本文選題：玉郎傘多糖 + 拉米夫定　； 參考：《廣西醫(yī)科大學(xué)》2014年碩士論文

【摘要】：目的：研究玉郎傘多糖(YLSP)及其與拉米夫定組(YLSP/3TC)體內(nèi)外抗乙型肝炎病毒的效果。 方法：(1)體外抗HBV作用：體外研究以HBV基因轉(zhuǎn)染的HepG2.2.15細(xì)胞株為細(xì)胞模型，MTT法檢測(cè)YLSP對(duì)HepG2.2.15細(xì)胞的半數(shù)毒性濃度(TC50)和最大無(wú)毒濃度(TC0)；采用直接加藥法進(jìn)行實(shí)驗(yàn)，在最大無(wú)毒濃度(TC0)下，研究不同濃度的含YLSP及YLSP/3TC的培養(yǎng)基，對(duì)HepG2.2.15細(xì)胞株進(jìn)行培養(yǎng)，于第72h和第144h分別收集不同組別的細(xì)胞培養(yǎng)上清液，以實(shí)時(shí)熒光定量PCR(FQ-PCR)技術(shù)檢測(cè)其HBV DNA的載量，并以ELISA法檢測(cè)其HBsAg和HBeAg的滴度。以HBV DNA，HBsAg和HBeAg作為藥物抗HBV的重要觀察指標(biāo)，對(duì)YLSP及YLSP/3TC體外抗HBV作用進(jìn)行評(píng)價(jià)。(2)體內(nèi)抗DHBV作用：體內(nèi)研究中，以1日齡感染DHBV廣西麻鴨作為動(dòng)物模型，7d后采用PCR法篩選出DHBV感染強(qiáng)陽(yáng)性鴨。隨機(jī)分為6組：YLSP高、中、低劑量組3個(gè)劑量組；YLSP/3TC劑量組；模型組和拉米夫定(3TC)陽(yáng)性對(duì)照組。每組10只，各組雛鴨均持續(xù)灌胃給藥14d。觀察用藥前(T0)、用藥后7d(T7)、14d(T14)及停藥3d(P3)后，鴨血清上清中DHBsAg、DHBeAg，DHBV DNA的表達(dá)以及轉(zhuǎn)氨酶(ALT，AST)活性變化；同時(shí)在停藥3d后，取新鮮肝組織，觀察HE染色肝組織病理變化，并用新鮮肝組織勻漿，觀察各劑量組肝勻漿液上清中超氧化物歧化酶(SOD)、谷胱甘肽過(guò)氧化物酶(GSH-PX)的活性以及丙二醛(MDA)、谷胱甘肽(GSH)的含量，從而評(píng)價(jià)YLSP及YLSP/3TC體內(nèi)抗DHBV作用。 結(jié)果：(1)體外抗HBV作用：①Y LSP在11.72mg/L濃度時(shí)，對(duì)HepG2.2.15細(xì)胞基本沒(méi)有毒性。②在最大無(wú)毒濃度(TC0)下，不同濃度組別的YLSP及YLSP/3TC對(duì)HepG2.2.15細(xì)胞HBsAg及HBeAg的分泌和HBV DNA的合成均有顯著的抑制作用，且抑制作用呈現(xiàn)明顯的時(shí)效及量效關(guān)系；與YLSP組和3TC對(duì)照組相比，YLSP/3TC各劑量組對(duì)HepG2.2.15細(xì)胞上清HBsAg、HBeAg和HBV-DNA的抑制作用有顯著性差異(P0.05或P0.01)。③YLSP對(duì)HBsAg和HBeAg的TI分別為29.27和15.05，揭示YLSP安全性較好。(2)體內(nèi)抗DHBV作用：用藥7d(T7)、14d(T14)后，YLSP高、中劑量及YLSP/3TC組與模型組比較，鴨血清上清中DHBV DNA的含量，DHBsAg和DHBeAg的滴度，AST及ALT的活性均明顯下降；且YLSP/3TC組與YLSP高劑量組和3TC對(duì)照組相比較，鴨血清上清中DHBV DNA的含量，DHBsAg和DHBeAg的滴度，AST及ALT的活性均明顯低于YLSP高劑量組和3TC對(duì)照組。停藥3d后，YLSP中、低劑量組及3TC組鴨血清上清中DHBV DNA的含量，DHBsAg和DHBeAg的滴度，AST及ALT的活性，均出現(xiàn)反跳現(xiàn)象，而反跳現(xiàn)象在YLSP/3TC組不明顯，YLSP高劑量沒(méi)有出現(xiàn)反跳現(xiàn)象。YLSP高、中劑量組及YLSP/3TC組與模型組對(duì)比，鴨肝勻漿上清中SOD和GSH-PX的活性與GSH的含量明顯升高，MDA的含量明顯降低。YLSP/3TC組鴨肝勻漿上清SOD和GSH-PX的活性與GSH的含量顯著高于YLSP高劑量組和3TC對(duì)照組，YLSP/3TC組鴨肝勻漿上清MDA的含量顯著低于YLSP高劑量組和3TC對(duì)照組；藥物的抗病毒作用與劑量大小及時(shí)間相關(guān)。此外，鴨肝臟組織病理學(xué)檢查發(fā)現(xiàn)YLSP及YLSP/3TC對(duì)DHBV引起的肝損傷有明顯的保護(hù)作用，YLSP/3TC組保護(hù)作用更顯著。 結(jié)論：體內(nèi)外，YLSP及YLSP/3TC抗HBV療效顯著，且毒性較低，，同時(shí)還可以緩解DHBV所引起的鴨肝損傷，改善肝功能。YLSP與3TC組合不僅比二者單用有更強(qiáng)的抗病毒效果，而且對(duì)DHBV所致的鴨肝損傷也有更強(qiáng)的保護(hù)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.
【學(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R96;R512.62

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