人釉原蛋白重组质粒PsecTaq2A-AMG在COS-1细胞系中表达

目的研究人釉原蛋白重组质粒PsecTaq2A-AMG在COS-1细胞系中的表达，为基因工程制备釉原蛋白奠定基础。方法采用脂质体载体法将釉原蛋白重组质粒PsecTaq2A-AMG导人COS-1细胞系，用Zeoin筛选得到稳定转染克隆，并经免疫组化及酶联免疫吸附实验（ELISA）检测细胞内和细胞培养液中重组釉原蛋白的表达。结果在未经转染的对照组细胞内和细胞培养液中均未检测到重组釉原蛋白的表达，而经转染的实验组不论细胞内或细胞培养液中均检测到重组釉原蛋白的较高表达，重组质粒PseeTaq2A．AMG转染组ELISA定量检测细胞内重组釉原蛋白浓度达0.877μg／ml。结论PsecTaq2A-AMG具有较强的在真核细胞系中表达和分泌重组釉原蛋白的能力，适宜基因工程体外制备重组釉原蛋白。     

大鼠釉原蛋白基因的融合表达及其纯化

目的：观察釉原蛋白（Ａｍｅｌｏｇｅｎｉｎ,Ａｍ） 基因ＰＣＲ 产物在大肠杆菌中的表达。方法：利用原核表达载体ＰＲＳＥＴ,转化大肠杆菌ＪＭ１０９ ,通过ＩＰＴＧ 诱导,收菌。聚丙烯酰胺凝胶电泳。过柱纯化。结果：电泳分析表明,釉原蛋白基因ＰＣＲ产物在大肠杆菌中成功的获得了表达。结论：表达的融合蛋白主要以包涵体的形式存在。利用Ｎｉ－ ＮＴＡ 金属螯合亲和层析在变性条件下对表达的蛋白进行纯化,获得纯化蛋白。     

通过真核细胞表达系统获得重组人釉原蛋白

目的构建重组人釉原蛋白基因的真核表达系统，并建立稳定表达该蛋白的细胞系。方法取26周龄引产胎儿的牙胚组织，提取总RNA，用RT—PCR技术扩增釉原蛋白基因片段，插入中间表达载体pGEM -T。经双酶切后，再与真核表达载体pcDNA3.1^TM／myc—His（-）B相连接，构成最终的表达质粒，将该重组表达质粒转染至HEK 293A细胞，用G418筛选出阳性细胞克隆，并建立稳定表达釉原蛋白的细胞系。结果通过测序表明，人釉原蛋白基因被成功地连接到了真核表达载体上。将该表达系统转染HEK293A细胞后，进行Western Blot检测，证明有相对分子质量约32000的釉原蛋白表达。结论本实验成功构建了重组人釉原蛋白真核表达系统，建立了稳定细胞系，为获得高纯度的釉蛋白，进一步研究蛋白质功能奠定了基础。     

人釉原蛋白重组质粒PcDNA3-AMG在COS-1细胞系中的表达

目的研究人釉原蛋白(AMG)重组质粒PcDNA3-AMG在COS-1细胞系中的表达。方法采用脂质体载体法将釉原蛋白重组质粒PcDNA3-AMG导入COS-1细胞系,用Zeoin筛选得到稳定转染克隆,并经ELISA检测细胞内和细胞培养液中重组釉原蛋白AMG的表达。结果在未经转染的对照组细胞内和细胞培养液中均未检测到AMG的表达,而经转染的实验组不论细胞内或细胞培养液中均检测到AMG的较高表达,重组质粒PcDNA3-AMG转染组细胞内AMG浓度达0.253μg/ml。结论PcDNA3-AMG具有较强的在真核细胞系中表达和分泌AMG的能力,适宜基因工程体外制备重组釉原蛋白。     

人釉原蛋白成熟肽基因的原核表达和纯化

目的 建立人釉原蛋白(amelogenin,AMG)成熟肽基因原核表达和纯化的技术路线,获得纯化的人AMG成熟肽蛋白,以期为牙周炎的基础治疗提供依据.方法 通过已构建并经鉴定的重组质粒pGEX-4T-1-AMG,转化BL21大肠杆菌,原核表达后利用谷胱甘肽-S-转移酶融合蛋白纯化系统(glutathione S-transferase fusion protein purification system,GSTrapFF)亲和层析柱进行重组人AMG的纯化.结果 十二烷基硫酸钠聚丙烯酰胺凝胶电泳图和蛋白质印迹法分析结果显示,获得纯化的相对分子质量为45 000大小GST-AMG融合蛋白和19 000大小的目的 蛋白AMG.结论 利用pGEX-4T-1-AMG-BL21体系成功获得了人AMG成熟肽基因在大肠杆菌中的原核表达和纯化.     

