【作者】 赵云利；

【导师】 宋礼华；

【作者基本信息】 安徽大学 ， 生物化学与分子生物学， 2006， 硕士

【摘要】 目的：分析聚乙二醇修饰对天花粉蛋白的安全性及其体内、体外活性的影响，以正确评价聚乙二醇修饰在天花粉蛋白研究中的意义和价值。方法：5～6周龄、体重为96±9g的SD大鼠随机分为四组，分别尾静脉注射不同剂量的受试物和溶媒，每周给药一次，四周后改为隔周给药，180d停止给药并解剖部分动物做常规毒理学观察和指标检测，剩余动物正常喂养，恢复30d后处死进行常规毒理学观察和指标检测；豚鼠药物致敏5次，末次致敏后14d、28d以双倍抗原攻击，观察0.5h内动物的过敏反应情况；大鼠皮内接种抗血清，24h后抗原加染料攻击，检测染料渗出程度；妊娠6～7d(早孕)或11～12d(中孕)的小鼠尾静脉给药，120h(早孕)或48h(中孕)解剖，记录死胎数、活胎数和吸收点数并计算引产有效率；细胞2～3×10~4／孔接种96孔板，培养24h后移去培养液，加入不同浓度药物继续培养24h，倾去上清液，染色30min，脱色10min，570nm测吸光度(630nm参比)，计算细胞存活率。结果：长毒试验中无动物死亡，大鼠外观活动正常；低剂量组MCH偏低，TRIG偏高，其余各指标与对照组无显著差异；中剂量组MCH、MCHC偏低，TRIG偏高，恢复期雌性大鼠的凝血时间延长、胸腺系数偏低，雄性大鼠的肾系数偏低；高剂量组WBC、MCH、MCHC偏低，HCT、ALT、ALP、TP、ALB、Glu等指标偏高，雄性大鼠的凝血时间延长，但至恢复期后各指标无明显差异其余各指标与对照组无显著差异；以PEG-TCS致敏和攻击的豚鼠仅有一只出现抓鼻、咳嗽现象，而且很快就恢复正常，而TCS致敏和攻击的动物在10m内全部死亡；与TCS相比，PEG-TCS引起大鼠被动皮肤过敏反应的程度也大大降低了，体外的细胞毒性和抗病毒活性却比修饰前降低了，但其体内引产活性却提高了，在较小的剂量下(0.5mg／ml)就能达到抗生育目的(引产率＞75％)。结论：PEG修饰能显著降低天花粉蛋白的毒性，提高其体内活性，安全可靠，在临床中有着广泛的应用前景。更多还原

【Abstract】 Objective: To analyze the effect of PEGylation on the safety and effect (in vivo and in vitro) of TCS, and estimate the significance and importance of PEG-TCS. Methods: Sprague-Dawley rats aging 5～6 weeks, weighing between 85～105g were used in the long-term toxicity. 160 rats were randomly divided into 4 groups. Each group were injected different dose PEG-TCS or only solvent, first once weekly, once two weeks one month later. Drugs were stopped, some rats were sacrificed 180 days later, the residual were subjected to 30 days recovery test. Clinical symptoms were observed and laboratory tests were examined in all rats. Guinea pigs were sensitized with antigens on days 1, 3, 5, fourteen days after the third sensitizing injection, the guinea pigs were challenged by an intravenous injection into the hind limb peripheral vein with the same antigen to evoke systemic anaphylaxis, signs were observed for 30 minutes immediately after injection. Middle or early gestation mice were injected drugs, and sacrificed 48 or 120 hours later. Dead and live fetuses were noted and the abortifacient rate was calculated. Rats were intradermal injected antiserum in back, antigens were intravenous injected through tail vein with Evens blue, then the seepage of the dye was observed by the diameters of blue points. Cells were inoculated into 96-well plates for 2～3 ×10~4 per well and incubated for 24 hours. Medium was changed with different drugs and incubated for another 24 hours. Medium was discarded, dye was added for 30 minutes, then dye was discarded and washed with lotion for 10 minutes, then the optical density of the plates was read on ELISA reader at 570 nm/630nm. Results: All rats survived after administering and no apparent changes induced by drugs were observed. Rats at the low dose showed decrease更多还原