論文名稱:

神經生長因子及其訊息核醣核酸在大白鼠牙齒形態發生時之表現

 

Spatiotemporal Patterns of Expression of Nerve Growth Factor and Its mRNA in Morphogenesis of Rat Tooth

研究生:

胡岳江  Hu, Yueh-Chiang

 

(以作者名查詢陽明大學館藏系統)

 

(以作者名查詢全國圖書書目資訊網)

指導教授:

楊世芳  Yang, Shin-Fang

        學校名稱:

國立陽明大學

系所名稱:

口腔生物研究所

          學年度:

84

關鍵字:

牙醫  DENTIST

 

牙醫學  DENTISTRY

 

口腔  ORAL-HEALTH

摘要:

神經生長因子被廣泛的研究於神經系統發育過程,已知其為痛覺神經相關之標的組織衍生因子,對於感覺神經元及交感神經元的存活是必要的。有許多文獻指出,神經生長因子亦參與器官形態發生(morphogenesis)的過程,在上皮與間葉組織間有著重要的調控角色。由於在牙齒發育過程中,其神經支配(innervation)為體內較晚建立之體系,而提供研究神經生長因子如何橫跨形態發生與神經支配這二種截然不同之時期,也提供良好題材來研究神經生長因子是否存在著特別的調控方式。本實驗利用免疫螢光法及反轉錄-聚合酉每鏈鎖反應原住雜交法(RT-PCR in Situ)研究大白鼠胚胎各時期牙齒上神經生長因子及其mRNA的表現時問與位置,並以物質P(substance P)的免疫螢光染色,以期了解神經生長情況。結果發現神經生長因子,在牙齒發育初期當作上皮與外間葉組織間的訊息傳遞者,在牙齒形態生成時則表現於控制牙冠外觀的牙上皮內,在造牙本質細胞從未分化的牙髓細胞中進行最終分化時,則開始於其內製造以幫助細胞分化過程,當神經支配時更扮演著標的組織衍生因子的角色,因此發現神經生長因子具有參與牙齒發育各時期的廣泛功能。另外也發現造牙本質細胞在神經支配前就已先分泌神經生長因子,不同於一般標的組織在神經末稍接時才開始製造之方式,這種現象可能與造成牙齒神經支配上許多不尋常特性有關。而今人意外的,牙齒發育時期物質P的表現位置均與神經生長因子類似,但其主要位於基底膜上故可能參與組織間的交互作用,將來研究其m RNA的表現以對其有進一步的了解。利用RT-PCR in situ研究組織切片上mRNA的表現為本實驗之特色,其在步驟上非常省時簡使且無放射污染,僅需更換引子即可定位不同的mRNA。其定位結果與過去文獻中所提及神經生長因子mRNA的表現位置均非常類似,而於試管中進行相同步驟之RT-PCR所得產物經電泳檢視,也發現的確具有相當高之特異性,就其可信度與正確性來看此為值得推廣之方法。

   
 

  Nerve growth factor (NGF) has been widely studied in the development of nerve system.  We have known that it is the target-derived factor concerning about the nociceptive neurons, and it is essential for the survival of sensory neurons and sympathetic neurons.  However, many evidences indicate that NGF also involves in the process of morphogenesis and plays an important regulatory role between epithelium and mesenchyrne. Because the innervation of the tooth is established later, it provides an excellent model to investigate how NGF adjusts between the two drastically different roles of morphogenesis and innervation.  And it also provides some special ways to study the regulation of NGF.  This experiment makes use of immunofluorescent method and RT-PCR in situ to investigate the expression site and time of NGF and its mRNA in tooth germ of rat embryo, and uses the immunofluoresent staining of substance P to understand the situation of the developing nerve.  As a result, we found that NGF acts as the signal transmitter between epithelium and ectomesenchyme in the initial period of tooth development; when morphogenesis it expresses in the dental epithelium which controls the shape of tooth crown; it begins to be produced in the odontoblasts that emerge from some undifferentiated pulp cells undergoing terminal differentiation; moreover, it plays the role of target-derived factor when the nerves innervate.  Therefore, NGF has very board functions of participating in different periods of tooth development.  In addition, it was found that odontoblasts have secreted NGF before the nerves innervate, which is different from the way that ordinary targets begin to produce NGF as soon as the nerve endings reach it.  This phenomena might concern about many unusual properties of tooth irmervation.  We were surprised to find that the expression of substance P is similar to NGF, but it mainly locates on the basement membrane, so it probably involves in the interactions between tissue layers.  We will study the expression of its mRNA in the future in order to understand more.  It is characteriezed to use RT-PCR in situ in this experiment. We can easily and fast proceed this study as well as without radio-labeling.  And we could localize another mRNA just using another pair of primers in this method.  The results of NGF mRNA localization were very similar to the expression sites that previous papers had mentioned.  When we examined by electrophoresis the product which was gained from the same procedure of RT-PCR and was developed in tubes, we also found that it really has very high level of specifisity.  As for its accuracy and confidence, RT-PCR in situ is a worth-introducing method.