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頁籤選單縮合
題名 | An in Vitro System for the Study of Hepatitis B Gene Expression=研究B型肝炎基因表達的新系統 |
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作者 | 胡志棠; | 書刊名 | 慈濟醫學 |
卷期 | 16:5 2004.10[民93.10] |
頁次 | 頁293-300 |
分類號 | 414.84 |
關鍵詞 | B型肝炎; 基因表達; HepG2細胞株; Hepatitis B; Gene expression; HepG2 cell line; |
語文 | 英文(English) |
中文摘要 | 目的:在於建構一個沒有B型肝炎病毒(HBV)複製,但能產生HBV蛋白的細胞系統以研究影響HBV的基因表達。材料與方法:載體pREP4主要為鼻咽癌過濾性病毒(Epstein-Barr Virus, EBV)的架構。能表達HBV蛋白的媒體為pKSV-HBV1(含有HBV 661 二聚物)。於轉染人類肝癌細胞HepG2以前,藉限制酶Sal I與Kpn I將pREP4內一段勞斯肉瘤病毒(Rous sarcoma virus, RSV)的引動子切除以容HBV 991插入,使得HBV蛋白的基因表達能藉HBV自己的引動子所驅策,並於定量時不受病毒複製的干擾。此外,一套細胞溶解液(cell lysate)可同時藉一改良過的乳酸脫氫酵素分析法(CytoTox lactate dehydrogenase assay)和酵素免疫測試劑(biokit ELISA)以評估細胞生長和做HBV抗原蛋白的定量。結果:完成兩個細胞株的建構,一為細胞內載體含有HBV基因組(HepG2/pREP4D/HBV991A),另一為對照組細胞,細胞內僅為載體(HepG2/pREP4D)。由ELISA及免疫沈澱合併西方墨點法證明HBV轉染細胞能產生HBsAg,也分泌至培養液中;另外,由LDH assay顯示兩種轉染細胞的生長速度相同(未因HBsAg的堆積而受影響),但比母細胞HepG2的生長速度緩慢。結論:此含母細胞HepG2、對照組細胞(HepG2/pREP4D)及能產生HBV表面抗原的細胞株(HepG2/pREP4D/HBV991A)可用以探討HBsAg與細胞生長的交互作用,其特色經深入研究後,可用以進一步探討HBV的基因表達。 |
英文摘要 | Objectives: This study was to develop an alternative in vitro system for the study of hepatitis B (HBV) gene expression in the absence of viral replication. Materials and Methods: Before transfecting HepG2 cells, the enhancer/promoter region in the Rous Sarcoma Virus long terminal repeat of the pREP4 expression vector was deleted. Thereafter, internal incorporation of a complete HBV991 genome with native HBV promoters conferred non-vector control over HBV gene expression from all HBV promoters without quantitative interference from replication. A Single lysate of the cells was used for both an improved CytoTox lactate dehydrogenase assay to follow the growth of both cell lines and a biokit ELISA followed by immunprecipitation with Western blotting to assay hepatitis B surface antigens. Results: A novel hepatitis B suface antigen producing cell line (HepG2/pREP4D/HBV991A) was constructed, which contained a whole linearised hepatitis B genome cloned into an episomal vector based on the Epstein-Barr virus replicon. In parallel, a control cell line containing only native vector (HepG2/pREP4D) was established. Initially with this novel system, we focused on the interaction between HBsAg and cell growth. Both new cell lines have been shown to grow at the same rate, despite the fact that one makes hepatitis surface antigens in quantities which were detected easily while the other did not. Conclusions: the availability of two such cell lines derived from a common parent stock should make possible for the first time direct studies of the interaction between HBsAg and cell growth in addition to HBV gene expression after meticulous characterizations. |
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