胰蛋白酶及其制剂的毒理学试验研究

发布时间：2018-04-15 01:00

本文选题：急性毒性 + 亚慢性毒性　；参考：《南京农业大学》2015年硕士论文

【摘要】：胰蛋白酶(Trypsin)是最早发现于胰液消化蛋白质中的酶。胰蛋白酶在脊椎动物消化系统中是一种重要的消化酶,其在胰脏中先以胰蛋白酶原的形式而被合成,然后随胰液从胰岛管进入小肠,在小肠内经肠激酶激活后活化为胰蛋白酶。胰蛋白酶能特异性的切断多肽链中赖氨酸和精氨酸残基的羧基侧。胰蛋白酶还能激活其他一些酶的前体,例如羧肽酶原、糜蛋白酶原等,是一种特异性非常强的蛋白酶。胰蛋白酶作为一种蛋白水解酶,能降解变性的蛋白质,对未变性的蛋白质则不起作用。因此,胰蛋白酶能分解脓液、血凝块等,利于脓液的引流排除,从而使坏死创面净化速度加快,并能促进肉芽组织的新生。在人医临床上主要用于脓胸、血胸、瘘管等所产生的局部水肿、脓肿的治疗,也用于毒蛇咬伤治疗;在生物科技上常用于细胞培养。目前关于胰蛋白酶在兽医临床上的应用报道鲜见,胰蛋白酶尚未作为兽用药物在国内进行注册。依照农业部兽药评审中心兽药注册资料汇编的毒理学试验规定,本研究对胰蛋白酶原料药进行了包括小鼠急性毒性试验、大鼠30天喂养亚慢性毒性试验、小鼠骨髓细胞微核试验,以及复合蛋白酶乳房注入剂在家兔的皮肤和黏膜刺激试验、皮肤致敏试验在内的一般毒理学试验研究。给小鼠灌服剂量分别为 40 mg/kg,200 mg/kg,1000 mg/kg,5000 mg/kg 和8000 mg/kg的胰蛋白酶原料药进行急性毒性试验,结果显示,胰蛋白酶的LD505000 mg/kg,根据化学物质急性毒性剂量分级表判定胰蛋白酶原料药属于实际无毒物质;给大鼠灌服为期30 d的剂量分别为高剂量(800 mg/kg)、中剂量(400mg/kg)、低剂量(200mg/kg)的胰蛋白酶原料药进行亚慢性毒性试验,试验结果显示,各个剂量组的大鼠均未出现明显中毒情况,受试大鼠的血液常规、血液生化指标中尽管有个别指标与对照组相比有显著差异,但数值均处于正常范围内,脏器系数指标与对照组均无显著差异,病理组织学检查也未见异常。将复合蛋白酶乳房注入剂涂在家兔背部完整皮肤,观察其对皮肤的刺激和致敏情况;同时将复合蛋白酶乳房注入剂注入家兔阴道,观察其对黏膜刺激性,试验结果显示,复合蛋白酶乳房注入剂对家兔皮肤和阴道黏膜未产生明显刺激性,同样也未引起家兔皮肤的过敏反应;给小鼠灌服剂量分别为LD50(5000mg/kg)、1/2LD50(2500 mg/kg)、1/4 LD50(1250 mg/kg)的胰蛋白酶原料和阳性对照药物环磷酰胺(40 mg/kg)及等量蒸馏水进行小鼠骨髓细胞微核致突变试验,结果显示骨髓细胞微核致突变试验为阴性,表明胰蛋白酶原料对体细胞无致突变作用。
[Abstract]:Trypsin is the first enzyme found in pancreatic juice to digest proteins.Trypsin is an important digestive enzyme in the digestive system of vertebrates. Trypsin is synthesized in the form of trypsinogen in the pancreas, then enters the small intestine with the pancreatic fluid from the islet duct, and activates into trypsin after activation by enterokinase in the small intestine.Trypsin can specifically cut the carboxyl side of lysine and arginine residues in the polypeptide chain.Trypsin also activates precursors of other enzymes, such as carboxypeptidase, chymotrypsinogen, and is a highly specific protease.As a proteolytic enzyme, trypsin degrades denatured proteins, but does not act on undenatured proteins.Therefore, trypsin can decompose the pus, blood clot and so on, which is conducive to the drainage and exclusion of the pus, thus speeding up the purification of necrotic wounds and promoting the regeneration of granulation tissue.It is mainly used in the treatment of local edema and abscess caused by empyema, hemothorax, fistula and so on. It is also used in the treatment of venomous snake bite, and is often used in cell culture in biotechnology.At present, there are few reports on the application of trypsin in veterinary clinic. Trypsin has not been registered as a veterinary drug in China.In accordance with the provisions of toxicological tests compiled by the Veterinary Drug Evaluation Center of the Ministry of Agriculture, this study conducted acute toxicity tests on trypsin raw materials, including acute toxicity tests in mice, and subchronic toxicity tests on 30 days feeding rats.The micronucleus test of mouse bone marrow cells, the skin and mucous membrane irritation test of rabbits with complex protease breast implants, and the general toxicological test including skin sensitization test.Acute toxicity tests were conducted in mice at doses of 40 mg / kg ~ (200 mg / kg ~ (-1) mg / kg ~ (-1) mg 路kg ~ (-1) ~ (-1) mg 路kg ~ (-1)) of trypsin for 5000 mg/kg and 8000 mg/kg, respectively.The LD505000 mg / kg of trypsin, according to the acute toxic dose scale of the chemical substances, the trypsin raw material was classified as the actual nontoxic substance.The subchronic toxicity test of trypsin raw drug was carried out in rats at high doses of 800 mg / kg, middle dose of 400 mg / kg and low dose of 200 mg / kg of trypsin respectively for 30 days. The results showed that there was no obvious toxicity in each dose group.The blood routine and blood biochemical indexes of the tested rats were significantly different from those of the control group, but the values were within the normal range, and there was no significant difference between the viscera coefficient index and the control group.Histopathological examination was not abnormal.In order to observe the irritation and sensitization of the skin in the back of the rabbit, the compound protease breast injection agent was applied to the intact skin of the rabbit back to observe the irritation of the mucous membrane by injecting the compound protease breast injection agent into the vagina of the rabbit.There was no obvious irritation to rabbit skin and vagina mucosa after breast injection of complex protease, and no allergic reaction of rabbit skin was also caused.The micronucleus mutagenicity test of bone marrow cells in mice was carried out by injecting the trypsin raw material and positive control drug cyclophosphamide 40 mg / kg and distilled water at the dose of LD50 5000mg / kg 1 / 2LD50,500mg / kg 2500mg / kg respectively. The results showed that the micronucleus mutagenicity test of bone marrow cells was negative.The results showed that trypsin had no mutagenicity on somatic cells.
【学位授予单位】：南京农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S859.7

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