell.
2. APC expresses in the whole stages of the tooth germ for the most part in the inner enamel epithelium, but the expression displays an down-regulation tendency(.P<0.01)The result suggests it participates in the formation of early enamel organ, and the masculine in the dental papilla. It is supposed that it gives nutrition and sustain to the formation.
3. Coincidance of the two factors staining site is found, and according to the statistics, there is sig correlation between them(P<0.01), which confirmes a negative correlation between APC and beta-catenin, it suggest Wnt/ beta-catenin signal transduction pathway participate in the development of tooth germ espically in the early stage.
目的:本实验欲通过观察小鼠牙胚发育过程中WNT经典通路因子β-catenin、APC的表达情况,了解其在牙胚发育过程中的调控作用;并对各因子的表达情况进行相关性分析,探讨WNT信号家族与牙胚发育的关系,为进一步了解牙胚发育过程中的分子调控机制打下基础。为体外培养牙胚,牙齿再生工程提供基础研究依据。
材料和方法:培养获得怀孕昆明小鼠,采用免疫组化SP法和DAB显色法检测不同时期胎鼠下颌第一磨牙胚内β-catenin和APC蛋白的表达情况,以细胞出现黄色颗粒为阳性表达。
结果:
1.β-catenin在蕾状期时在口腔及牙板上皮中均弱阳性表达,牙蕾末端突起阳性表达,邻近间充质细胞为弱阳性,着色部位主要在细胞膜、细胞质及细胞核;帽状期在口腔上皮、成釉器外釉上皮和星网状层表达弱阳性,内釉上皮阳性表达,尤其在内釉上皮中央处原发性釉结为强阳性,邻近牙乳头细胞中见弱阳性表达,着色在细胞膜、细胞质、细胞核;钟状期在外釉上皮表达阳性,内釉上皮强阳性表达,星网状层弱阳性表达;牙乳头内前成牙本质细胞阳性表达,着色在细胞膜、细胞质。
2. APC蕾状期时在口腔上皮,牙蕾上皮中均强阳性表达,着色在细胞膜、细胞质,在牙蕾周围间充质细胞的细胞膜可见弱阳性表达;帽状期时在口腔上皮和星网状层表达阳性,内釉上皮表达强阳性,外釉上皮为弱阳性,牙囊细胞中可见弱阳性表达,着色在细胞膜、细胞质。钟状期在内釉上皮阳性表达,在牙囊中的表达弱阳性,着色在细胞膜、细胞质;在牙乳头中各期均无表达。
3.β-catenin和APC的表达有统计学相关性。
结论:
1.β-catenin随着牙胚发育其表达逐渐增强,尤其以内釉上皮及釉结处的表达更为明显,而且早期位于细胞核及细胞质中这一事实推测其参与了牙胚早期的细胞增殖作用。
2. APC随着牙胚发育其表达逐渐减弱,而且在同一期内,内釉上皮及牙囊的表达较其它部位较强,这一现象提示其主要参与了牙胚早期细胞增殖,随着细胞增殖活性下降其作用减弱。
3.β-catenin与APC在牙胚发育过程的时空表达有重叠现象,表达结果分析有显著负相关性,这符合WNT经典信号通路中两者的作用关系,推测WNT经典信号通路参与了牙胚的发育,并主要作用在发育早期。
Background: Tooth morphogenesis is a process that is regulated by sequential and reciprocal interactions between the epithelial and mesenchymal tissues,during which the simple oral ectoderm thickens, buds, grows and folds to form the complex shape of the tooth crown. During tooth initiation the ectoderm thickens and forms a placode that buds to the underlying neural-crest-derived mesenchyme. The epithelium signals to the mesenchyme, which then condenses around the epithelial bud. During subsequent morphogenesis the epithelium folds and grows to surround the dental papilla mesenchyme (cap stage).It is known that there are a serious of signals including WNT which plays an essential roal during tooth development with the research level from cell to melocular. In recent years, more and more reaearchers have confirmed the function of WNT and much correlatation factor’s expression within the tooth germ. Beta-catenin and APC(adenomatous polyposis coli),two key factors in the WNT signal pathway have been frequently found in the tumorigenesis. But there are few study of them about tooth germ in domestic.
Objective: In this research, through examining the gene distributions of and APC in the tooth germ. The message of function of the WNT signal pathway in the tooth development is obtained, and the functional relationship between beta-catenin and APC is also discussed.
Method: Embryos are obtained by mating km male and female mouse.The age is determined by the day of apperance of the vaginal plug. Moalr tooth germ are dissected from day 13.5 to17.5. The distributions of beta-catenin and APC are examined in the paraffin-embedded sections by immunohistochemistry methods. Yellow staining in the cell are masculine.
Result:
1. beta-catenin: During the bud stages, weakly positive is found in the oral epithelium and the dental lamina, masculine expression are found in the top of the tooth bud .The staining were whole cell; During the cap stages, except the strong masculine staining in the middle of the inner dental epithelium (primary enamel knot),there are all weakly positive in other location. In the dental papilla,only adjacent cell have weakly positon. The staining were whole cell. In the bell stage, the outer enamal epithelium expresses the beta-catenin gene in the normal level, the inner dental epithelium is strong expression, and the stratum intermedium is weakly expression. No stainging in the dental papilla except for the cell that differented into odontoblast. The staining in the enmel organ were not found in cell nucleus.
2. APC: During the bud stages, strong positive is found in the oral epithelium and the dental lamina, and weakly expression is found in the cytomembrane of mesenchymal cell nearby; During the cap stages, there is strong masculine staining in the inner dental epithelium and masculine in the oral epithelium and the stratum intermedium. In the outer enamal epithelium and the cytomembrane of mesenchymal cell in the dental follicle, there is weakly staining. In the bell stage, the inner enamal epithelium expressed the APC gene in the normal level, the expression in the dental follicle were weakly. There is no stainging in the dental papilla in all the stages.
3. There is statistics sig correlation between beta-catenin and APC.
Conclusion:
1. Beta-catenin expression in the whole stages of tooth germ is in an up-regulation tendency.(P<0.01). In both the inner enamel epithelium and the enamel knot, the expression of beta-catenin is stronger than the other place. The staining in the mesenchymal cell is masculine .The result suggests its contribution in the early development of enamel organ and the proliferation of cell.
2. APC expresses in the whole stages of the tooth germ for the most part in the inner enamel epithelium, but the expression displays an down-regulation tendency(.P<0.01)The result suggests it participates in the formation of early enamel organ, and the masculine in the dental papilla. It is supposed that it gives nutrition and sustain to the formation.
3. Coincidance of the two factors staining site is found, and according to the statistics, there is sig correlation between them(P<0.01), which confirmes a negative correlation between APC and beta-catenin, it suggest Wnt/ beta-catenin signal transduction pathway participate in the development of tooth germ espically in the early stage.
引文
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