骨涎蛋白在人牙胚和下颌骨发育中表达的研究

目的：观察骨涎蛋白（BSP）在人牙胚发育和分化过程中的分布情况。方法：采用免疫组化和原位杂交的方法进行人牙胚和下颌骨中BSP的定位研究。结果：钟状期牙胚中成牙本质细胞BSP为强阳性，成釉细胞、前期牙本质和釉质为阳性；骨组织和成骨细胞呈强阳性；外釉上皮、星网状层、中间层、内釉上皮和牙乳头为阴性。免疫组化与原位杂交的结果基本一致。结论：BSP与矿化组织如牙本质、釉质、骨组织的形成和矿化有关。     

釉基质蛋白对人牙周膜细胞合成骨桥蛋白、骨涎蛋白的影响

目的:观察釉基质蛋白对体外培养的人牙周膜细胞合成骨桥蛋白、骨涎蛋白能力的影响.方法:乙酸法提取猪釉基质蛋白,改良组织块法原代培养人牙周膜细胞,免疫细胞化学方法和图像分析方法观察细胞合成骨桥蛋白、骨涎蛋白的能力.结果:人牙周膜细胞胞浆骨桥蛋白、骨涎蛋白染色阳性,200、100、50mg/L釉基质蛋白作用下可以使细胞胞浆骨桥蛋白、骨涎蛋白染色不同程度地加深.人牙周膜细胞在釉基质蛋白作用下,最早从第3d开始骨桥蛋白表达增加、从第7d开始骨涎蛋白表达增加.结论:一定浓度的釉基质蛋白在特定的时间可以促进牙周膜细胞合成骨桥蛋白、骨涎蛋白.     

矿化相关蛋白在牙周组织中的分布及意义

目的：研究矿化相关蛋白在牙周组织中的分布并探讨其意义。方法：取正常成年杂种狗第二磨牙及其牙周组织,利用免疫组化SP法对矿化相关蛋白在牙周组织中的表达进行定位研究。结果：骨桥素OPN在牙周韧带基质及细胞中表达阳性,牙龈结缔组织中表达弱阳性,牙槽骨中邻近牙周韧带的部位表达阳性,其余部位及牙骨质中表达阴性;骨钙素OC在牙周韧带中表达阳性,牙龈结缔组织中表达弱阳性,牙槽骨中染色较弱且不均匀,牙骨质中表达阴性;骨唾蛋白BSP在牙周韧带中表达阳性,牙槽骨中染色集中在哈佛氏管周围,牙骨质和牙龈表达阴性。结论：矿化相关蛋白在牙周韧带基质及细胞中阳性表达,提示牙周韧带细胞在矿化组织的形成与再生中具有重要作用;矿化相关蛋白在牙周韧带细胞和牙龈成纤维细胞中的不同表达可作为鉴别牙龈成纤维细胞与牙周韧带细胞的标志。     

维甲酸体外诱导鼠磨牙牙胚骨涎蛋白基因表达的研究

目的 :观察维甲酸 (RA)对小鼠磨牙牙胚BSPmRNA表达的影响 ,探讨RA在牙胚早期发育中的作用。方法 :将 16d胎龄的鼠胎下颌第一磨牙牙胚置于含外源性RA(5× 10 -8mol/L)的RPMI16 40半固态培养基表面 ,分别培养 4、5、6d。培养结束后提取总RNA ,经RT -PCR扩增 30循环后 ,采用Southern印迹法检测BSPmRNA在牙胚组织中的表达。结果 :体外培养 4d后 ,实验组 (含外源性RA )和对照组 (不含外源性RA)的牙胚组织中BSPmRNA的表达均为阴性。培养 5d后 ,实验组的牙胚开始表达BSPmRNA ,对照组的牙胚仍为阴性。培养 6d后 ,实验组牙胚BSPmRNA的表达明显增强 ,对照组的牙胚也开始表达BSPmRNA ,但表达强度明显低于实验组的牙胚。结论 :RA具有诱导牙胚组织细胞分化的功能     

