臺灣博碩士論文加值系統

　　法布瑞氏症為X染色體性聯遺傳疾病，發生率約為五萬分之一，而研究發現在台灣的發生率極高（男性約1/1,500；女性約1/800）。其致病原因主要為GLA基因發生突變，導致酵素蛋白功能異常。缺乏α-半乳糖苷酶酵素使得細胞無法正常代謝鞘糖脂，導致鞘糖脂大量堆積於細胞溶酶體當中，進一步使細胞受損而影響器官功能產生疾病。
　　對於法布瑞氏症最有效的方法為酵素替代療法，目前我國衛生福利部已核准兩種用於治療法布瑞氏症的酵素蛋白藥物，分別為 Agalsidase alfa（Replagal®, Shire）與Agalsidase beta（Fabrazyme®, Sanofi）。酵素治療可有效延緩症狀的發生，但是現行的酵素替代療法價格昂貴、且患者須長期輸注，因此如何開發新的治療策略提高療效、改善長期輸注的模式便顯得相當重要。酵素治療主要是將α-半乳糖苷酶酵素透過靜脈輸注到患者體內，透過細胞膜上的甘露糖六磷酸根受體進入溶酶體，分解過度堆積的糖鞘脂。先前研究發現第二型胰島素生長因子（IGF2）也可以被非陽離子依賴型甘露糖六磷酸根受體辨識結合，因此後續研究便開始嘗試將蛋白酵素與IGF2部分胜肽片段結合，發現此嵌合蛋白確實能增加進入細胞溶酶體內的效率。在本研究中，我們藉由建構第二型胰島素生長因子α半乳糖苷酶嵌合重組蛋白，並測試其是否能提高細胞的吸收效率，進而提高治療的效果。
　　先前本實驗室已經利用CRISPR/Cas9的技術成功建立GLA基因剔除HEK293細胞株，同時我們也建構不同形式的嵌合蛋白。接著轉染至GLA基因剔除HEK293細胞株使其過度表現嵌合蛋白，最後發現將IGF2部分胜肽片段嵌於GLA的羧基端為最具有酵素活性。在細胞吸收的實驗中，我們發現此形式的嵌合重組蛋白與野生型重組蛋白相比，其被細胞吸收的效率有9.8倍的提升。我們進一步利用GLA基因剔除HEK293細胞株製作GLA-IGF2嵌合式蛋白的穩定表現細胞株（stable clone），避免內生性蛋白的干擾，做為大量製備嵌合式重組蛋白的來源。接下來將大量純化嵌合式重組蛋白，探討嵌合式重組蛋白對於細胞內Gb3的清除能力。期許藉由嵌合式重組蛋白的開發，提升法布瑞氏症患者現行酵素治療的療效。

Fabry disease is an X-linked inherited disease with the incidence rate 1/50000 and was reported at an high frequency in Taiwan. The major cause of the disease is due to the mutation of alpha-Galactosidase A (GLA). Deficient of GLA activity results in the accumulation of globotriaosylceramide (Gb3) in cells and induce cell damages.
The most effective therapy so far is enzyme replacement therapy (ERT). At present, Ministry of Health and Welfare has approved two protein drugs for Fabry disease treatment, Agalsidase alfa (Replagal®, Shire) and Agalsidase beta (Fabrazyme®, Sanofi), respectively. Although ERT can delay the onset of Fabry symptoms, the cost is very expensive. Develop a new treatment strategy to increase efficiency is highly demanded. The main concept of ERT is the intravascular injection of GLA which can accesses lysosomes through binding to mannose-6-phosphate receptor (M6PR) and help break down Gb3. Previous study showed that IGF2 can be recognized by cation-independent mannose-6-phosphate receptor (CI-M6PR) as well. Furthermore, following studies demonstrated that fusion of IGF2 with enzyme can enhance uptake efficiency. In this study, we develop a fusion protein contained a partial IGF2 peptide and GLA. The fusion protein will be examined of uptake efficiency and therapeutic effect.
First, we generated GLA-deficient HEK293 cell lines using CRISPR/Cas9 technique. Meanwhile, we constructed the most active form of fusion protein by measuring enzyme activity. We found out this GLA-IGF2 fusion protein is more efficient up-taken by cells compare with recombinant GLA. We have generated a GLA-IGF2 stable clone and now purifying recombinant protein to investigate its Gb3 clearence ability. We speculated that the development of GLA-IGF2 recombination protein would provide a better treatment for Fabry patients.

