1.利用短暫性殖入系統探討carbonic anhydrase與肝癌的關係 2. silibinin對GST isoforms表現之影響

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Title:	1.利用短暫性殖入系統探討carbonic anhydrase與肝癌的關係 2. silibinin對GST isoforms表現之影響 1.Study of the relationships between carbonic anhydrase and liver cancerous cells, the use of a transient expression system. 2.Effect of silibinin on the expression of GST isoforms
Authors:	許瑞玲 Jui-Ling Hsu
Contributors:	中山醫學大學：生物化學研究所;周芬碧
Keywords:	carbonic anhydrase;肝癌;GST isoforms;silibinin carbonic anhydrase;liver cancerous cells;GST isoforms;silibinin
Date:	2001
Issue Date:	2010-04-22T03:40:58Z (UTC)
Abstract:	1.CA是一種含鋅的金屬酵素，其分子量約為30kDa左右，廣泛分佈於動植物中，是屬於六大酉每裡面的Lyase。在二氧化碳的輸送或分泌過程扮演重要的角色。在之前的研究，發現接種肉瘤細胞Sarcoma 180的老鼠，肝臟組織之細胞質carbonic anhydrase III之表現，在六天後逐漸減少。此外也有報告指出在直腸大腸癌的病人，他們的CA I,II的表現是減少的。是什麼原因導致此現象並不清楚？所以本實驗乃使用短暫性殖入系統以探討carbonic anhydrase 與肝癌的關係，期望能找出一個解答。實驗利用RT-PCR放大CA的基因序列，再接至GFP載體以得到CA的cloning，之後短暫性殖入肝癌細胞( Hep 3B )。結果發現CA的曲線經校正後有下降的趨勢；且在螢光顯微鏡下，殖入GFP-HCA II的細胞形狀與殖入GFP (控制組)的細胞形狀有些不同。隨時間增加，殖入GFP-HCA II細胞形狀呈現圓形的增多，且綠色螢光亮度增強，此圓亮的細胞最後會死亡。作TUNEL實驗，發現轉殖GFP-HCA II細胞，呈現螢光反應，表示細胞可能有apoptosis的現象。結果顯示過度表現CA II可能對細胞造成傷害，必須作更多實驗來確認細胞的死亡是apoptosis或necrosis。 2.Glutathione S-transferase（GST）體內的解毒酵素在化學預防中扮演了重要的角色。由於當某些癌症形成時，GST pi form有明顯的大量表現，而治療癌症時GST alpha form又與藥物產生抗藥性有關，所以使用天然藥物能降低(抑制)GST pi及GST alpha form的表現，對於治療癌症是有益的，可使得抗癌藥的效果增強而達到治療癌症的目的。在本實驗中是利用肝癌細胞(Hep G2、3B cell)，以Western blotting及RT-PCR的方法探討silibinin，EGCG，PCA，geniposide等天然藥物對於Glutathione-S Transferase 基因表現的影響，結果G2及3B細胞以EGCG和silibinin處理後，發現GST alpha mRNA的量都是下降的，且GST mu mRNA的量是上升的(3B細胞)。另外以silibinin處理，GST alpha mRNA的量呈現dose-dependent，time-dependent。所以初步認為silibinin在預防及治療癌症上是不錯的天然藥物。進一步還須合併抗癌藥，證明silibinin的確可以幫助癌症的治療。 Carbonic anhydrases are zinc-containing metalloenzymes with molecular weights about 30 kDa that are widely distributed in animals and plants. CAs belong to lyases. CAs play an important role in transportation or secretion of carbon dioxide. In previously study, we found that mouse inoculated with sarcoma 180 cells showed a gradual decrease in the expression of CA III in the liver 6 days after the inoculation. Other reports also demonstrated that the expressions of CA I and II were reduced in colorectal tumors. In order to understand the role of CAs in cancer development, a transient expression system was used to explore the effect of over-expressed CA II on liver cancerous cells. 1.Hepatoblastoma cells (Hep 3B) were transfected with cDNA of CA II that was amplified by RT-PCR and cloned into pGFP vector. The results, after normalized with transfection efficiency, showed that the number of cells transfected with CA II gradually decreased as the culture time increased. Furthermore, the cells transfected with CA II were morphologically different from that of control cells. The number of cells that were round in shape and bright in green fluorescence increased along with the increasing culture time. These cells were not viable. The results of TUNEL assay found that the cells transfected with GFP-HCA II presented fluorescence indicating that these cells may be apoptotic cells. These results indicated that over-expression of CA II might be toxic to cells. Further study will be needed to clarify the cause of their death, apoptosis or necrosis. 2.Glutathione S —transferases are detoxifying enzymes and play important roles in chemoprevention. However, the expression of GST pi subunits were linked to the development of several cancers, and the increases in GST alpha subunits were related to the drug-resistance of cancers. Therefore, a compound that could decrease the expression of GST pi or alpha would be beneficial in treating cancers, such as enhancing the efficiency of anticancer drugs by decreasing drug-resistance. In this study, we tested the effect of several natural compounds, including silibinin, EGCG, PCA and geniposide on the gene expressions of GST subunits. Using Western blotting and RT-PCR analyses, we found that the treatment of EGCG and silibinin on hepatoblastoma cells (Hep G2 and 3B) reduced the mRNA levels of GST alpha in both cell lines, and increased the transcripts of GST mu in 3B cells. The reduction of GST alpha mRNA level by silibinin was dose- and time-dependent. These results suggested that silibinin would be advantageous in cancer treatment. Further study using combination of silibinin and anticancer drug will be needed to demonstrate the actual effect of the compound in helping cancer treatment.
URI:	http://140.128.138.153:8080/handle/310902500/1204
Appears in Collections:	[The Institute of Biochemistry, Microbiology and Immunology ] Electronic Theses of Dissertation

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