BSP在破骨细胞分化和骨改建过程中的作用

骨涎蛋白(bone sialoprotein,BSP)作为小整合素结合配体N端联结糖蛋白家族(SIBLING)的成员,参与调控骨改建过程的多个环节,并与破骨细胞分化和过度活跃的骨吸收密切相关。该文就BSP在破骨细胞和骨改建过程中的作用及其细胞和分子生物学机制作一综述。     

过量氟对大鼠切牙骨涎蛋白表达的影响

目的:研究过量氟对牙硬组织发育过程中骨涎蛋白(bonesialoprotein,BSP)时空表达的影响,从蛋白水平上探讨氟牙症的发病机制。方法:20只Wistar大鼠随机分为对照组(饮用蒸馏水)和实验组(100mg/LF-)2组。饲养8周后处死动物,通过免疫组织化学染色观察并比较BSP在对照组与实验组大鼠牙胚上皮中的表达,采用计算机图像分析系统对免疫组化染色阳性结果进行计算机图像分析,采用SASv6.12统计软件对2组图像分析结果进行t检验。结果:对照组,各期成釉细胞排列均匀整齐,细胞形态正常,成熟的成釉细胞、成牙本质细胞、成牙骨质细胞中BSP表达为阳性;实验组,大鼠切牙成釉细胞由原有的高柱状变矮,细胞排列成多层,釉基质形成混乱,大鼠牙胚上皮中BSP表达显著低于对照组,P<0.01。结论:氟化物能抑制大鼠牙胚上皮BSP的表达,提示氟可能通过抑制BSP在牙胚发育过程中的表达,从而抑制牙胚上皮细胞(成釉细胞、成牙本质细胞、成牙骨质细胞)的增殖分化及随后的基质合成与分泌,导致氟斑牙的形成。     

Immunolocalization of bone extracellular matrix proteins (type I collagen,osteonectin and bone sialoprotein) in human dental pulp and cultured pulp cells

AIM: To simultaneously analyse the expression of type I collagen, osteonectin and bone sialoprotein (BSP) in human dental pulp of different ages. METHODOLOGY: Cultured dental pulp fibroblasts (FP1 cell line), pulps from dental germs with incomplete root formation (n = 4) and pulps of erupted teeth with total root formation (n = 4) were used. Bone proteins were searched by immunohistochemistry and immunofluorescence using polyclonal antibodies and compared among the three groups assessed. RESULTS: Immunohistochemistry detected the three proteins in dental pulp tissue, as it labelled extracellular matrix, predentine and odontoblasts. The BSP label was weaker, when compared to both type I collagen and osteonectin. The presence of type I collagen was more evident in pulps from erupted teeth, when compared to germ dental pulps. On the other hand, a strong expression of osteonectin in germ dental pulps was observed. CONCLUSIONS: Regardless of the degree of maturation, dental pulps present type I collagen, osteonectin and BSP in the extracellular matrix (ECM) and in the odontoblastic layer. Thus, the results suggest that these proteins are related to the production and mineralization of dentine.     

Early in vivo and in vitro effects of amelogenin gene splice products on pulp cells

Recombinant amelogenin gene splice products A+4 and A-4, implanted in the pulp, induce the recruitment, proliferation, and differentiation of reparative cells. Our aim was to investigate the precocious events occurring in the pulp 1 d and 3 d after implantation of agarose beads alone or loaded with A+4 or A-4. Proliferation and cell recruitment towards an odonto/osteogenic phenotype were visualized by detection of the proliferation cell nuclear antigen (PCNA) and RP59. After implantation of beads alone or loaded with A+4, at day 3, pulp cells were moderately immunopositive for osteopontin (OP), whereas labeling was strongly positive upon treatment with A-4. Dentin sialoprotein (DSP) labeling was not detectable. Parallel in vitro studies were carried out on odontoblastic and mesenchymal progenitor cells in order to evaluate the effect of the amelogenin peptides on the expression of a series of marker genes involved in the odontoblastic/osteogenic/chondrogenic differentiation pathways. Altogether, our results suggest that the 'signaling' effects of the amelogenin peptides A+4 and A-4 may differ according to the type of target cells, their stage of differentiation, the time of treatment, and the type of amelogenin peptide (A+4 or A-4).     

