- 學位論文
利用組織免疫染色法觀察局部切除兔子關節板後顳顎關節中第一型及第二型膠原蛋白的變化:(一)第一型及第二型膠原蛋白抗體的製造及純化
The Changes of Type I and Type II Collagens in The Rabbit Temporomandibular Joint After Partial Discectomy-An Immunohistochemical Study:(I)Production and Purification of Type I and Type II Collagen Ab
摘要
本實驗室先前利用重組膠原蛋白模板（collagen template）植入局部切除顳顎關節板（p artial discectomy）的兔子顳顎關節（temporomandibular joint）中，在組織學上觀察 到有良好的癒合及組織再生的現象。膠原蛋白（collagen）為構成結締組織骨架（framew ork）的重要成分，其在組織上的分佈（distribution）及型式（type）可以用來評估傷 口癒合（wound healing）及組織再生（regeneration）的過程及結果。為了進一步確認 及評估顳顎關節中膠原蛋白的變化及機轉，因此嘗試利用免疫組織染色法（immunohistoc hemistry）來作確認。但是經實驗發現市售六種膠原蛋白抗體皆不能有效地和膠原蛋白產 生反應，因此本實驗之重點即進行抗兔子第一型及第二型膠原蛋白多株抗體（polyclonal antibody）的製造及純化。首先由兔子肌腱及肋骨分別萃取第一型及第二型膠原蛋白。 在反覆酸溶、鹽析、透析及最後冷凍乾燥處理得到經SDS-PAGE電泳法分析出含有(2(I)、( 1(I)、((1,2)、((1,1)及(等帶（band）的第一型膠原蛋白及(1(II)及((1,1)及(的第二型 膠原蛋白。以同樣8% SDS-PAGE的膠片（gel）轉移（transfer）蛋白質作西方點墨分析法 （Western blot）染色來觀察抗原和抗體之間的反應時在相對於(1(1)的帶上有明顯的DAB 呈色反應，因此可知純化出來的豬抗兔第一型膠原蛋白抗體確實會和第一型膠原蛋白作用 。至於豬抗兔第二型膠原蛋白抗體的反應，約可見帶（band）只出現在第二型膠原蛋白(1 (II)上，而不會對第一型反應，和其他對照組比較，可確定豬抗兔第二型膠原蛋白抗體確 實會和第二型膠原蛋白作用。因此可見我們已成功地製造及分離出抗兔子第二型膠原蛋白 的抗體。進一步即可利用此抗體進行組織免疫染色法（immunohistochemistry）來觀察局 部切除兔子顳顎關節關節板後，將重組膠原蛋白模板植入兔子顳顎關節關節板中，第一型 及第二型膠原蛋白的變化。以確認及評估顳顎關節中膠原蛋白的變化及傷口癒合、修補及 再生機轉。
並列摘要
Previous study showed that a potential regenerative material, reconstituted co llagen template, enabled the disc to regenerate in the rabbit temporomandibula r joint after partial discectomy. The subsequent evaluation of macromolecular mechanisms of collagen changes in the TMJ disc by immunohistochemical method was further investigated. Six commercial collagen antibodies were tested to d etermine whether there a cross-reaction between these antibodies and rabbit ty pe I or type II collagens is. The results showed no antigen-antibody reaction among those collagen antibodies and the rabbit type I and type II collagen. An attempt to establish an animal model-porcine to produce antibodies against the rabbit type I and type II collagens was required.Type I and type II collag ens were extracted and purified from rabbit tendons and ribs respectively acco rding to H. L. Lin's method. The type II collagen was further purified to rem ove the type I collagen by immunoadsorption method. After emulsified with adj uvant, injected at multiple sites into the back skin of 60-70 Kg Lan-Yu minipo rcines. Bleeds were performed after enough title of antisera tested by ELISA. IgG was isolated by 40% saturated (NH4)2SO4 precipitation and liquid chromat ography on DEAE-Sephadex (Bio-Rad Econo system). Gradient SDS-PAGE showed one major band of 60 KDa (heavy chain) and one minor band of 23 KDa (light chain) . Furthermore, Western blot analysis showed that the porcine anti-rabbit type collagen I antibody recognized the (1(I) chain whereas porcine anti-rabbit ty pe II collagen antibody recognized the (1(II) chain. The results demonstrated that the polyclonal porcine anti-rabbit type I and type II collagen antibodie s were specific against the rabbit type I and type II collagen respectively.Th is porcine model for producing polyclonal antibody against the rabbit collagen may be used as a tool to characterize the structural and pathological propert ies of collagens.In this proposed study, collagen alternation in the joint hea ling after collagen template implantation is evaluated by using the immunohist ochemical method. The expecting results will yield the information to underst and the mechanism of joint diseases.Keywords: temporomandibular joint, collage n, polyclonal antibody
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