藉由報導基因EGFP探討DMP1在發育過程中的組織專一性表現及構築DMP1啟動子啟動Shh過量表現之結構體

Expression pattern of Dentin Matrix Protein 1 (DMP1) during Development via EGFP Reporter Gene and Construction of DMP1 promoter-Shh cDNA Over-expression Vector

林麗芬(Li-Fen Lin)

指導教授 ： 張百恩

國立臺灣大學/醫學院/口腔生物科學研究所/碩士(2007年)

https://doi.org/10.6342/NTU.2007.01499

摘要

在骨形成及牙本質形成的過程中成骨細胞與齒質母細胞會分泌一種非膠原性蛋白，並在細胞外形成基質，其中一類非膠原性的蛋白稱為SIBLING家族。而牙本質基質蛋白(DMP-1)是屬於這個家族蛋白裡的成員之ㄧ。

DMP-1是一種酸性磷酸化的蛋白質，它的生化功能和骨頭、軟骨以及牙齒的發育有關。在牙齒和骨骼的分化過程中，DMP-1在牙本質母細胞、齒釉質母細胞、齒質母細胞、頭頂以及長骨都有表現。

以綠色螢光蛋白EGFP為報導基因，在斑馬魚及小鼠的動物模式中分析人類DMP-1啟動子的活性可發現人類DMP-1啟動子的上游2.783 kb片段對基因的調控在斑馬魚和小鼠間具有保留性(余俐亭，2004)。本研究發現，此結構體可以驅動綠色螢光蛋白在胚胎15.5天小鼠的手臂、腳掌、毛囊、上顎骨、下顎骨和尾巴表現。在一個月小鼠的骨頭、氣管、胸腺、腋下淋巴結、鼠蹊部淋巴結、脾臟、腎臟、peyer’s patch、睪丸、副睪的結締組織、胃、支氣管、食道及舌的腹面都具有綠色螢光蛋白之表現，並且利用反轉錄聚合酶鏈鎖反應及原位雜合法發現DMP-1的表現並非侷限在礦化的組織，在非礦化的組織中也有DMP-1的表現。另一方面，構築以人類DMP-1啟動子驅動Sonic Hedgehog(Shh)異位過量表現的結構體並以顯微注射的方式來觀察此結構體對斑馬魚骨頭發育過程的影響。

關鍵字

牙本質基質蛋白 ； 反轉錄聚合酶鏈鎖反應 ； 原位雜合法 ； 牙齒 ； 骨頭 ； 綠色螢光蛋白

並列摘要

In the process of osteogenesis and dentinogenesis, osteoblasts and odontoblasts secrete non-collagenous proteins to form extracellular matrix (ECM). One group of the non-collagenous proteins is SIBLING family (SIBLING family; Small-Integrin-Binding Ligand, N-linked Glyco- protein). DMP-1 (Dentin Matrix Protein 1; DMP-1) is a member of the family.
DMP-1, an acidic phosphorylated protein, is expressed biofunctionally in bone and teeth calcification. Previous studies have shown that DMP-1 is expressed in odontoblasts, ameloblsats, cementoblasts, calvaeia, and long bone during teeth and bone differentiation.
Upstream 2,783 Kb fragment of human DMP-1 promoter is functional by the expression of the reporter gene (EGFP, enhanced green fluorescent protein) in transgenic zabrafish and mice (Yu, 2004). In this study, I found that Hu-DMP-1-GFP-2.783K-36 can drive the expression of the reporter gene (EGFP) in lymph node, palm, hair follicle, maxilla, mandible, and tail at the stage of 15.5 dpc (day post coitum). Moreover, Hu-DMP- 1-GFP-2.783K-36 also can drive the expression of the reporter gene (EGFP) in the bone, trachea, thymus, spleen, kidney, peyer’s patch, testis, stomach, esophagus and tongue in the transgenic mice at the postnatal development (one month). The overall results suggest that location of the DMP-1 is not restricted to mineralized tissues and may be present in non-mineralized tissue. Furthermore, the expression pattern of DMP1 was further confirmed by RT-PCR and in situ hybridization.
I have constructed the pDMP-shh-IRES-hrGFP chimeric gene, and the constructs were micro-injected into the zebrafish eggs. In this way, Shh can be over-expressed in the bone, and simultaneously the transgenic fish containing the construct can be screened by the expression of GFP. However, no transgenic stable line was obtained.

並列關鍵字

DMP-1 ； RT-PCR, ； In situ hybridization, ； teeth, ； bone ； GFP

參考文獻

余俐亭。2004。人類DMP-1基因啟動子之選殖及分析。國立台灣大學醫學院口腔生物科學研究所口腔細胞生物學組碩士論文。

王偉庭。2005。利用基因轉殖異位超量表現方法分析Sonic Hedgehog在斑馬魚眼睛視網膜發育過程中所扮演之功能。國立台灣大學醫學院口腔生物科學研究所碩士論文。

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Akira Yamaguchi, Toshihisa Komori, and Tatsuo Suda, 2000. Regulation of Osteoblast Differentiation Mediated by Bone Morphogenetic Proteins, Hedgehogs, and Cbfa1. Endocr. Rev. 21, 393–411

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