玉郎伞多糖及其与拉米夫定组合抗乙型肝炎病毒及其作用机制研究

发布时间：2018-07-06 11:12

本文选题：玉郎伞多糖 + 拉米夫定　；参考：《广西医科大学》2014年硕士论文

【摘要】：目的：研究玉郎伞多糖(YLSP)及其与拉米夫定组(YLSP/3TC)体内外抗乙型肝炎病毒的效果。方法：(1)体外抗HBV作用：体外研究以HBV基因转染的HepG2.2.15细胞株为细胞模型，MTT法检测YLSP对HepG2.2.15细胞的半数毒性浓度(TC50)和最大无毒浓度(TC0)；采用直接加药法进行实验，在最大无毒浓度(TC0)下，研究不同浓度的含YLSP及YLSP/3TC的培养基，对HepG2.2.15细胞株进行培养，于第72h和第144h分别收集不同组别的细胞培养上清液，以实时荧光定量PCR(FQ-PCR)技术检测其HBV DNA的载量，并以ELISA法检测其HBsAg和HBeAg的滴度。以HBV DNA，HBsAg和HBeAg作为药物抗HBV的重要观察指标，对YLSP及YLSP/3TC体外抗HBV作用进行评价。(2)体内抗DHBV作用：体内研究中，以1日龄感染DHBV广西麻鸭作为动物模型，7d后采用PCR法筛选出DHBV感染强阳性鸭。随机分为6组：YLSP高、中、低剂量组3个剂量组；YLSP/3TC剂量组；模型组和拉米夫定(3TC)阳性对照组。每组10只，各组雏鸭均持续灌胃给药14d。观察用药前(T0)、用药后7d(T7)、14d(T14)及停药3d(P3)后，鸭血清上清中DHBsAg、DHBeAg，DHBV DNA的表达以及转氨酶(ALT，AST)活性变化；同时在停药3d后，取新鲜肝组织，观察HE染色肝组织病理变化，并用新鲜肝组织匀浆，观察各剂量组肝匀浆液上清中超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-PX)的活性以及丙二醛(MDA)、谷胱甘肽(GSH)的含量，从而评价YLSP及YLSP/3TC体内抗DHBV作用。结果：(1)体外抗HBV作用：①Y LSP在11.72mg/L浓度时，对HepG2.2.15细胞基本没有毒性。②在最大无毒浓度(TC0)下，不同浓度组别的YLSP及YLSP/3TC对HepG2.2.15细胞HBsAg及HBeAg的分泌和HBV DNA的合成均有显著的抑制作用，且抑制作用呈现明显的时效及量效关系；与YLSP组和3TC对照组相比，YLSP/3TC各剂量组对HepG2.2.15细胞上清HBsAg、HBeAg和HBV-DNA的抑制作用有显著性差异(P0.05或P0.01)。③YLSP对HBsAg和HBeAg的TI分别为29.27和15.05，揭示YLSP安全性较好。(2)体内抗DHBV作用：用药7d(T7)、14d(T14)后，YLSP高、中剂量及YLSP/3TC组与模型组比较，鸭血清上清中DHBV DNA的含量，DHBsAg和DHBeAg的滴度，AST及ALT的活性均明显下降；且YLSP/3TC组与YLSP高剂量组和3TC对照组相比较，鸭血清上清中DHBV DNA的含量，DHBsAg和DHBeAg的滴度，AST及ALT的活性均明显低于YLSP高剂量组和3TC对照组。停药3d后，YLSP中、低剂量组及3TC组鸭血清上清中DHBV DNA的含量，DHBsAg和DHBeAg的滴度，AST及ALT的活性，均出现反跳现象，而反跳现象在YLSP/3TC组不明显，YLSP高剂量没有出现反跳现象。YLSP高、中剂量组及YLSP/3TC组与模型组对比，鸭肝匀浆上清中SOD和GSH-PX的活性与GSH的含量明显升高，MDA的含量明显降低。YLSP/3TC组鸭肝匀浆上清SOD和GSH-PX的活性与GSH的含量显著高于YLSP高剂量组和3TC对照组，YLSP/3TC组鸭肝匀浆上清MDA的含量显著低于YLSP高剂量组和3TC对照组；药物的抗病毒作用与剂量大小及时间相关。此外，鸭肝脏组织病理学检查发现YLSP及YLSP/3TC对DHBV引起的肝损伤有明显的保护作用，YLSP/3TC组保护作用更显著。结论：体内外，YLSP及YLSP/3TC抗HBV疗效显著，且毒性较低，，同时还可以缓解DHBV所引起的鸭肝损伤，改善肝功能。YLSP与3TC组合不仅比二者单用有更强的抗病毒效果，而且对DHBV所致的鸭肝损伤也有更强的保护作用。
[Abstract]:Objective: To study the effect of Yu Lang umbrella polysaccharide (YLSP) and its lamivudine group (YLSP/3TC) on anti HBV in vivo and in vitro.
Methods: (1) the effect of anti HBV in vitro: in vitro, the HepG2.2.15 cell line transfected with HBV gene was used as the cell model, and half toxic concentration (TC50) and maximum non-toxic concentration (TC0) of HepG2.2.15 cells were detected by MTT, and the experiment was carried out by direct addition method, and the YLSP and YLSP/3TC of different concentrations were studied under the maximum concentration of TC0 (TC0). Culture medium, culture of HepG2.2.15 cell lines, cell culture supernatants of different groups were collected at 72h and 144H respectively. The load of HBV DNA was detected by real-time fluorescence quantitative PCR (FQ-PCR) technique and ELISA method was used to detect the titer of HBsAg and HBeAg. The anti HBV effect of SP/3TC in vitro was evaluated. (2) the anti DHBV effect in the body: in the body study, DHBV Guangxi ducks were infected with 1 days of age as animal model, and DHBV infected ducks were screened by PCR method after 7d. They were randomly divided into 6 groups: YLSP high, middle and low dose group; YLSP/ 3TC dose group; model group and lamivudine (3TC) positive control Group of 10 rats in each group, all ducks in each group were given