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研究生:盧曉鈴
研究生(外文):Hsiao-Ling Lu
論文名稱:中草藥對肝炎之研究探討
論文名稱(外文):The effect of Chinese herbal on hepatitis with in vivo and in vitro model
指導教授:黃惠宇黃惠宇引用關係
指導教授(外文):Hui-Yu Huang, Ph.D.
學位類別:碩士
校院名稱:實踐大學
系所名稱:食品營養研究所
學門:醫藥衛生學門
學類:營養學類
論文種類:學術論文
論文出版年:2005
畢業學年度:93
語文別:中文
論文頁數:92
中文關鍵詞:慢性B型肝炎慢性C型肝炎樟芝冬蟲夏草干安能干擾素敗醬草免疫反應
外文關鍵詞:Chronic Hepatitis B、Chronic Hepatitis C、Antrodia camphorata、Cordyceps camphorate、Lamivudine、Interferon、Herba Patrini
相關次數:
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  • 收藏至我的研究室書目清單書目收藏:4
根據衛生署公佈之國人十大癌症死因疾病,顯見肝病已成為我國之國病,尤其以B肝及C肝為威脅國人健康之首,因此,提出新型治療方式,有效降低血清病毒量、使肝功能恢復正常並改善患者免疫功能,將會是治療肝病之重大突破。
本研究主要分為三個部分,第一部份是探討樟芝併用干安能是否具有加乘慢性B型肝炎的治療效果,實驗對象,來自台中沙鹿光田綜合醫院B型肝炎門診病患,並分為兩組:A組給予干安能併用樟芝治療,B組單獨給予干安能治療,分別於治療3個月及6個月時,分析患者之肝功能(GPT)、病毒量(HBV DNA)及免疫功能之變化。實驗結果顯示,A組及B組治療6個月時,其GPT值都大幅下降至正常,其值分別為25.91U/L及24.07U/L。另外,治療6個月時,A組有50 %的患者測不到HBV DNA的量,41.6 %的患者產生e抗原轉陰(指e抗原消失且產生e抗體);而單獨服用干安能的B組,有14.28 %的患者測不到HBV DNA的量,12.5 %的患者產生e抗原轉陰。在免疫功能方面,合併樟芝治療的A組,治療3個月及6個月時,其毒殺型T細胞(CD8)及B細胞與控制組相比,分別上升了19.05 %、14.11 %及13.04 %、11.85 %,而且具有統計上意義。此外,治療6個月時,活化型T細胞(HLA-DR)與治療3個月時相比,減少了約28.50 %,並具統計上的明顯意義。結果顯示合併樟芝治療的A組,比單獨使用一般性治療的B組,出現有意義且明顯的改善,由此可推測得知,併用樟芝治療將會藉由提升B細胞及毒殺型T細胞之免疫反應,進而使B型肝炎患者減少HBV DNA病毒量,e抗原轉陰率的比例亦提升。
第二部分是探討干擾素/ Ribavirin併用冬蟲夏草是否具有加乘C型肝炎的治療效果,實驗對象,來自台中沙鹿光田綜合醫院C型肝炎門診病患,並分為兩組:A組給予短效型干擾素合併抗病毒藥及冬蟲夏草治療,B組給予長效型干擾素合併抗病毒藥治療,分析治療3個月及6個月時,患者之肝功能(GPT)、病毒量(HCV RNA)及免疫功能之變化。實驗結果顯示,A組及B組治療6個月後,其GPT值都大幅下降至正常,其值分別為23.35 U/L及22.32 U/L。另外,治療6個月時,A組有92.8 %的患者測不到HCV RNA的量;而給予長效型干擾素α-2b/ribavirin的B組,有25 %的患者測不到HCV RNA的量。在免疫功能方面,合併冬蟲夏草治療的A組,治療3個月及6個月時,其T細胞及輔助型T細胞(CD4)與控制組相比,分別上升了2.29 %、2.73 %及7.64 %、8.73 %,具有統計意義。此外,毒殺型T細胞(CD8)及活化型T細胞(HLA-DR),治療3個月時與控制組相比,分別下降了5.85 %及33.03 %,具有統計意義。實驗結果顯示,合併冬蟲夏草治療的A組比單獨使用一般性治療的B組,出現有意義且明顯的改善,由此可推測得知,併用冬蟲夏草治療可能會藉由提升輔助型T細胞的功能,進而使C型肝炎患者減少HCV RNA病毒量。
第三部分是評估敗醬草抗病毒之能力,利用中國草藥-敗醬草(Herba Patriniae)處理可持續分泌出高量的B型肝炎表面抗原、e抗原及病毒顆粒體之Hep-ES2肝癌細胞株,實驗分別以酵素連結免疫吸附法(ELISA)、DPPH測定法及MTT分析法,測定B型肝炎表面抗原(HBsAg)、e抗原 (HBeAg)、清除自由基能力及毒殺能力,藉以評估敗醬草抗病毒之能力。實驗結果顯示,給予Hep-ES2肝癌細胞株甲醇萃及水萃敗醬草處理48小時,在無細胞毒害的劑量(noncytotoxic dose) 250 μg/mL下,敗醬草甲醇萃取液與敗醬草水萃取液,對B型肝炎表面抗原及e核心抗原之抑制率相對於控制組(0 %)分別為24 %、17 %及2 %、12 %。而在清除DPPH自由基的能力方面,亦發現敗醬草甲醇萃取液大於敗醬草水萃取液,且隨著濃度的上升,其清除DPPH自由基的能力也會跟著上升。由以上結果可知,敗醬草具有對抗B型肝炎病毒能力,而且以敗醬草甲醇萃取液優於水萃取液,而敗醬草抗病毒能力與其清除DPPH自由基的能力並不是成正比的。
According to the Department of Health (DOH) proclaimed that ten-major cancer disease in R.O.C. Obviously, that liver disease has became an important health issue, especially menaced in health with Hepatitis B and C is primacy. Hence to bring up a new mode in liver disease therapy about effective in reduces serum HBV DNA level, and liver function.
This dissertation is divided into three parts: the first part is to investigate the effect of combination therapy of lamovudine and Antrodia camphorate in chronic hepatitis B patients. The subjects of experiments are outpatients of Chronic Hepatitis B from Kuang Tien General Hospital in Shalu Town, Taichung. The subject was separated into two groups: group A used lamivudine and Antrodia camphorate treatment for six months, and group B used lamivudine monotherapy for six months. The serum level of GPT, HBV DNA, and immune function were determined by peripheral blood sample at three and six months, respectively. The results indicate that both group A and B decreased GPT value to normal after six months treatment, and its value was 25.91U/L and 24.07U/L, respectively. From the result of HBeAg seroconversione, group A showed the better result than group B, which were 41.6% and 12.5%, respectively. The patients with undetectable HBA DNA value after six months treatment were50% of total Group A patient, 14.28% of total group B patient. The result of flow cytometry showed that the cytoxicity T cell and B cell of have significant increase when we compared the data of group A after three and six months treatment with control group, the value was 19.05%, 14.11%, and 13.04%, 11.85%, respectively. The HLA-DR showed significant decrease after six months treatment, when we compared with after three months treatment. Therefore to conjecture that combination therapy with Antrodia camphorate could regulated immune response by enhancing B cell and CD8, decrease virus quantity and increase the ratio of HBeAg seroconversione in Hepatitis B patients .
The second part of this experiment is to investigate the possibility of an additive effect of Interferon/Ribavirin plus Cordyceps camphorate combination therapy for the chronic hepatitis C patient. The subjects of experiments were outpatients of Chronic Hepatitis C from Kuang Tien General Hospital in Shalu Town, Taichung. The subject was separated into two groups: group A used PEG-Intron/Ribavirin and Cordyceps camphorate combination therapy for six months, and group B used Intron-A/Ribavirin therapy for six months. The level of GPT, HCV RNA in serum, and immune function were determined by peripheral blood sample at three and six months treatment. The results indicated the GTP value of group A and group B decreased to normal after six months treatment, its value was 23.35U/L and 22.32U/L, respectively. The patients with undetectable HCA RNA value after six months treatment were 92.8% of total Group A patient, 25% of total group B patient .The T cell and help T cell number have significant increase, which compared the data of group A after three and six months treatment with control group, the value is 2.29 %、2.73 % and 7.64 %、8.73 % respectively. Also, cytoxicity T cell number showed significant decrease after three months treatment when we compared with control group. In conclusion, the investigate on combination Intron-A/Ribavirin and Cordyceps camphorate therapy could promoted immune response for chronic hepatitis C patients by regulation of T cell activity to decrease quantity of HCV RNA.
The third part was to evaluate anti-Hepatits B virus ability of Herba Patriniae extracts. The Hep-ES2 hepatoblastoma cell line were treated with methanol or liquid
extract from Herba Patriniae in noncytotoxic dose, 250μg/mL, for 48hrs. The result showed that inhibitory rates of HBsAg and HBeAg secretion were 24%, 17% (methanol extract), and 2%, 12% (liquid extract), when we compared with control group (0%). The antioxidant ability of Herba Patriniae was increase following the does increase in does-dependently range (250-1000μg/mL), and methanol extract showed the better result than liquid extract. In conclusion, Herba Patriniae extract possesses anti-HBV activity, and methanol extract shown the better antivirus effect than liquid extract. The anti-Hepatitis B virus ability of Herba Patriniae extract in not positively correlate with free radical scavenging ability.
中文摘要 i
英文摘要 iii
誌謝 vi
目錄 vii
圖目錄 xi
表目錄 xii