甲壳素及其衍生物用于骨缺损修复的研究进展

颌面部骨缺损造成的畸形与功能障碍主要通过骨移植来修复,寻求理想的骨缺损修复材料是相关研究的热点.甲壳素是天然高分子化合物,具有独特的分子结构和优越的生物学特性,有望成为理想的骨修复材料.本文就目前国内外对甲壳素及其衍生物应用于骨缺损修复方面的主要研究及进展作一综述.     

Human β‐defensin‐3 up‐regulates cyclooxygenase‐2 expression and prostaglandin E2 synthesis in human gingival fibroblasts

Chotjumlong P, Khongkhunthian S, Ongchai S, Reutrakul V, Krisanaprakornkit S. Human β‐defensin‐3 up‐regulates cyclooxygenase‐2 expression and prostaglandin E ₂ synthesis in human gingival fibroblasts. J Periodont Res 2010; 45: 464–470. © 2010 John Wiley & Sons A/S Background and Objective: Oral epithelial cells express three antimicrobial peptide human β‐defensins (hBDs) that have previously been demonstrated to exert proinflammatory effects on various immune cells. We wanted to examine whether hBDs could induce cyclooxygenase‐2 (COX‐2) expression and prostaglandin E₂ (PGE₂) synthesis in non‐immune cells, such as human gingival fibroblasts. Material and Methods: Cultured fibroblasts were treated with different concentrations of hBD‐1, ‐2, ‐3 or interleukin‐1β, as a positive control, for various times, in the presence or absence of NS‐398, a specific COX‐2 inhibitor. The levels of COX‐1 and COX‐2 mRNA expression were analyzed using RT‐PCR and real‐time PCR. Whole cell lysates were analyzed for COX‐1 and COX‐2 protein expression by western blotting. Cell‐free culture supernatants were assayed for PGE₂ levels by ELISA. The lactate dehydrogenase assay was performed to determine the cytotoxicity of hBDs. Results: Ten and 40 μg/mL of hBD‐3 up‐regulated COX‐2 mRNA and protein expression, consistent with COX‐2 up‐regulation by interleukin‐1β, whereas hBD‐1 and hBD‐2 did not. However, COX‐1 mRNA and protein were constitutively expressed. The time‐course study revealed that hBD‐3 up‐regulated COX‐2 mRNA and protein expression at 6 and 12 h, respectively. Consistent with COX‐2 up‐regulation, 10 and 40 μg/mL of hBD‐3 significantly increased PGE₂ levels in cell‐free culture supernatants (p < 0.05), and this was inhibited by NS‐398 in a dose‐dependent manner. Neither of the hBD concentrations tested in this study was toxic to the cells. Conclusion: These findings indicate that epithelial human β‐defensin‐3 functions as a proinflammatory mediator in controlling arachidonic acid metabolism in underlying fibroblasts.     

hBMP2基因转染对人牙龈成纤维细胞生物学特性的影响

目的分析hBMP2基因转染人牙龈成纤维细胞的生物学特性.方法用脂质体转染法将hBMP2基因转入人牙龈成纤维细胞内,经G418筛选后,获得阳性克隆.以原位杂交和免疫组化对转染细胞进行鉴定;进一步观察细胞形态、生长特性、碱性磷酸酶活性、骨钙素合成以及体外形成矿化结节的能力.结果hBMP2基因转染后,人牙龈成纤维细胞有hBMP2 mRNA的转录和蛋白表达;部分细胞由原来的长梭形转化为多角形,碱性磷酸酶活性和骨钙素合成均明显高于对照组(两组均P＜0.01);在矿化液作用下,能形成体外矿化结节.但细胞的增殖特性无明显变化.结论外源性hBMP2基因能够在人牙龈成纤维细胞表达,并促进其向成骨细胞方向转化,而对细胞的生长特性无明显影响.