重组促卵泡素-Fc融合蛋白的安全性评价

发布时间：2018-02-14 12:09

本文关键词： 重组促卵泡素-Fc融合蛋白大鼠小鼠安全性评价　出处：《扬州大学》2017年硕士论文　论文类型：学位论文

【摘要】：促卵泡素(Follicular Stimulating Hormone,FSH)是一种由动物脑垂体合成并分泌的激素,属于糖基化蛋白质激素,对机体的发育、生长、性成熟和繁殖等一系列生理活动都具有重要的调节作用,因此促卵泡素不论是在畜牧生产还是在人类不育症的治疗上均有重要应用价值。在实际生产和应用中,重组促卵泡素具有更高的活性,更稳定的结构和更低的价格。为客观评价重组促卵泡素-Fc融合蛋白的毒性,本研究对重组促卵泡素-Fc融合蛋白分别进行了急性毒性试验、重复染毒试验和致突变试验,为随后进行的临床应用研究提供科学依据。1.急性毒性试验20只清洁级ICR小鼠,雌雄各半,采用最大给药量法,肌内注射染毒。结果表明,广州威生医药科技有限公司研制的重组促卵泡素-Fc融合蛋白肌注LD50大于2g/kg b.w.。根据WTO有关化学物急性毒性分级标准进行评价,重组促卵泡素-Fc融合蛋白属实际无毒。2.重复染毒试验80只Wister大鼠,随机分成高、中、低剂量组和对照组,每组20只,雌雄各半,受试物重组促卵泡素-Fc融合蛋白设1000U/kg b.w./d、500U/kg b.w./d和50U/kg b.w./d三个剂量组;另设一生理盐水对照组。在大鼠后肢大腿外侧肌肉内注入药液,每天一次,连续给药14d。在最后一次染毒结束后24h内采集所有存活大鼠血样测定血常规、血清生化指标,并进行病理解剖及病理组织学检查。结果表明,试验组各项指标均在正常范围内,与对照组无显著差异。在试验条件下,重组促卵泡素-Fc融合蛋白连续14d重复肌内注射不会对实验动物大鼠产生明显毒性副作用。3.致突变试验3.1精子畸形试验60只清洁级ICR小鼠随机分为五组,每组12只。重组促卵泡素-Fc融合蛋白给药剂量分别设为375 U/kg b.w.、187.5U/kgb.w.、93.75 U/kg b.w.,另分别设环磷酰胺阳性对照组和无菌水阴性对照组。按常规方法进行小鼠精子畸形试验,腹腔注射给予,连续5天,从首次给予受试物计第35d颈椎脱臼处死动物,取双侧附睾制片,每只动物计数1000个精子中的畸形数,计算畸形率。试验结果显示,重组促卵泡素-Fc融合蛋白对小鼠精子无影响,表明重组促卵泡素-Fc融合蛋白对生殖细胞无遗传毒性。3.2微核试验选用50只清洁级ICR小鼠随机分为五组。每组10只。重组促卵泡素-Fc融合蛋白给药剂量分别设为375U/kgb.w.、187.5U/kgb.w.、93.75U/kgb.w.,另分别设环磷酰胺阳性对照组和无菌水阴性对照组。于第二次给药后6h取材、制片,计算微核率。试验结果显示,重组促卵泡素-Fc融合蛋白对小鼠骨髓细胞的微核率无影响。3.3 Ames 试验通过对重组促卵泡素-Fc融合蛋白进行SD小鼠伤寒沙门氏菌回复突变试验,评价该药的潜在致突变性。用重组促卵泡素-Fc融合蛋白制备混悬液进行试验,采用平板掺入法,计数TA97、TA98、TA100和TA102标准测试菌株在4个不同浓度下,于37℃培养48h后的回复突变菌落数。结果显示重组促卵泡素-Fc融合蛋白在50180.U～80 U范围内,无论有或无代谢活化系统(S-9)时,各剂量组均未引起测试菌株回复突变菌落数的明显增加,因此试验结果为阴性。表明重组促卵泡素-Fc融合蛋白对鼠伤寒沙门氏菌无致突变性。综上所述,急性毒性试验、重复染毒试验和致突变试验结果表明,重组促卵泡素-Fc融合蛋白对实验动物无毒性和致突变作用,具有较高的安全性。
[Abstract]:FSH (Follicular Stimulating Hormone, FSH) is a hormone synthesized and secreted by the pituitary gland animal, belonging to the glycosylated protein hormone, growth of the development of the body, and has an important role in the regulation of a series of physiological activities of maturation and reproduction, so whether FSH in animal production were in the treatment of human infertility. The important value of application in the actual production and application of recombinant FSH has higher activity, more stable structure and lower price. For the objective evaluation of recombinant FSH -Fc fusion protein on the toxicity, acute toxicity test was carried out respectively on the fusion protein of recombinant FSH -Fc, repeated exposure test and mutagenicity test, clinical application study is then conducted to provide scientific basis for the acute toxicity test of.1. 20 clean level ICR mice, male and female, using the large dosage method, Intramuscular injection of exposure. The results show that the development of Guangzhou vesun Pharmaceutical Technology Co. Ltd. the recombinant FSH -Fc fusion protein than 2g/kg b.w.. Intramuscular injection of LD50 was evaluated according to the acute toxicity of WTO on chemical classification standard, recombinant FSH -Fc fusion protein is actually non-toxic.2. exposure test of 80 Wister rats were randomly divided into high. In the low dose group and control group, 20 rats in each group, male and female, the test of recombinant FSH -Fc fusion protein with 1000U/kg b.w./d, 500U/kg b.w./d and 50U/kg b.w./d three dose group; another