壹. 緒論
一. 文獻回顧
(一). B型肝炎
1.盛行率及成因 1
2. B型肝炎病毒結構 1
3. B型肝炎自然病史 3
4. B型肝炎病毒複製生活史 4
5. B型肝炎目前治療方法 4
6.中草藥對B型肝炎的治療 8
(二). C型肝炎
1.盛行率及成因 11
2. C型肝炎病毒結構 12
3. C型肝炎自然病史 14
4. C型肝炎目前治療方法 14
5.中草藥對C型肝炎的治療方法 15
(三). 樟芝
1.樟芝的產地 16
2.樟芝的型態 17
3.樟芝的歷史 18
4.樟芝之成分分析
4.1 一般成分分析 18
4.2 樟芝之有效成分 19
5.樟芝之藥理作用 20
(四). 冬蟲夏草
1.冬蟲夏草的產地 24
2.冬蟲夏草的型態 24
3.冬蟲夏草的歷史 25
4.冬蟲夏草的化學成分 25
5.冬蟲夏草之藥理作用 26
(五). 敗醬草
1.敗醬草的產地 28
2.敗醬草的型態 29
3.敗醬草的化學成分 30
4.敗醬草之藥理作用 30
二. 本實驗之研究動機
(一). 中草藥對慢性肝炎之免疫調節 32
(二). 敗醬草對抗B型肝炎病毒之能力 33
(三). 研究架構 34