誌謝　i
中文摘要　ii
英文摘要　iii
目錄　iv
圖目錄　vii
表目錄　viii
附錄目錄　ix
1.緒論　1
1.1　溶酶體功能簡介　1
1.2　溶酶體儲積症　1
1.3　台灣新生兒篩檢　2
1.4　法布瑞氏症簡介與文獻回顧　3
　　　人類GLA酵素功能介紹　3
　　　台灣的法布瑞氏症常見基因突變點　4
　　　法布瑞氏症的症狀與分類　5
　　　法布瑞氏症的診斷　6
　　　法布瑞氏症的致病機轉　6
1.5　法布瑞氏症的治療　8
　　　酵素替代療法　8
　　　藥理伴護子療法　10
　　　化學修飾　11
　　　基因治療　11
1.6　甘露糖六磷酸根受體（M6PR）簡介　12
　　　甘露糖六磷酸根受體（M6PR）的結構　13
　　　非陽離子依賴型甘露糖六磷酸根受體（CI-M6PR）在治療上扮演的角色　13
　　　以甘露糖六磷酸根受體（M6PR）為標的蛋白質酵素研究　14
1.7　研究動機　16
2.材料與方法　17
2.1　實驗材料　17
　　　試藥列表　17
　　　試劑套組　20
　　　儀器列表　21
2.2　載體的建構　22
　　　GLA-myc-6xHis載體建立　22
　　　IGF2-GLA-myc-6xHis載體建構　22
　　　IGF2-GLA-myc-6xHis spd載體建構　22
　　　IGF2-GLA-myc-6xHis scd載體建構　23
　　　GLA-IGF2-myc-6xHis載體建立　23
2.3　細胞株相關實驗　24
　　　細胞培養　24
　　　細胞轉染　24
　　　細胞蛋白質萃取　24
　　　蛋白質定量　25
　　　酵素活性測試　25
2.4　西方墨點法　26
　　　膠體配置　26
　　　膠體電泳　26
　　　轉漬與抗體染色　27
2.5　穩定表現GLA和GLA-IGF2的 HEK293細胞株建立　27
　　　載體的建立　27
　　　穩定細胞株的建立　28
　　　培養液中酵素活性的測量　29
2.6　免疫螢光染色　29
2.7　GLA-IGF2蛋白質純化　30
2.8　M6P抑制細胞吞噬能力測試　30
2.9　統計分析　31
3.　結果　32
3.1　建立具有功能的GLA-IGF2酵素嵌合蛋白　32
3.2　細胞吞噬效率測試　36
3.3　細胞Gb3清除能力測試　37
3.4　穩定表現GLA、GLA-IGF2的HEK293細胞株建立　38
3.5　細胞吞噬抑制實驗測試　39
3.6　GLA-IGF2蛋白質純化　39
4.　討論　43
5.　參考文獻　48
圖　58
表　74
附錄　79

圖目錄
圖一、將IGF2-GLA-myc-6xHis載體轉染至GLA基因剔除HEK293細胞株中　59
圖二、將IGF2-GLA-myc-6xHis spd載體轉染至GLA基因剔除HEK293細胞株中　60
圖三、將IGF2-GLA-myc-6xHis scd載體轉染至GLA基因剔除HEK293細胞株中　61
圖四、將GLA-IGF2-myc-6xHis載體轉染至GLA基因剔除HEK293細胞株中　62
圖五、細胞對於GLA和GLA -IGF2重組蛋白的吞噬能力測試（I）　63
圖六、細胞對於GLA和GLA -IGF2重組蛋白的吞噬能力測試（II）　64
圖七、GLA-IGF2酵素重組蛋白對於細胞內Gb3清除能力　65
圖八、GLA基因剔除HEK293穩定表現GLA細胞株　66
圖九、GLA基因剔除HEK293穩定表現GLA-IGF2細胞株　67
圖十、穩定表現GLA和GLA-IGF2細胞株酵素活性和西方墨點法分析　68
圖十一、GLA-IGF2細胞吞噬抑制實驗　69
圖十二、GLA-IGF2酵素融合蛋白純化　70
圖十三、Imidazole影響GLA酵素活性測試　71
圖十四、測試鎳離子管柱Elution時所需Imidazole濃度　72
圖十五、利用Imidazole濃度為70mM的Elution緩衝液執行蛋白質純化　73

表目錄
表一、穩定表現GLA的HEK293 細胞株　75
表二、穩定表現GLA-IGF2的HEK293細胞株　76
表三、GLA-IGF2 純化酵素活性分析（I）　77
表四、GLA-IGF2 純化酵素活性分析（II）78

附錄目錄
附錄一、溶酶體的結構　80
附錄二、溶酶體的主要功能　81
附錄三、溶酶體儲積症的分類　82
附錄四、Glycosphingolipid代謝流程圖　83
附錄五、27個在台灣常見的GLA基因突變點　84
附錄六、心臟型法布瑞氏症特異突變(GLA IVS4+919 G>A)造成mRNA剪輯異常　85
附錄七、CD-M6PR和CI-M6PR結構　86
附錄八、抗體列表　87
附錄九、pcDNA3.1/myc-His B載體　88附錄十、利用 CRISPR/Cas9 技術剔除GLA基因　89
附錄十一、各種溶酶體儲積症的酵素蛋白作用方式　90

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