normal saline control group. The injection of liquid in the lateral muscle of rat hind leg, once a day. Continuous administration of 14D. in the last time after the end of the exposure of 24h collected in all survival rats blood determination of blood, serum biochemical indexes and pathological anatomy and histopathological examination. The results showed that the test group refers to the The subject was in the normal range, and the control group had no significant difference. Under the experimental conditions, the recombinant FSH -Fc fusion protein for 14d repeated intramuscular injection not have obvious toxic side effects caused by.3. mutation test 3.1 sperm malformation test 60 Sprague ICR mice were randomly divided into five groups of animal experimental rats. 12 rats in each group. The recombinant FSH -Fc fusion protein dosage was set to 375 U/kg b.w., 187.5U/kgb.w. U/kg, 93.75 b.w., respectively. Another positive control group and negative control group. The sterile water mouse sperm abnormality test were used according to the conventional method, the abdominal cavity injection, 5 consecutive days, from the first time to give the subjects the matter 35d were killed by cervical dislocation animal, the bilateral epididymis deformity rate in each animal production, counting 1000 sperm, calculated the rate of abnormality. The test results show that the recombinant FSH -Fc fusion protein on mouse sperm showed no effect. Recombinant FSH -Fc fusion protein has no genetic toxicity of.3.2 micronucleus test using 50 male ICR mice were randomly divided into five groups of germ cells. 10 rats in each group. The recombinant FSH -Fc fusion protein dosage were divided into 375U/kgb.w., 187.5U/kgb.w., 93.75U/kgb.w., respectively. Another positive control group and negative control of sterile water in the second group. After administration of 6h were calculated. The micronucleus rate of production, the test results show that the recombinant FSH -Fc fusion protein on mice bone marrow cell micronucleus rate had no effect on.3.3 Ames test by FSH -Fc fusion protein of SD mouse Salmonella typhimurium reverse mutation test of recombinant, mutagenicity evaluation the medicine. The recombinant FSH -Fc fusion protein for preparation of mixed suspension test, using the plate incorporation method, count TA97, TA98, TA100 and TA102 standard test strains in 4 different concentrations, At the temperature of 37 48h after culture reverse mutation. The results showed that the recombinant FSH -Fc fusion protein in 50180.U ~ 80 U range, either with or without metabolic activation system (S-9), each dose group were not caused by the tested strains significantly increased the number of colonies back mutation, so that test results are negative. Recombinant FSH -Fc fusion protein had no mutagenicity in Salmonella typhimurium. In summary, the acute toxicity test, repeated exposure test and mutagenicity test results showed that the recombinant FSH -Fc fusion protein on experimental animal toxicity and mutagenic action, has high safety.

【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S859.7

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