臺灣博碩士論文加值系統

霍山石斛為名貴的傳統眼科中草藥，本實驗室研究霍山石斛的作用機轉，發現水萃取物可促進視網膜色素上皮(RPE)細胞一氧化氮(NO)的生成及肝生長因子(HGF)的基因表現，且這與RPE吞吃作用相關。此外，也發現粗萃取物可抑制RPE細胞bFGF、VEGF、TGF-β基因的表現。糖尿病視網膜病變為眼底異常而產生血管新生的病變。首先我們以霍山石斛的眼科應用為出發點，探討其對眼底病變的療效。
已知蛋白質醣化與糖尿病併發症密切相關。在實驗管內以醣化的蛋白作為單一的致病因子，發現在1g/ml的醣化白蛋白(AGE-BSA)處理下即會使RPE細胞VEGF與bFGF的基因表現量增加，此二因子與視網膜血管病變相關，也發現處理醣化白蛋白亦會使視網膜微血管內皮細胞增生。實驗中以FITC標定AGE-BSA處理RPE細胞時發現，培養的RPE細胞會與AGE-BSA明顯結合。對取自新鮮牛眼之RPE細胞相較於視網膜其他部位的細胞具有較強的蛋白質水解活性，能有清除醣化白蛋白的作用，而霍山石斛甲醇粗萃物能提高RPE 細胞對醣化蛋白質降解活性。
我們建立一個由醣化白蛋白引起的全身性的糖尿病併發症模型，來觀察全身及視網膜的病變。首先自小鼠血清中大量純化白蛋白，並在試管中進行60天的醣化作用後，再以靜脈注射方式給予不同劑量醣化的白蛋白，在持續給予醣化白蛋白的六週觀察發現，在血糖及尿糖的量並未發生太大變動。進行組織病理的觀察發現肝臟在靠中央靜脈部位的肝細胞有壞死情形，並且觀察到血清肝生長因子量會隨之上升。尿液中的蛋白質成分有些微變化，但並不顯著。在腎臟絲球體中會有物質堆積形式，推測此模型亦對腎臟功能會有所影響。
石斛在古中醫書中屬上品，亦有調節生體機能的記載。我們與新藥研究中心合作，對霍山石斛的水萃層部分進行多糖成分的萃取，於動物體的實驗中觀察到餵食石斛多糖三天的小鼠會增強自然殺手細胞毒殺活性，同時也能增強骨髓細胞受L929培養液共同刺激的增生作用。在抗體免疫上，發現口服石斛多糖對口服以ovalbumin或Pneumovax 23多糖疫苗為抗原，在血清中的IgM、IgG量無明顯變化，而對黏膜免疫具增強效果，能增加在腸道中的IgA抗體量。
本研究中發現，醣化白蛋白會使RPE細胞表現血管新生因子，而RPE具結合與降解AGE-BSA活性，霍山石斛對此有促進效果。我們也建立醣化白蛋白誘發糖尿病併發症動物研究模型，可進一步提供藥物評估的平台。我們也發現口服餵食霍山石斛多糖的免疫調節功能。相信這些結果能對霍山石斛的效用有更進一步的佐證。

Dendrobii Caulis is a famous Chinese herbal medicine for traditional ophthalmological use. Previous study in our laboratory showed that water extract of D. Caulis could enhance phagocytic activity of PRE cell by a mechanism involving nitric oxide (NO) production and HGF gene expression. Further study showed that water extract inhibited the gene expression of bFGF, VEGF and TGF-β. Diabetic retinopathy is a complication had abnormality on retinal cells, then it often caused neovascularization. The first part of my study focused on the effect of D. Caulis about amelioration of retinal complication.
Glycation of physical proteins were found highly related to diabetic complications. We studied the effect of protein after glycation in vitro. It increased gene expression of VEGF and bFGF when RPE cell treated 1g/ml AGE-BSA for 6 hours. It also could cause proliferation of retina capillary endothelial cell. We found RPE cells could bind FITC labeled AGE-BSA specifically. RPE cells isolated from fresh bovine eye had higher proteolytic activity than cells isolated from neuronal retina. Proteoliyic activity was enhanced when RPE cells treated with methanol extraction of D. Caulis.
For studying the diabetic complications of whole body or eye, we tried to estiblish an animal model of diabetic complications caused by AGE modified serum albumin. We purified large amount of albumin from mice serum by cold ethanol precipitation. Mice serum albumin glycated in vitro via incubated with glucose for 60 days. Mice were administrated different amount glycated albumin by tail vein injection. In the period of experiment, the concentration of glucose in serum and urine were maintained in normal level. Hepatocytes were damaged at the site of near the central vein. The amount of serum HGF increased during experiments. Proteins in urine had slight change. We also found that some molecules were accumulated at glomerule, and thought it might also affect the function of kidney.
Dendrobii Caulis was classified to the top grade of Chinese herb and also described as body’s regulatory herb on ancient medicine books. Collaborating with the Research Center for Drug Discovery, we extracted and separated polysaccharides from the water extraction of D. Caulis. The cytotoxic effect of nature killer cell was enhanced after mice orally administrated D. Caulis polysaccharides for 3 days. It also enhanced proliferation of bone marrow cells costimulating with L929 condition medium. Mice challenged with ovalbumin or Pneumovax 23 vaccine as protein or polysaccharide antigen by gastric intubation accompanies with long term taken D. Caulis polysaccharides was performed. It has no obvious effect on serum IgG and IgM, but it enhanced mucosal immunity. Ovalbumin specific IgA antibody elevated in intestine.
In our study, RPE cell expression the genes of neovascularization factors caused by AGE-BSA. RPE cell has the activity of binding and degradation AGE-BSA. We build an animal model on studying complications caused by AGE modified albumin. This model could serve a platform for evaluating the AGE-induced complication eliminating drugs. We also proved the immunomodulating activity via oral administration of D. Caulis polysaccharides. We do think these results could provide some evidence on the therapeutic effects of D. Caulis.

縮寫表 ------------------------------------------------------------------- IV
中文摘要 ---------------------------------------------------------------- 1
英文摘要 ---------------------------------------------------------------- 3
緒論 ---------------------------------------------------------------------- 5
試藥及器材 ------------------------------------------------------------- 17
實驗方法 ---------------------------------------------------------------- 24
結果 ---------------------------------------------------------------------- 41
討論 ---------------------------------------------------------------------- 48
參考文獻 ---------------------------------------------------------------- 57
圖表說明 ---------------------------------------------------------------- 68
附圖------------------------------------------------------------------------ 99

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