胡務亮2006-07-262018-07-112006-07-262018-07-111998http://ntur.lib.ntu.edu.tw//handle/246246/22824纖維芽細胞生長激素(fibroblast growth factor, FGF)及其受體是生物體中 很重要得的一套訊息傳遞系統，然而纖維 芽細胞生長激素受體(FGFR)和人類遺傳疾 病之關係，直到最近才被發現。由基因連 鎖分析建立基因圖譜後，發現軟骨形成不 全侏儒症(achonchoplsia)，一種常見顯性 遺傳侏儒，在圖譜上的位置和FGFR3 基因 相同。後來果然找到了軟骨形成不全侏儒 症的突變點，在台灣我們也證實了軟骨形 成不全侏儒症， Apert ， Cruzon ， hypochondroplasia,thanatophoric dysplasia 等骨骼疾病都和這一群受體 (FGFR1, FGFR2,and FGFR3)有關。 這些疾病多半是顯性遺傳，軟骨形成 不全侏儒的突變後來竟然被証實是激發性 的。因此我們計畫以抗體去阻斷受體的激 發，以期達到治療的酵果。我們首先自 Dr.Hayman 處得到了FGFR3 的cDNA(Keegan 1991)。我們接著把cDNA 中決定細胞外受 體的一段，放入pRSET 載體中，並以表現 出的蛋白質，注射兔子得到了FGFR3 抗體。 這個抗體可以在人類皮膚纖維芽細胞及一 些細胞株的Western blot 分析中，偵測到 約130kDa 的FGFR3 蛋白。然而這個抗體無 法在免疫沉澱實驗中抓下FGFR3 蛋白。由 於免疫沉澱實驗是研究細胞表面受體表 現、功能及活化不可缺少的工具，因此後 續的實驗就無法進行。 我們試圖將pRSET 表現出的FGFR3 蛋 白質，注射小鼠製造單株抗體。我們可以 挑選到不少可以和FGFR3 蛋白質作用的抗 體株，但是其效價在培養的過程中很快的 降低。我們試圖表現其他位置的FGFR3 蛋 白質來製造抗體，FGFR3 蛋白質細胞外的部 分表現量都很低，細胞內部分則順利表現 出來。利用這個蛋白，我們製造了一個新 的抗體，不幸的，這個抗體在Western blot 分析中，偵測到不只一個蛋白質，所以也 沒有幫助。 本實驗的困難應該是因為FGFR3 是一 個細胞膜蛋白質， 而且有大量堂醣化 (glycosylation)的現象。這樣的蛋白質由 細菌表現較難，所製造出之抗體也未必能 有效偵測細胞膜蛋白質，尤其是做免疫沉 澱實驗時。用FGFR3 細胞內的部分做抗體 就簡單得多，可是tyrosine kinase domain 相似性太高，抗體的特異性就不夠了。This Fibroblast growth factors and their receptors are an important set of signal transduction system in animals. the association between this system and human diseases was understood recently. Achondroplasia, a common type of dominantly inherited drawfism, was found to be linked to FGFR3, and patients were found to have the FGFR3 mutations. Initiated by this finding, the FGF receptors (FGFR1, FGFR2 & FGFR3) was found to be related to several human bone dysplastic diseases including hypochondroplasia, thanatophoric dysplasia, Apert and Crouzon syndromes. The mutations causing achondroplasia and thanatophoric dysplasia were stimulating mutations. Therefore, it is possible to modify or to block the activation of the mutated receptors by antibody or other methods. We got FGFR3 cDNA frome Dr. Hayman. We cloned a portion of the cDNA responsive for the extracellular domain of the receptor into 2 pRSET vector. The expressed recombinant protein was used to immunized rabbit for FGFR3 antibody. This antibody detected a 130 kDa protein in both human skin fibroblasts lysate and in many cell lines by western blot analysis. However, this antibody was unable to capture FGFR3 protein in immunoprecipitation (IP) experiment. Since IP is the indispensable tools in the study of membranous receptors, either for the expression, function or activation. We tried to raise monoclonal antibody with this pRSET protein. We got some clones at the initial stage of screening, but the potencies of these clones dropped rapidly during continuing culture. In order to solve the problem, we tried to express other fragments of FGFR3. It is pretty difficult to express any of the extramembranous domains of FGFR3, but the intracellular domain was smoothly expressed. With the expressed intracellular domain, a second antibody was raised. However, this antibody detected more than one protein in western blot analysis. The difficulty in this study may arise from the character of FGFR3. It is a membranous receptor with intense glycosylation. E coli expressed protein may not reflect the structure of FGFR3, and thus difficult to capture the receptor in IP study. The intracellular domain, the tyrosine kinase domain, in the way is homologous to other tyrosine kinase proteins. However, we got a lot of experience in this study which will help us in future studies.application/pdf97018 bytesapplication/pdfzh-TW國立臺灣大學醫學院小兒科纖維芽細胞生長激素受體軟骨形成不全株儒抗體Fibroblast growth factor receptor 3 (FGFR3)achondroplasiaantibody[SDGs]SDG3reporthttp://ntur.lib.ntu.edu.tw/bitstream/246246/22824/1/872314B002142.pdf