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研究生:謝艷玉
研究生(外文):Hsieh, Yen-Yu
論文名稱:以視網膜色素上皮細胞為眼底疾病及藥物開發模型的建立
論文名稱(外文):Establishment of RPE cells as a model for ocular disease and its drug development
指導教授:吳榮燦
指導教授(外文):Wu, Rong-Tsun
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:生物藥學研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:1997
畢業學年度:85
語文別:中文
論文頁數:88
中文關鍵詞:生物科技藥學視網膜眼底疾病
外文關鍵詞:BIOTECHNOLOGYPHARMACOLOGY
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高齡化社會的到臨,視力的保健也愈形重要,然而目前西方所發展的眼科用藥僅限於抗生素、類固醇等供暫時控制病情的藥物,對視網膜有關病變的治療藥物,則付之厥如。中藥及民間藥中有許多常用的眼科中藥,但其藥效則缺乏科學的佐證,因此,本研究擬以與眼睛機能息息相關的視網膜色素上皮細胞(RPEE; Retinal pigmnent epithelialcells)為對象,建立眼科藥物開發模型,期能以此模型來研發新藥及探討其作用機轉。
RPR的最主要機能為吞噬錐狀細胞及桿狀細胞受光線刺激而脫落的外節。有許多由RPRPR病變所引起的視力障礙與疾病,如:增殖性視網膜病變,老年性黃斑部病變,糖尿病導致的視網膜病變等,其bFGF,VEGF,IGF-I,TGF-β等生長因子會呈現不同的表現。另一方面,一氧化氮((NOO; Nitric oxide)在眼內由約各種機能上扮演重要的角色也在近幾年被廣為認知,然而,NO在RPE約正常吞吃機能中扮演的角色仍然不明,因此我們以RPRPR的吞吃機能為指標,輔以NO產生的量及基因的調節來探討藥物的藥效與作用機轉。
我們首次發現以胎牛血清處理RPRPR後可促進其微量NO的產生、增強其正常的吞噬機能,這個作用會被NOS inhhibitor抑制,同時我們也發現在吞噬的過程中是須要消耗微量的NO,我們認為RPE微量NO的產生應可提供藥物研發的新指標。
由於中醫書上常提及一些中草藥處方具有『清肝明目』的作用,因此我們選用肝臟細胞生長因子(HGF; Hepatocyte Growth Factor)探討其對RPE的作用。我們的初步的研究發現,HGF具有促進RPE的吞噬功能。為進一步目前研究HGF對RPE的機能及作用機轉作更深入的探討,目前已完成以基因工程生產recombinant HGF。
石斛是目前民間最名貴的眼科用藥,我們發現石斛可能是經由促進RPE產生微量的NO而增強其吞吃機能,証實了石斛之藥效,為此傳統眼科中藥的藥效提供合理的理論基礎。我們進一步發現,石斛可以選擇性調節在眼底疾病扮演重要角色的基因,如bFGF、VEGF、TGF-β等mRNA的表現。目前石斛約有效成份經超臨界流體萃取,分子篩層析,高速液態層析純化後,正待進行結構分析。
在本實驗中,我們建立以RPE為眼底疾病試管內細胞及分子生物學研究的模型,提供藥物研發基礎。我們相信這些發現將來能有效地應用於眼科疾病的治療,對視力的維持能有重要的貢獻。
To date, no drugs for ocular disease are shown to regulate the function of RPE (retinal pigment epithelial cells) which plays an important role in vision transduction. In present study, the RPE cells were used as an experimental model to develop drugs for the treatment of ocular disease and exploring its mechanism of action.
The predominant function of RPE is the phagocytosis of rods and cones outer segments. Evidence is now emerging that NO (Nitric oxide) is a mediator of physiological and pathological processes in the retina. However, the role of NO in the normal phagocytic activity of RPE is unknown. In PVR (proliferative vitreoretinopathy), PDR (proliferative diabetic retinopathy) and AMD (aged-macular degeneration), the expression of bFGF (basic fibroblast growth factor), VEGF (vascular endothelial growth factor), IGF (insulin-like growth factor) and TGF-β (transforming growth factor-beta) are increased. Accordingly, we established the bioassay system of phagocytosis with FITC-ROS as an indicator in bovine RPE and evaluated its correlation to the production of NO and the expression of growth factors which concerning the PVR and PDR.
We are the first group to find that FCS stimulated the slight production of NO, and enhanced the phagocytic activity of RPE. This effect is inhibited by NOS inhibitor. We also found that the consumption of nitric oxide correlates the phagocytosis in RPE. The benefit of slightly enhanced production of nitric oxide may offer a new approach for drug development in most of the ocular diseases.
We also found that HGF (Hepatocyte growth factor) enhanced the phagocytic activity of RPE. To obtain the recombinant HGF, we have cloned the cDNA by polymerase chain reaction from MRC-5 cells. The recombinant HGF was expressed in insect cells by baculovirus expression system, and detected by Western analysis.
Dendrobii Caulis is a famous Chinese herbal medicine in maintenance of eyesight. We found that Dendrobii Caulis enhanced phagocytic activity and the production of NO in RPE slightly. Besides, we also found that Dendrobii Caulis down-regulated the expression of bFGF, VEGF and TGF-β selectively. The purification and identification of the active fractions from Dendrobii Caulis were under SFE, Sephadex-LH20 and HPLC chromatography. The structure of the active components is still remains to be determinated.
In this studies, we established the in vitro bioassay models for developing the drug which might regulate the functions of RPE. We believed that these results might be useful in improving the therapy for ocular diseases.
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