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題名 | Recombinant Retinal Pigment Epithelium-Derived Factor:Its Purification from Overexpressing Escherichia Coli and Survival Effect on Induced Apoptosis in Cerebellar Granule Neurons=基因重組網膜色素上皮細胞因子:由大腸桿菌中選殖表現及純化和其對於小腦顆粒神經元誘導性程式化死亡之救活效應 |
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作者姓名(中文) | 葉則祥; 蔡長; 李淑美; 劉榮宏; 吳燕琴; | 書刊名 | 中華民國眼科醫學會雜誌 |
卷期 | 34:3 1995.09[民84.09] |
頁次 | 頁1-12 |
分類號 | 416.746 |
關鍵詞 | 基因重組; 網膜色素; 上皮細胞因子; 大腸桿菌; 選殖; 純化; 小腦顆粒神經元; 死亡; 救活效應; Pigment epithelium-derived factor; PEDF; Neurotrophic factor; Apoptosis; Programmed cell death; Cell survival; |
語文 | 英文(English) |
中文摘要 | 網膜色素上皮因子最初純佔自胎兒網膜色素上皮細胞之培養基,而為一 具神經滋養性之蛋白質。因其在培養基中含量稀少,前人乃經由人類胎兒補體 DNA基因廣建立基因重組的截斷架構(夭門冬素44一普羅林418),以基因工程技術 殖入表現於大腸菌中。經由引導含有此截斷架構之載體,我們自菌體之包含體得 到一預期大小(42.8kDa)的蛋白質。溶解後,此蛋白質經陽離子交換色層分析和超 過濾可被高度純佔且在西方墨點試驗中被色素上皮因子抗體特定檢出。於神經突 生長檢驗中,這基因重組色素上皮因子以促進Y一79視網膜胚母細胞瘤絀胞株之 分化來表現其神經滋養活性。因此這基因重組蛋白有類似天然色素上皮細胞因子 的生物活性。 網膜色素上皮因子也在源自中樞神經的小腦顆粒神經元土表現神經滋養救活作 用;它可明顯增加活存神經元數目。進一步,我們在以低鉀引導小腦顆粒神經元 發生程式化死亡的模式中,檢驗重組色素上皮因子的作用。我們發現其可經由抑 制DNA斷裂來延緩細胞的程式化死亡。有鑑於細胞程式化死亡無論於發育中的神 經系統(包括視網膜)和神經退化性疾病都很常見,於該系統,尤其是視網膜,細 胞程式化死亡的發生及其受存活因子所抑制的機轉的釐清就顯得非常值得。畢竟 色素上皮細胞因子是感光細胞間質組成中唯一能引導Y-79細胞產生神經突分化 的。 |
英文摘要 | Pigment epithelium-derived factor (PEDF), first purified from the culturemedium of retinal pigment epithelial cells of human fetus, is a neurotrophicprotein. Since it is present in low amount in conditioned medium, a truncatedrecombinant expression construct (Asp44-Pro418), based on PEDF cDNAcloned from a fetal human cDNA library, has been expressed in Escherichiacoil. By induction from a vector containing the truncated PEDF version, weproduced a protein with expected size of 42.8 kDa associated with inclusionbodies in the host. After solubilization, the protein was highly purified bycation exchange chromatography and ultrafiltration, and specificallyrecognized on Western transfers by polyclonal antibodies to PEDF. In neuriteoutgrowth assays, the recombinant PEDF (rPEDF) demonstratedneurotrophic activity by potentiating the differentiation of human Y-79retinoblastoma cells as reported for the native PEDF. Thus, the recombinantprotein retains a potent biological activity. PEDF has shown to be a neurotrophic survival factor of the primaryculture of neurons from the central nervous system, the cerebellar granuleneurons (CGNs), by significantly increasing the number of viable neurons.To extend the study, we further tested the survival effect of rPEDF on amodel of low k+-induced apoptosis (programmed cell death) in CGN. rPEDFcould delay the induced cell death by inhibiting DNA fragmentation. Giventhat apoptosis is extensively observed in the developing nervous systemincluding the retina as well as in neurodegenerative diseases, it is worthwhile |
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