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靈芝醇可溶萃取物對活化腎膈細胞之效應及相關機轉 Effects and mechanisms of Ganoderma lucidum alcohol-soluble extracts on activated mesangial cells
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| 作者 |
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方明瑜 |
| 學校系所 |
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國立陽明大學醫學生物技術研究所 |
| 地點 |
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全臺 全部
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| 研究內容 |
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[ 摘要 ]
一氧化氮(Nitric oxide, NO)為自由基，在多種類型的腎絲球腎炎中扮演重要的致病角色。細菌內毒素脂多醣體 (lipopolysaccharide, LPS)和細胞激素可誘發腎絲球腎膈細胞(mesangial cells)中誘導型一氧化氮合成酶(inducible nitric oxide synthase, iNOS) 表現及大量NO生成，直接參與腎絲球發炎反應。除此，NO在體內可經由扮演訊息傳遞者之角色來調控促發炎基因或轉錄因子的表現，多層面地參與腎絲球腎炎之發生。在動物模式中已證實，專一抑制iNOS表現可減少腎絲球趨化激素(chemokines)產生、白血球浸潤以及半月體形成。因此抑制iNOS表現或活性可能是治療腎絲球腎炎的有效策略。靈芝是我國廣泛使用的中草藥，它曾被報導具有抗氧化、抗腫瘤以及免疫調節等功效。有研究指出靈芝在RAW264.7巨噬細胞中可抑制LPS所誘發的NO生成，故其很有可能具有治療腎絲球腎炎的潛力。因此本研究以LPS及Interferon-γ( IFN-γ) 活化小鼠腎膈細胞作為模擬急性腎絲球腎炎的體外模式，再以此模式探討靈芝醇可溶萃取物(Ganoderma lucidum alcohol-soluble extracts, GL)是否可抑制iNOS表現及其相關調控機轉。實驗發現在小鼠腎膈細胞以LPS ( 1μg/ml )及IFN-γ ( 100U/ml )共同處理下會刺激NO生成、iNOS mRNA、蛋白質表現增加，pro-inflammatory基因表現增加以及細胞增生。GL則呈劑量效應抑制NO生成，iNOS表現和iNOS promoter活性。這些結果暗示GL可能是透過調控iNOS promoter上相關的轉錄因子，而達到抑制iNOS基因表現的效應。除此之外，GL也可抑制LPS所誘發的pro-inflammatory gene表現，例如IL-1β、TNF-α、MIP-2等。綜合以上結果顯示GL在in vitro確實可經由抑制小鼠腎膈細胞iNOS表現而達到其抗發炎效果，進一步需以in vivo實驗證明，可為治療腎絲球腎炎提供新的方向。
[ 英文摘要 ]
The hepatitis B virus (HBV) posttranscriptional regulatory element (PRE) is a cis-sequence required for high level expression of viral surface transcripts and appears to function by promoting the export of the surface RNA from the nucleus. Functional studies of PRE have been heavily relied on the RNA export system pDM138. As a result, PRE sequences, such as RNA stem-loop �� (SL��), stem-loop ��1 (SL��1) and polypyrimidine tract binding protein (PTB) binding sites were found to be important for PRE function. On the other hand, recent report on the study of patients’ HBV isolates found a single G�莧 alteration within the surface gene at position 458 (G458A) acting at posttranscriptional level to reduce surface RNA expression. The nucleotide 458 is at �{1 position to a potential splice donor site SD459 on the HBV genome where G458A mutation was shown to prevent surface RNA splicing from position 458 to 1305. The finding raised an issue whether the delicate balance between splicing and export is essential in HBV surface RNA biogenesis.
In this study, we re-investigated the functional significance of PRE cis-elements defined by pDM138 reporter system under the nature context of HBV surface gene. We found that the surface RNA level decreased when the structure of SL�� was disrupted and otherwise not affected when SL��1 or putative PTB sites were mutated. It is noted that PRE was initially defined by serial truncations of the 3’ end of the HBV genome where no functional PRE sequences were found to locate in regions SL��1 or putative PTB binding sites mapped. Thus, the discrepancy of our findings to the previous arose by the systems chosen. We also reproduced and confirmed the effect of G458A mutation on surface RNA production in expression system employed in this study. Moreover, we found that surface RNA expression could be partially restored when a heterologous intron from the rabbit ��-globin gene was included into surface expression construct carrying G458A mutation, suggesting the function of splice donor site SD459 involves in surface RNA export. We also showed that the action of RNA splicing was rested at the early stage of spliceosome formation since elimination of all known splice acceptor sites on the surface RNA had no effect on its production. Furthermore, analysis of the effect of the splice donor site sequence on surface RNA expression indicates that the stability of splice donor site sequence bonds to U1 snRNA is more important than whether such site is functional in splicing. The results of this study show that the step of U1 snRNA binding to splice donor site SD459 is instrumental in HBV surface RNA biogenesis.
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