【作者】 宋芸娟；

【导师】 义祥辉； 徐庆；

【作者基本信息】 广西师范大学 ， 应用化学， 2005， 硕士

【摘要】 乙型肝炎是由乙型肝炎病毒(HBV)引起的传染性疾病,我国属乙肝高发区,乙型肝炎传染途径复杂,发病率高,病程长,易反复,特别是它与肝硬化和肝癌密切相关,危害性极大,因此寻找安全有效的抗HBV 药物已成为当今医药学界一项迫切任务。为了更准确、更方便的寻找低毒高效的抗HBV 药物, 我们建立了体外、体内抗HBV 药物筛选体系,并进行中草药抗HBV 的研究,本课题的研究内容如下: 1. 对荔枝核、紫玉盘、蚕蛾、田基黄、叶下珠、大黄等药物进行提取,并运用HepG2.2.15细胞株进行抗乙肝病毒的体外研究,实验结果表明:广西产的荔枝核对乙肝病毒有较好的抑制作用。2. 建立HepG2.2.15 细胞株培养体系,同时使用SRB(suforhodamine B 磺基罗丹明B)细胞染色技术测定药物对细胞的毒性、并采用PCR 方法(聚合酶链反应法)结合荧光探针的体外扩增和检测技术测定HBV-DNA 的含量。3. 运用PCR 技术筛选出先天感染鸭乙肝病毒(DHBV)的桂林地区麻鸭,以先天感染DHBV的桂林麻鸭作为实验动物模型对体外筛选成功的抗HBV 药物进行体内实验。4. 采用光密度测定法和和定量PCR 技术研究荔枝核提取物对HepG2.2.15 细胞株HBsAg 与HBeAg 表达的抑制作用与HBV-DNA 含量的影响。96 孔板试验:实验第3、6 天,荔枝核提取物(800,400,200,100 μg/mL)对HepG2.2.15 细胞HBsAg 与HBeAg 的表达有明显的抑制作用(与对照组比较P<0.01)。24 孔板试验:实验第3、6、9 天荔枝核提取物(400,200,100,50 μg/mL)对HepG2.2.15 细胞HBsAg 与HBeAg 的表达有明显的抑制作用(与对照组比较P<0.01);浓度为400 μg/mL 时,于实验第6、9 天使HepG2.2.15 细胞培养液中的HBV-DNA 转阴,说明荔枝核提取物在体外有较强的抗乙肝病毒的作用。5. 对荔枝核进行工业化提取分离,并对所得的荔枝核提取物再次进行体内实验和体外实验。6. 研究荔枝核工业提取物对HepG2.2.15 细胞株HBsAg 与HBeAg 的表达的抑制作用,其结果表明荔枝核工业提取物(800,400,200,100 μg/mL)对HepG2.2.15 细胞HBsAg 与更多还原

【Abstract】 Hepatitis B is one kind of infective disease which caused by HBV and difficult to be cured. The morbidity rate is very high in china. Hepatitis B could lead to cirrhosis and liver cancer. The disease is harmful to human beings. It is an urgent task for us to look for both safe and efficacious anti-HBV drugs. The high efficiency anti-HBV drugs-screening system both in vitro and in vivo have been established and applied. The contents of this article are as follows: 1. Nine kinds of nature durgs were extracted and tested by HepG2.2.15 cells the anti-HBV drugs screening system in vitro. The results show that extraction from the Semen Litchi has strong function in anti-HBV in vitro. 2. Cytotoxicity was determined by SRB(suforhodamine B Protein dye-binding)assay and the content of HBV-DNA was determined by polymerase chain reaction(PCR)method in the anti-HBV drugs screening system in vitro. 3. Throught PCR method, the Guilin brown spot ducklings congenitally infected by duck hepatitis B virus(DHBV)were Screened out, then the effect of durgs on DHBV in vivo was studied. 4. Optical density mensuration, cell culture and FQ-PCR technique were used to study the inhibitory effects of the extraction from the Semen Litchi on the expression of HBsAg, HBeAg and the content of HBV-DNA in HepG2.2.15 cells. In 96 holes plate test, i(t800,400,200,100 μg/mL)had evident action to inhibition on expression of HBsAg and HBeAg in HepG2.2.15 cells on the thirth and sixth experiment day,compared with contral group(P<0.01). In 24 holes plate test, it(400, 200, 100, 50 μg/mL)had evident action to inhibitiong on expression of HBsAg and HBeAg in HepG2.2.15 cells on the thirth, sixth and ninth experment day,compared with contral group(P<0.01). FQ-PCR showed that the extraction(400 μg/mL)turned HBV-DNA in HepG2.2.15 cells medium negative.The exraction from the core of Semen Litchi has strong function in anti-HBV in vitro. 5. The anti-HBV effect of extraction from the core of Semen Litchi. By industrial produced way was also investigated both in vitro and in vivo examination. 6. Inhibitory effects on expression of HBsAg, HBeAg in HepG2.2.15 cells with the industrial extraction from the core of Semen Litchi were stdudied. The extraction(800,400,200,100 μg/mL)had evident action to inhibition on expression of HBsAg and HBeAg in HepG2.2.15 cells on the thirth and sixth experiment day,compared with contral group(P<0.01).Inhibition rate to HBsAg of industrial extraction(400 μg/mL)was 73.00 % and 94.00 % in 3 day and in 6 day. Inhbition rate to HBeAg was 49.00 % and 62.00 % in 3 day and in 6 day. The result showed that the industrial extraction from the core of Semen Litchi has also obvious function to anti-HBV in vitro. 7. Guilin brown spot ducklings congenitally infected with DHBV was used as animal modal to study the anti-HBV action of industrial extraction from the core of Semen Litchi. After medicated with the industrial extraction(2 g/kg/day)5 day, 10 day ang 15 day. DHBV-DNA in ducks sera were detected with Optic density valus(OD value)DHBV-DNA in ducks sera of the industrial extraction treatment group was obviously lower than before treatment. There was significant difference(compared with virus control group P<0.05, compared with before treatment P<0.01), The inhibition rate was 62.5 % in after medicated with the industrial extraction 5 day, The result showed the industrial extraction(2 g/kg/day)can inhibit the replication of DHBV-DNA in GuiLin brown spot ducklings. GuiLin brown spot ducklings of DHBV-modal were killed to observe pathological in liver tissue. The resuts showed that industrial extraction(2 g/kg/day)has obvious action to protest livers(compared with control group P<0.05).更多还原