人釉原蛋白基因重组慢病毒载体的构建及在293T细胞中的表达

目的:构建含人釉原蛋白(human Amelogenin,hAm)成熟肽编码区基因的慢病毒载体FuAmw,并观察在重组慢病毒感染的293T细胞中釉原蛋白的表达.方法:以构建好的重组质粒PQE30-Am为模板,采用PCR技术体外扩增hAm成熟肽编码区.将扩增产物与慢病毒载体转移质粒FUGW分别用BamH Ⅰ和EcoR Ⅰ双酶切后连接,构建重组慢病毒载体,转移质粒FUAmW,并进行酶切及测序鉴定.通过聚乙烯亚胺(polytheylenimine,PEI)法将三质粒共转染293T细胞,包装得到重组慢病毒FUGW和FUAmW,然后分别离体感染293T细胞,培养 72h后,经荧光显微镜下观察和流式细胞计数检测感染效率,采用RT-RCR及Western印迹法检测釉原蛋白基因的表达.结果:重组质粒经测序证实.插入片段与人釉原蛋白成熟肽基因序列完全一致.流式细胞仪测得重组病毒感染293T细胞绿色荧光蛋白(green fluoresence protein,GFP)的阳性率为67.38%.RT-RCR和Western印迹均证实,重组慢病毒FUAmW感染的293T细胞能够表达人釉原蛋白.结论:成功构建携带人釉原蛋白成熟肽基因的重组慢病毒载体质粒,以293T细胞包装得到的重组人釉原蛋白病毒能感染真核细胞并获得表达.     

大鼠成釉器细胞的分离纯化

目的：寻找一种大鼠成釉器细胞分离纯化的简捷方法。方法：分离出生后6dSD大鼠上下颌第一和第二磨牙完整牙胚，剥离牙囊和成釉器，酶消化法原代混合培养。采用多次差别消化法去除成纤维样细胞，纯化上皮样细胞，并进行抗角蛋白染色和RT—PCR检测釉原蛋白、成釉蛋白mRNA表达。结果：原代细胞为混杂细胞，经2～3次差别消化可完全去除成纤维样细胞。上皮样细胞呈多角形，铺路石样生长，抗角蛋白染色阳性，表达釉原蛋白、成釉蛋白mRNA。结论：本研究通过多次差别消化获得了纯化的大鼠成釉器细胞。     

冷冻电子显微镜观察人釉原蛋白的自组装

目的:采用冷冻电子显微镜(cryo TEM)体外观察人釉原蛋白的自组装过程,分析自组装各阶段蛋白的聚集状态及细微结构.方法:提取青少年下颌第三磨牙牙胚细胞总RNA,用RT-PCR获得人釉原蛋白基因全长,再与pMD19-T载体构建重组质粒,转入宿主菌E.coli Top10中诱导表达并纯化.冷冻电子显微镜观察pH值从3....     

人重组成釉蛋白C端肽在大肠杆菌中的表达及初步纯化

目的 :在大肠杆菌中表达人重组成釉蛋白C端肽 ,并对重组蛋白进行纯化 ,为进一步制备抗人成釉蛋白抗体和功能研究奠定基础。方法 :将编码人成釉蛋白C端肽的基因片段克隆到大肠杆菌表达载体pRSET -A上 ,构建表达质粒 pRSET -A -AMBN ,以大肠杆菌E .coliBL2 1(DE3)为宿主菌进行诱导表达 ,表达产物经镍 -次氮基三乙酸 (Ni -NTA)柱进行亲和层析纯化。结果 :工程菌在IPTG诱导 3h后 ,在SDS -PAGE上出现一条新的蛋白带 ,相对分子量 (Mr)为 5 2× 10 3 ,以可溶形式存在 ,经Ni-NTA柱纯化后获得纯度大于 95 %的人重组成釉蛋白C端肽。结论 :获得Mr为 5 2× 10 3 的人重组成釉蛋白C端肽。     

