六月青多糖及其皂苷对鸭乙型肝炎的治疗作用及分子作用机制研究

发布时间：2018-06-19 16:40

本文选题：六月青多糖 + 六月青皂苷　；参考：《广西医科大学》2013年硕士论文

【摘要】：目的：观察六月青多糖(LYQP)及其皂苷(LYQS)体外抗乙型肝炎病毒(HBV)及体内抗鸭乙型肝炎病毒(DHBV)的作用。方法：(1)体外抗HBV作用：采用MTT法检测LYQP及LYQS对转染HBV DNA全基因组的HepG2.2.15细胞的半数毒性浓度(TC50)及最大无毒浓度(TCo)；采用直接加药法观察在TCo条件下不同浓度的药物对HepG2.2.15细胞的作用,分别在第72h和第144h收集细胞培养上清液,采用ELISA法检测上清液中HBsAg和HBeAg的滴度,采用荧光定量PCR(FQ-PCR)法检测上清液中HBV DNA的含量。(2)体内抗DHBV作用：用1日龄广西麻鸭感染DHBV,7d后用普通PCR法筛选出DHBV感染强阳性鸭。随机分成8组：LYQP高、中、低剂量组；LYQS高、中、低剂量组；模型组和阿昔洛韦(ACV)阳性对照组。每组10只,每组雏鸭均连续灌胃14d。分别于用药前(To)、用药后7d(T7)、14d(T14)及停药3d(P3)后采血,采用ELISA法检测血清中DHBsAg和DHBeAg的滴度,用FQ-PCR法检测血清中DHBV DNA的含量。同时检测血清中丙氨酸氨基转移酶(ALT)和门冬氨酸氨基转移酶(AST)活性,HE染色观察鸭肝脏组织的病理学变化。结果：(1)体外抗HBV作用：①LYQP及LYQS对HepG2.2.15细胞的毒性较低,TC50分别为111.77mg/mL;90.46mg/L; TC0分别为11.88mg/mL;1.59mg/L。②无毒浓度下不同浓度的含药培养液在HepG2.2.15细胞的培养中可有效的抑制HBsAg及HBeAg的分泌和HBV DNA的合成,抑制作用有明显的时效及量效关系。③TI都大于2,是高效低毒的抗HBV药物。(2)体内抗DHBV作用：给药后,与模型组比较,LYQP及LYQS各剂量组鸭血清中DHBV DNA的含量显著降低(P0.∞或P0.01),停药3d后,中、低剂量组及ACV组血清中DHBV DNA的含量有回升现象,高剂量组DHBV DNA的回升现象不明显。与模型组比较,LYQP及LYQS各剂量组能降低鸭血清中AST及ALT的活性,给药前后血清中DHBsAg及DHBeAg的OD值变化与DNA的含量改变相似；药物的抗病毒效果与剂量大小及时间有一定关系。此外,鸭肝脏组织病理学检查发现LYQP及LYQS对DHBV引起的肝损伤有明显的保护作用。结论：LYQP及LYQS体内外均有显著的抗HBV作用,同时能减轻DHBV所致的鸭肝损伤,改善肝功能。
[Abstract]:Aim: to observe the effects of LYQP) and its saponins (LYQS) on anti-hepatitis B virus (HBV) in vitro and anti-duck hepatitis B virus (DHBV) in vivo. Methods in vitro anti-HBV effect: MTT assay was used to detect the median toxic concentration (TC50) of LYQP and LYQS on HepG2.2.15 cells transfected with the whole genome of HBV DNA, and the maximum non-toxic concentration (TCoC) to detect the toxicity of LYQP and LYQS to HepG2.2.15 cells transfected with HBV DNA. The effects of different concentrations of drugs on HepG2.2.15 cells were observed by direct addition method. The supernatants of cell culture were collected at 72h and 144h respectively. The titers of HBsAg and HBeAg in the supernatants were detected by enzyme-linked immunosorbent assay (Elisa). The anti-DHBV activity in the supernatant was detected by fluorescence quantitative PCR- FQ-PCR method. The DHBV positive ducks were screened by normal PCR method after 1 day old Guangxi duck infected with DHBV for 7 days. They were randomly divided into 8 groups: high, middle and low dose groups with high, middle and low doses of LYQS, model group and acyclovir ACV-positive control group. Ten ducklings in each group were fed continuously for 14 days. The serum DHBsAg and DHBeAg titers were detected by Elisa and the contents of DHBV DNA in serum were detected by FQ-PCR. The activities of alanine aminotransferase (alt) and aspartate aminotransferase (AST) in serum were also detected. The pathological changes of duck liver were observed by HE staining. Results the antiHBV effects of 1: 1LYQP and LYQS on HepG2.2.15 cells in vitro were lower. TC50 was 111.77 mg / mL1 / mL 90.46 mg / L, respectively, and TC0 was 11.88 mg / mL1.59mg / L.2 in HepG2.2.15 cells, respectively, which could effectively inhibit the secretion of HBsAg and HBeAg and the synthesis of HBV DNA in HepG2.2.15 cells. The inhibitory effect has obvious time-effect and dose-effect relationship. 3TI is more than 2, which is a high efficiency and low toxicity anti-HBV drug. Compared with the model group, the serum DHBV DNA content of the DHBV DNA in the DHBV DNA of the DHBV group and the LYQS group was significantly lower than that of the model group. After 3 days of withdrawal, the serum DHBV DNA content in the low dose group and the ACV group was increased, but the DHBV DNA level in the high dose group was not obvious. Compared with the model group, LYQP and LYQS could decrease the AST and alt activity in duck serum, and the changes of OD value of DHBsAg and DHBeAg in serum were similar to the change of DNA content before and after administration, and the antiviral effect of the drug was related to the dose and time. In addition, duck liver histopathology showed that LYQP and LYQS had obvious protective effects on DHBV induced liver injury. Conclusion both in vivo and in vitro, WW LYQP and LYQS have significant anti-HBV effects, and can alleviate the damage of duck liver induced by DHBV and improve liver function.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R285.5;R512.62

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