本文關(guān)鍵詞： 重組促卵泡素-Fc融合蛋白 大鼠 小鼠 安全性評價　出處：《揚(yáng)州大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：促卵泡素(Follicular Stimulating Hormone,FSH)是一種由動物腦垂體合成并分泌的激素,屬于糖基化蛋白質(zhì)激素,對機(jī)體的發(fā)育、生長、性成熟和繁殖等一系列生理活動都具有重要的調(diào)節(jié)作用,因此促卵泡素不論是在畜牧生產(chǎn)還是在人類不育癥的治療上均有重要應(yīng)用價值。在實際生產(chǎn)和應(yīng)用中,重組促卵泡素具有更高的活性,更穩(wěn)定的結(jié)構(gòu)和更低的價格。為客觀評價重組促卵泡素-Fc融合蛋白的毒性,本研究對重組促卵泡素-Fc融合蛋白分別進(jìn)行了急性毒性試驗、重復(fù)染毒試驗和致突變試驗,為隨后進(jìn)行的臨床應(yīng)用研究提供科學(xué)依據(jù)。1.急性毒性試驗20只清潔級ICR小鼠,雌雄各半,采用最大給藥量法,肌內(nèi)注射染毒。結(jié)果表明,廣州威生醫(yī)藥科技有限公司研制的重組促卵泡素-Fc融合蛋白肌注LD50大于2g/kg b.w.。根據(jù)WTO有關(guān)化學(xué)物急性毒性分級標(biāo)準(zhǔn)進(jìn)行評價,重組促卵泡素-Fc融合蛋白屬實際無毒。2.重復(fù)染毒試驗80只Wister大鼠,隨機(jī)分成高、中、低劑量組和對照組,每組20只,雌雄各半,受試物重組促卵泡素-Fc融合蛋白設(shè)1000U/kg b.w./d、500U/kg b.w./d和50U/kg b.w./d三個劑量組;另設(shè)一生理鹽水對照組。在大鼠后肢大腿外側(cè)肌肉內(nèi)注入藥液,每天一次,連續(xù)給藥14d。在最后一次染毒結(jié)束后24h內(nèi)采集所有存活大鼠血樣測定血常規(guī)、血清生化指標(biāo),并進(jìn)行病理解剖及病理組織學(xué)檢查。結(jié)果表明,試驗組各項指標(biāo)均在正常范圍內(nèi),與對照組無顯著差異。在試驗條件下,重組促卵泡素-Fc融合蛋白連續(xù)14d重復(fù)肌內(nèi)注射不會對實驗動物大鼠產(chǎn)生明顯毒性副作用。3.致突變試驗3.1精子畸形試驗60只清潔級ICR小鼠隨機(jī)分為五組,每組12只。重組促卵泡素-Fc融合蛋白給藥劑量分別設(shè)為375 U/kg b.w.、187.5U/kgb.w.、93.75 U/kg b.w.,另分別設(shè)環(huán)磷酰胺陽性對照組和無菌水陰性對照組。按常規(guī)方法進(jìn)行小鼠精子畸形試驗,腹腔注射給予,連續(xù)5天,從首次給予受試物計第35d頸椎脫臼處死動物,取雙側(cè)附睪制片,每只動物計數(shù)1000個精子中的畸形數(shù),計算畸形率。試驗結(jié)果顯示,重組促卵泡素-Fc融合蛋白對小鼠精子無影響,表明重組促卵泡素-Fc融合蛋白對生殖細(xì)胞無遺傳毒性。3.2微核試驗選用50只清潔級ICR小鼠隨機(jī)分為五組。每組10只。重組促卵泡素-Fc融合蛋白給藥劑量分別設(shè)為375U/kgb.w.、187.5U/kgb.w.、93.75U/kgb.w.,另分別設(shè)環(huán)磷酰胺陽性對照組和無菌水陰性對照組。于第二次給藥后6h取材、制片,計算微核率。試驗結(jié)果顯示,重組促卵泡素-Fc融合蛋白對小鼠骨髓細(xì)胞的微核率無影響。3.3 Ames 試驗通過對重組促卵泡素-Fc融合蛋白進(jìn)行SD小鼠傷寒沙門氏菌回復(fù)突變試驗,評價該藥的潛在致突變性。用重組促卵泡素-Fc融合蛋白制備混懸液進(jìn)行試驗,采用平板摻入法,計數(shù)TA97、TA98、TA100和TA102標(biāo)準(zhǔn)測試菌株在4個不同濃度下,于37℃培養(yǎng)48h后的回復(fù)突變菌落數(shù)。結(jié)果顯示重組促卵泡素-Fc融合蛋白在50180.U～80 U范圍內(nèi),無論有或無代謝活化系統(tǒng)(S-9)時,各劑量組均未引起測試菌株回復(fù)突變菌落數(shù)的明顯增加,因此試驗結(jié)果為陰性。表明重組促卵泡素-Fc融合蛋白對鼠傷寒沙門氏菌無致突變性。綜上所述,急性毒性試驗、重復(fù)染毒試驗和致突變試驗結(jié)果表明,重組促卵泡素-Fc融合蛋白對實驗動物無毒性和致突變作用,具有較高的安全性。
[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.

【學(xué)位授予單位】：揚(yáng)州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S859.7

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