人釉原蛋白基因在大肠杆菌中的融合表达

目的建立人釉原蛋白（AMG）成熟肽基因在大肠杆菌中融合表达和纯化的技术路线。方法利用已构建并经鉴定的重组质粒pGEX- 4T- 1/AMG转化大肠杆菌BL21,分别对诱导时间、异丙基- β- D硫代半乳糖苷（IPTG）浓度和诱导温度进行优化,在最佳诱导表达条件下,分别对菌液上清、周质、胞质和包涵体中的目的蛋白表达进行分析,在可溶性蛋白中发现大量目的蛋白,随后利用GSTrapFF亲和层析柱进行人AMG融合蛋白的过柱纯化。结果pGEX- 4T- 1/AMG重组质粒的双酶切凝胶电泳鉴定结果和测序鉴定结果和预期一致。最佳诱导时间为14.5 h、最佳诱导剂浓度为1.0 mmol/L、最佳诱导温度为20 ℃,在此条件下目的蛋白的表达量达到峰值。在最佳诱导条件下,胞质蛋白和包涵体中都有大量的目的蛋白。提取大量胞质蛋白,经GSTrapFF亲和层析柱纯化,收集纯化液,进行SDS- 聚丙烯酰胺凝胶电泳分析,显示成功纯化了AMG融合蛋白,在提取液洗涤2次后,可获得高纯度的融合蛋白。结论利用pGEX- 4T- 1/AMG原核表达体系成功获得纯化的人AMG融合蛋白。     

Putative signaling action of amelogenin utilizes the Wnt/β-catenin pathway

Background and Objective:  While it has long been known that amelogenin is essential for the proper development of enamel, its role has generally been seen as structural in nature. However, our new data implicate this protein in the regulation of cell signaling pathways in periodontal ligament cells and osteoblasts. In this article we report the successful purification of a recombinant mouse amelogenin protein and demonstrate that it has signaling activity in isolated mouse calvarial cells and human periodontal ligament cells.
Material and Methods:  To determine the regulatory function of canonical Wnt signaling by amelogenin, we used TOPGAL transgenic mice. These mice express a β-galactosidase transgene under the control of a LEF/TCF and β-catenin-inducible promoter. To investigate in greater detail the molecular mechanisms involved in the β-catenin signaling pathway, isolated osteoblasts and periodontal ligament cells were exposed to full-length recombinant mouse amelogenin and were evaluated for phenotypic changes and β-catenin signaling using a TOPFLASH construct and the LacZ reporter gene.
Results:  In these in vitro models, we showed that amelogenin can activate β-catenin signaling.
Conclusion:  Using the TOPGAL transgenic mouse we showed that amelogenin expression in vivo is localized mainly around the root, the periodontal ligament and the alveolar bone.     

人釉原蛋白真核表达载体体外表达产物生物学活性的检测

目的探讨人釉原蛋白基因重组质粒PcDNA3．1-AMG的真核细胞转染表达产物是否具有促进牙周组织再生的作用。方法脂质体介导PcDNA3．1-AMG体外转染COS1细胞．ELISA法检测转染细胞内及其培养上清液中重组釉原蛋白的表达：建立犬牙周组织缺损模型．局部使用转染表达产物冻干粉．8周后通过组织学观察牙周组织再生的情况。结果PcDNA3．1-AMG转染的COS1细胞内重组釉原蛋白浓度为（0．253±0．075）μg／ml。培养液中浓度为（0．065±0．011）μg／ml；使用转染表达产物8周后．牙周缺损区牙骨质、牙槽骨均有显著再生，并且新生牙骨质为有胶原纤维穿通的无细胞牙骨质。结论重组质粒PcDNA3．1－AMG在体外转染哺乳动物细胞后能够表达重组人釉原蛋白．并且表达产物具有促进牙周组织再生的生物学活性。     

Effects of C‐Terminal Amelogenin Peptide on Proliferation of Human Cementoblast Lineage Cells

