| Abstract: | 第一部分:
葡萄糖-六-磷酸鹽去氫缺乏症(Glucose-6-phosphate dehydrogenase; G6PD)是一種很常見的性連(X-link)遺傳,對偶基因的突變將會導致G6PD的活性降低甚至缺乏，當遭遇感染或使用例如抗瘧疾藥物如primaquine；quinine或磺胺類藥物都容易增加氧化物生成，皆可能使紅血球細胞膜破壞而造成溶血性傷害，已有文獻指出碳酸酐異構 (carbonic anhydrase isoenzymes; CA)在許多的貧血中的表現上升，因此在本實驗中將進行1. CA及麩胱甘轉移(glutathione S-transferase; GST)在葡萄糖-六-磷酸鹽去氫缺乏病人紅血球之量與活性的探討。2. GST在G6PD缺乏病人中所扮演的角色。本研究結果顯示在G6PD缺乏病人與正常個體比較CA activity分別為27.2±2.1 U/gHb及22.9±1.69 U/gHb，有明顯上升，具有統計學上的差異(P<0.01)，以iimmunoblot 技術分析顯示CAII的蛋白表現亦有上升的現象(P<0.05)，而CAI及CAIII的蛋白表現則是明顯下降，具有極顯著的差異(P<0.001，P<0.001)。在GST的分析過程中本研究使用去除血紅素的血液樣本進行GST活性分析，數據顯示Total GST及 alpha-GST活性上升，在統計學上有顯著的差異(P<0.001，P<0.01)，而Mu-GST及Pi-GST則無差異存在(P>0.05)。綜合上述的結果，G6PD缺乏病人因氧化性傷害產生變性血紅素之病理現象產生時，紅血球中量僅次於血紅素的CAI及膜上的band 3會因紅血球破壞導致流失，同一家族的CAII在生理功能上會形成代償作用，為了調節細胞內的酸鹼平衡及氣體的轉送，CAII蛋白合成也跟著上升，因此認為CAI可作為溶血性貧血的指標。CAIII在G6PD缺乏病人的紅血球中明顯下降，已知CAIII參與蛋白質的硫化作用，而硫化作用是細胞抗氧化機轉的初期反應。G6PD缺乏病人在抗氧化機轉的初期CAIII形成蛋白質的硫化作用就已減少，後期更因G6PD缺乏無法產生足夠的NADPH，使外來氧化物氧化GSSH無法還原成GSH，因此認為G6PD缺乏病人若是伴隨著CAIII下降將會使溶血症狀更嚴重。以上的數據支持我們認為CAIII在紅血球中可作為抗氧化的蛋白的推論。G6PD缺乏病人體內存在著氧化傷害，先前已有文獻指出氧化傷害產生會誘發GST基因表現，與在本研究中觀察到GST活性上升的現象符合。
第二部分:
本研究主要探討在台灣發生的非小型細胞肺癌組織中碳酸酐異構及金屬依賴型基質分解中各種蛋白的表現，再深入進行CAs及MMPs的相關性分析。本研究以CA activity，immunoblotting，immunohistochemistry，gelatin zymography，casein zymography，reverse transcription polymerase chain reaction (RT-PCR)等技術進行分析，結果顯示肺癌組織與其周圍正常組織比較CA activity及CAII蛋白的表現有明顯下降，具有顯著的差異(p<0.01，p<0.001)，而CAII mRNA的表現下降亦有差異(p<0.05)，因此推論CAI及CAII在細胞內的不正常表現，可能使二氧化碳無法完成水解而產生滯留，導致胞內的pH值偏向酸性，而促進癌細胞生長。MMP-9及MMP-2在基底膜的破壞扮演著重要角色，與癌症的侵襲及轉移最有關，實驗結果顯示肺癌組織與其周圍正常組織比較可觀察proMMP-2； proMMP-9及active MMP-2 (P<0.01)，但祇有15.8%的肺癌組織中同時具有活化態的MMP-9，因此推論肺癌組織中缺乏活化態的MMP-9。u-PA之分析在統計學上有顯著的差異(P<0.001)，因u-PA可活化MMPs，屬於MMPs活性層面的調節，在此認為proMMP-2確實透過u-PA活化為active MMP-2。在相關性分析實驗中，CAII蛋白表現及CAII mRNA與MMPs system的蛋白包括active/latent MMP-2及u-PA皆有不同程度的相關，推論CAII的表現下降確實可促使癌組織細胞內pH值偏向酸性而營造一個適合MMP-2的活化的環境，幫助癌細胞的生長、侵襲及轉移。CAII與MMP-2的關係清楚釐清之後，使用CA inhibitor等藥物抑制癌細胞的生長、侵襲及轉移，我們認為將對癌症的治療方向將會是一大貢獻。
Part I:
Glucose-6-phosphate dehydrase (G6PD) deficiency, an X-linked abnormality, is caused by one of a multitude of allelic G6PD variants with reduced enzyme activity. This defect is usually well tolerated, except for acute haemolytic crises associated with an oxidative stress, such as drugs, fava beans ingestion, and infection. The concentration of carbonic anhydrase (CA) has been demonstrated to be elevated in several types of anemia. This study was designed to evaluate the alternations in quantities and activities of cytosolic CA isoenzymes and glutathione S-transferase (GST) isoenzymes in the erythrocytes of G6PD deficiency individuals and to investigate the altered expression of erythrocyte GST, representing the ability for removing circulating xenobiotics in G6PD deficiency. The total CA activity from human erythrocytes of 30 controls and 30 G6PD deficiency individuals, was 22.9±1.69 U/g Hb and 27.2±2.1 U/g Hb, respectively showing that the total CA activity was significantly higher in G6PD deficiency individuals (P<0.01). The quantitative analyses by