a continuous perfusion of 14D. (T0), 7d (T7), 14d (T14) and 3D (P3) after drug withdrawal. The expression of DHBsAg, DHBeAg, DHBV DNA, and the changes in the activity of aminotransferase were observed. To observe the activity of superoxide dismutase (SOD), glutathione peroxidase (GSH-PX) and the content of malondialdehyde (MDA) and glutathione (GSH) in the supernatant of the liver homogenate, and to evaluate the anti DHBV effect of YLSP and YLSP/3TC in the dosage groups.
Results: (1) the effect of anti HBV in vitro: (1) Y LSP has no toxicity to HepG2.2.15 cells at the concentration of 11.72mg/L. (2) at the maximum non-toxic concentration (TC0), YLSP and YLSP/3TC of different concentration groups have significant inhibitory effect on the secretion of HBsAg and HBeAg and the synthesis of HBV. Compared with the YLSP group and the 3TC control group, there were significant differences in the inhibition of HBsAg, HBeAg and HBV-DNA in the HepG2.2.15 cell supernatant (P0.05 or P0.01) in each dose group of YLSP/3TC and the control group (P0.05 or P0.01). (3) YLSP to HBsAg and HBeAg were 29.27 and 15.05 respectively. Compared with the model group, the content of DHBV DNA, the titer of DHBsAg and DHBeAg, the activity of AST and ALT in the duck serum supernatant decreased obviously, and the content of DHBV DNA in the duck serum supernatant and the titer of the YLSP/3TC group were significantly lower than those of the high dose group and the 3TC control group, and the activity of the YLSP/3TC group was significantly lower than that of the high dose group and the 3TC control group. In the dose group and the 3TC control group, the content of DHBV DNA in the low dose group and the 3TC group, the titer of DHBsAg and DHBeAg, the activity of AST and ALT in the YLSP, the low dose group and the 3TC group were all reverse jump phenomenon, but the rebound phenomenon was not obvious in the YLSP/3TC group, and the high dose of YLSP did not appear to be high, the middle dose group and the group were compared with the model group. The activity of SOD and GSH-PX and the content of GSH in the supernatant of duck liver homogenate increased significantly. The content of MDA was significantly lower in the SOD and GSH-PX activities in the.YLSP/3TC group of duck liver homogenate and the content of GSH. The content of MDA in the YLSP/3TC group of duck liver homogenate was significantly lower than that of the high dose group and the control group. The antiviral effect was related to the dose and time. In addition, the histopathological examination of duck liver found that YLSP and YLSP/3TC had obvious protective effect on DHBV induced liver injury, and the protective effect of YLSP/3TC group was more significant.
Conclusion: the anti HBV effect of YLSP and YLSP/3TC in vivo and in vivo is significant, and the toxicity is low. At the same time, it can also relieve the injury of duck liver caused by DHBV, and the combination of.YLSP and 3TC can not only have stronger antiviral effect than those of the two, but also have a stronger protective effect on the liver injury caused by DHBV.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R96;R512.62

【参考文献】