Background: Extracts of enamel matrix proteins are used to regenerate periodontal tissue; amelogenin, the most abundant enamel protein, plays an important role in this regeneration. Studies have demonstrated that amelogenin fragments promote tissue regeneration, but the bioactive site of amelogenin remains unclear. This study explores the functional domain of amelogenin by investigating effects of four amelogenin species on cementoblast proliferation. Methods: Four amelogenin species based on amelogenin cleavage products were investigated: 1) recombinant human full‐length amelogenin (rh174); 2) amelogenin cleavage product lacking the C‐terminal (rh163); 3) amelogenin cleavage product lacking the N‐terminal (rh128); and 4) the C‐terminal region of rh174 (C11 peptide), which was synthesized and purified. Human cementoblast‐like cell line (HCEM) cells were cultured and treated with rh174, rh163, rh128, or C11 peptide. Cell proliferation was evaluated using 3‐(4,5‐dimethylthiazol‐2‐yl)‐5‐(3‐carboxymethoxyphenyl)‐2‐(4‐sulfophenyl)‐2H‐ tetrazolium assay and cell proliferation enzyme‐linked immunosorbent assay. Mitogen‐activated protein kinase (MAPK)–extracellular signal‐regulated kinase (ERK) (MAPK–ERK) pathway was examined by Western blot analysis. Results: Proliferation of HCEM cells was significantly enhanced on treatment with rh174, rh128, or C11 peptide. However, rh163 had no effect compared with the untreated control group. Western blot analysis revealed enhanced phosphorylated ERK1/2 signaling after addition of rh128 or C11 peptide and reduced phosphorylated ERK1/2 signaling after blocking with a specific MAPK inhibitor (U0126). Conclusion: C‐terminal amelogenin cleavage product increased proliferation of HCEM through MAPK–ERK signaling pathway, indicating possible application of C11 peptide for periodontal tissue regeneration.     

Amelogenins in human developing and mature dental pulp

Amelogenins are a group of heterogenous proteins first identified in developing tooth enamel and reported to be present in odontoblasts. The objective of this study was to elucidate the expression and function of amelogenins in the human dentin-pulp complex. Developing human tooth buds were immunostained for amelogenin, and mRNA was detected by in situ hybridization. The effects of recombinant amelogenins on pulp and papilla cell proliferation were measured by Brd U immunoassay, and differentiation was monitored by alkaline phosphatase expression. Amelogenin protein was found in the forming dentin matrix, and amelogenin mRNA was localized in the dentin, presumably in the odontoblast processes. Proliferation of papilla cells was enhanced by recombinant human amelogenin rH72 (LRAP+ exon 4), while pulp cells responded to both rH72 and rH58 (LRAP), with no effect by rH174. These studies suggest that odontoblasts actively synthesize and secrete amelogenin protein during human tooth development, and that low-molecular-weight amelogenins can enhance pulp cell proliferation.     

Full length amelogenin binds to cell surface LAMP-1 on tooth root/periodontium associated cells

Objectives

Lysosome-associated membrane protein-1 (LAMP-1) has been suggested to be a cell surface receptor for a specific amelogenin isoform, leucine-rich amelogenin peptide or LRAP. However, it is unclear if LAMP-1 is an amelogenin receptor for dental mesenchymal cells. The goal of this study was to determine if LAMP-1 serves as a cell surface binding site for full length amelogenin on tooth root/periodontium associated mesenchymal cells.

Design

Murine dental follicle cells and cementoblasts (OCCM-30) were cultured for 2 days followed by addition of full length recombinant mouse amelogenin, rp(H)M180. Dose-response (0-100 μg/ml) and time course (0-120 min) assays were performed to determine the optimal conditions for live cell surface binding using immunofluorescent microscopy. A competitive binding assay was performed to determine binding specificity by adding Emdogain^® (1 mg/ml) to the media. An antibody against LAMP-1 was used to detect the location of LAMP-1 on the cell surface and the pattern was compared to cell surface bound amelogenin. Both amelogenin and cell surface LAMP-1 were immuno-co-localized to compare the amount and distribution pattern.

Results

Maximum surface binding was achieved with 50 μg/ml of rp(H)M180 for 120 min. This binding was specific as demonstrated by competitive inhibition (79% lower) with the addition of Emdogain^®. The binding pattern for rp(H)M180 was similar to the distribution of surface LAMP-1 on dental follicle cells and cementoblasts. The high co-localization coefficient (0.92) for rp(H)M180 and LAMP-1 supports rp(H)M180 binding to cell surface LAMP-1.

Conclusions

The data from this study suggest that LAMP-1 can serve as a cell surface binding site for amelogenin on dental follicle cells and cementoblasts.