western blotting showed that CAI/CAII ratio of G6PD deficiency individuals (1.28±0.06) was significantly lower than that of controls (3.79±0.18) (P<0.001). This alteration could be obviously the consequence of the significant decrease of CAI and increase of CAII. Furthermore, the concentration of CAIII of G6PD deficient individuals was significantly lower than that of the normal subjects(P<0.001) and there were significant correlations between the concentrations of CAI, CAII, CAIII and the activity level of G6PD. Erythrocyte total GST and alpha-GST activities were significantly elevated in anemia patients (P<0.001 and P<0.01, respectively). Further analysis showed that mu-GST and Pi-GST activity was not significantly altered in G6PD deficient individuals as compared to healthy controls. Furthermore, we have improved the conventional GST assay by incorporating a chloroform treatment to remove the interference of hemoglobin. Different CA isoenzymes and GST isoenzymes may serve different roles in G6PD-deficient erythrocytes while CAI could be an indicator for hemolytic anemia. CAII is able to compensate for the functions of CAI and CAIII can provide the G6PD-deficient individuals with some extent of protection against oxidative damage. Erythrocyte total GST and alpha-GST activities were significantly elevated in G6PD-deficient patients and our GST assay may be feasible in the evaluation for the levels of circulating electrophiles/oxidant, as well as for other enzymes in erythrocytes.
Part II:
Lung cancer, including non-small cell lung cancer (NSCLC) and small cell lung cancer, is the leading cause of cancer death worldwide, with a extremely poor survival rate. Therefore there is an intense and urgent need for a better understanding of the molecular and cellular processes involved in this aggressive disease. The matrix metalloproteinases (MMPs), a large family of extracellular matrix degrading enzymes, are believed to be crucially involved in tumor invasion and metastasis. This study was designed to elucidate the possible relationships between the expression levels of cytosolic carbonic anhydrase (CA), MMP-2, -9, and u-PA, and NSCLC. Activity and expression level of CA, MMP-2, MMP-9 and u-PA of 120 NSCLC patients were analyzed by various techniques including CA activity analysis, immunoblotting, RT-PCR, immunohistochemical staining and zymography. The results showed that the activity, expression level and mRNA of CA were consistently and significantly decreased in NSCLC patients. A reciprocal correlation between expression of CA and MMP system related proteins (include MMP-2 and u-PA) was identified in the linear regression analysis, as demonstrated in the Regression Wizard analysis. High levels of MMPs and concomitant and significant reductions of CAII were identified in all patients. From our study, it was suggested that the reduction of CAII and high levels of MMPs in NSCLC patients may promote tumor cell motility and contribute to tumor growth and metastasis. |