貳. 材料與方法
一. 實驗材料
(一). 實驗對象 36
(二). 細胞株 36
(三). 樣品製備 36
(四). 藥品及試劑
1.人體試驗部分 37
2.細胞實驗部分 38
二. 實驗方法
(一). 人體實驗部分
1.實驗設計 38
2. GPT(ALT)、GOT(AST)的測定 39
3. HBsAg, HBeAg, Anti-HBsAg, Anti-HBeAg測定 39
4. HBV DNA、HCV RNA測定
4.1病毒DNA及RNA的製備 40
4.2 HCV RNA定量測定 40
4.3 HBV DNA定量測定 41
5.患者血液中淋巴細胞數之分析 41
(二). 細胞實驗部分
1.樣品配製 41
2.細胞培養 42
3.肝細胞再生試驗(MTT) 42
4.抗氧化能力試驗(DPPH) 42
5.抗HBV病毒試驗(HBsAg、HBeAg) 43
三. 方法原理介紹
(一). 流式細胞儀(flow cytometry) 44
(二). GOT (AST) assay 44
(三). GPT (ALT) assay 45
(四). MTT assay……………… 45
(五). 補捉DPPH自由基能力測試 46

参. 結果
一. 人體實驗部分
(一). 健康受試者與B及C型肝炎患者未治療前之免疫功能 47
(二). 樟芝和B型肝炎患者
1. B型肝炎患者治療前之基本資料 47
2.健康受試者其免疫功能之範圍 47
3.干安能併用樟芝及單獨給予干安能之B型肝炎患者淋巴細胞群數量
之影響 47
4.治療6個月時肝指數的變化 48
5.治療6個月時HBV DNA及HBeAg seroconversion的改變 48
(三). 冬蟲夏草和C型肝炎患者
1. C型肝炎患者治療前之基本資料 49
2.冬蟲夏草併用短效型干擾素/ribavirin及單獨使用長效型干擾素/ribavirin
之C型肝炎患者淋巴細胞群數量之影響 49
3.治療6個月後肝指數的變化 50
4.治療6個月後HCV RNA的變化 50
二. 細胞實驗部分
(一). 敗醬草對ES2肝癌細胞之毒性試驗 51
(二). 敗醬草對ES2肝癌細胞之抗氧化能力評估 52
(三). 敗醬草對ES2肝癌細胞之抗病毒能力評估 52

肆. 討論 53

伍. 結論 61

陸. 參考文獻 85
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