羧甲基壳聚糖锌多肽复合材料的生物安全性评价

发布时间：2018-01-31 00:28

本文关键词： 羧甲基壳聚糖锌多肽牙周膜成纤维细胞细胞毒性动物毒性　出处：《青岛大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:研究羧甲基壳聚糖锌多肽复合材料(carboxymethyl chitosan zinc and peptide,CMC-Zn+-P)的生物安全性,为材料的安全使用提供依据。方法:1.细胞毒性检测:在体外培养人牙周膜成纤维细胞(human periodontal ligament cells,HPDLCs),对其形态学进行观察,并进行免疫组织化学鉴定。制取材料浸提液,以不同的浓度与细胞培养液混合,用于培养细胞,并进行分组,1~4组为实验组,材料浸提液的浓度分别为100%、75%、50%、25%,第5组为对照组,不含材料浸提液,采用CCK-8法测定不同浓度的材料浸提液对细胞增殖活性的影响,通过计算细胞的相对增殖率(RGR),对细胞毒性进行评级。2.急性毒性检测:选取20只Wistar大鼠,随机分为实验组与对照组,实验组单次灌胃5 g/kg复合材料,对照组单次灌胃相同剂量的生理盐水。给药以后,大鼠正常饮食饮水,连续观察一周,记录大鼠的行为活动状态,7天后对其进行称重、取血,检测大鼠血常规及生化指标,处死后进行尸检,完整取出大鼠各脏器,计算大鼠体重相对增长率和脏器指数,并进行病理切片检查。3.口腔黏膜刺激试验:选取10只Wistar大鼠,雌雄各半,麻醉后,将复合材料涂抹于大鼠的左侧口腔黏膜,将生理盐水涂抹于大鼠的右侧口腔黏膜,每侧均涂5 min,观察24 h后,将大鼠处死,并取用药部位的口腔黏膜,用10%福尔马林固定,进行HE染色,并于光学显微镜下观察。4.急性皮肤刺激试验:选取10只Wistar大鼠,雌雄各半。试验前24 h剔除背部的被毛,约3 cm×5 cm大小区域,将5 g/kg体重剂量的复合材料均匀涂抹于大鼠的背部,将药物用纱布盖住,使用无刺激性的胶布将其固定。24 h后去除纱布及药物,观察试验区皮肤是否出现红斑、水肿等情况,并观察大鼠是否有中毒表现及死亡情况,连续观察一周。5.长期毒性检测:选取健康的Wistar大鼠40只,将它们随机分为4组,高、中、低剂量组分别给予大鼠灌胃5 g/kg、2 g/kg、0.5 g/kg复合材料,对照组灌胃给予相同剂量的生理盐水,每天1次,连续给药3个月。3个月后,取血检测大鼠血常规及生化指标,处死后进行尸检,完整取出大鼠各脏器,计算大鼠体重相对增长率和脏器指数,并进行病理检查。6.统计学处理:采用SPSS 17.0软件包对数据结果进行统计学分析。结果:1.细胞毒性检测:体外培养的HPDLCs呈成纤维细胞的形态;免疫组化结果示细胞抗波形丝蛋白染色呈阳性,抗角蛋白染色呈阴性,培养的细胞来源于中胚层组织;不同浓度的材料浸提液对体外培养的HPDLCs均有促进增殖的作用,各组细胞的RGR均大于100%,细胞毒性分级为0级。2.急性毒性检测:单次灌胃给药后,实验组与对照组的试验大鼠行为状态良好,均未出现大鼠死亡的现象,两组大鼠在体重增长、脏器指数、血常规及血液生化指标各方面的差异均无统计学意义,病理检查各组大鼠的内脏器官,未发现明显异常。3.口腔黏膜刺激试验:试验用鼠未出现死亡,双侧口腔黏膜均无充血、水肿、糜烂等情况,病理切片也未见明显异常。4.急性皮肤刺激试验:试验用鼠皮肤未出现红斑、充血、水肿及糜烂等异常情况,也未出现实验动物的死亡。5.长期毒性检测:高、中、低剂量组与对照组相比,大鼠的体重变化、脏器指数无明显差异,各脏器无肉眼可见的病损,病理切片检查也未见明显异常,血常规结果在四组之间也无统计学差异,在血液生化指标的检测中,谷丙转氨酶(AST)、谷草转氨酶(ALT)、尿素(BUN)、葡萄糖(GLU)及肌酐(CREA)在四组中的结果差异无统计学意义,但总胆固醇(CHOL)结果出现组间差异,进一步进行两两比较发现,高剂量组与对照组之间差异显著,P0.001,高剂量组的CHOL含量明显高于对照组;高剂量组与中剂量、低剂量组相比,CHOL含量偏高,P=0.015,0.001;中剂量组与对照组相比,CHOL含量偏高,P=0.003;中剂量组与低剂量组以及低剂量组与对照组之间,差异无统计学意义结论:CMC-Zn+-P复合材料对体外培养的HPDLCs不仅无细胞毒性,而且还可促进细胞的增殖,对大鼠无急性毒性、无急性皮肤刺激毒性、无口腔黏膜刺激毒性、无长期毒性。
[Abstract]:Objective: To study the effect of carboxymethyl chitosan zinc composite (carboxymethyl chitosan zinc peptide and, peptide, CMC-Zn+-P) of the biological safety, provide the basis for the safe use of materials. Methods: to detect 1. cell toxicity of cultured human periodontal ligament fibroblasts in vitro (human periodontal ligament cells, HPDLCs), the morphology was observed, and were identified by immunohistochemistry. The preparation of material extract, with different concentration and cell culture fluid mixing for cultured cells, and were divided into two groups, 1~4 group for the experimental group, material extract concentration was 100%, respectively, 75%, 50%, 25%, fifth for the control group, without material extracts were determined by CCK-8 method, the material effects of extracts on cell proliferation, cell proliferation rate calculated by the relative rating (RGR),.2. acute toxicity test on the cell toxicity: 20 Wistar rats were randomly divided into As the experimental group and control group, experimental group, single dose 5 g/kg composite materials, the control group normal saline single oral administration of the same dose. After administrated rats with normal diet and water, continuous observation of the week, recording the activity status of rats, 7 days after the blood sampling, weighing, large blood routine and biochemical indexes of rats were sacrificed after detection, autopsy, complete removal of the organs of rats, the body weight of rats relative growth rate and organ index calculation, and pathological examination of.3. oral mucosa irritation test: a total of 10 Wistar rats, male and female, after anesthesia, the left buccal composite smear in rats the right of oral mucosal saline smear in rats, each side is coated with 5 min, 24 h was observed after rats were killed, and the oral mucosal administration area, fixed with 10% formalin, HE staining, and observed under optical microscope in acute skin.4. Stimulation test: a total of 10 Wistar rats, half male and half female. Before the test 24 h to eliminate the back coat, about 3 cm * 5 cm in size, the composite 5 g/kg body weight dose evenly on the back of a rat, the drug with gauze cover, use non irritating tape to fix the.24 h after the removal of gauze and drugs, to observe whether test area of skin erythema, edema, and rat are manifestations of poisoning and death, continuous observation of the long-term toxicity detection a week.5.: select 40 healthy Wistar rats, they were randomly divided into 4 groups, high, low dose group the rats were given intragastric administration of 5 g/kg, 2 g/kg, 0.5 g/kg composite, saline control group were lavaged with the same dose, 1 times a day, continuous administration of 3.3 months after the blood serum of rats and biochemical indicators were examined, the complete removal of various organs in in the calculation The body weight of rats relative growth rate and organ index, and pathological examination of.6. statistical packages for statistical analysis of data using SPSS 17 software. Results: the detection of 1. cell toxicity: HPDLCs fibroblast cell morphology in vitro; immunohistochemistry showed cell anti vimentin staining was positive, anti keratin staining was negative, cultured cells derived from the mesoderm tissue; different concentrations of material extract on cultured HPDLCs could promote the proliferation of the cells in each group, the RGR was more than 100%, cell toxicity was graded as acute toxicity test: single level 0.2. after intragastric behavior of experimental rats the experimental group and control group, there was no death in rats, the rats in the two groups in body weight, organ index, the differences of blood routine and blood biochemical index were not statistically significant, pathological examination of the Rats of the visceral organs, found no obvious abnormal.3. oral mucosa irritation test: test rats caused no death, no bilateral oral mucosal hyperemia, edema, erosion, pathological sections showed no obvious abnormal.4. acute skin irritation test with rat skin not appear erythema, hyperaemia, edema and erosion etc. abnormal situation, long-term toxicity of death.5. detection does not appear in the experimental animal: high, low dose group, compared with the control group, the changes of body weight of rats, no significant difference between the organ index of each organ, no visible lesions, pathological examination showed no obvious abnormalities, blood test results had no significant difference in the four between the groups, in the detection of blood biochemical index, alanine aminotransferase (AST), aspartate aminotransferase (ALT), urea (BUN), glucose (GLU) and creatinine (CREA) in the four groups showed no significant difference, but the total cholesterol (CHOL) results There differences between groups, further comparison showed that in 22, the difference between high dose group and the control group was P0.001, CHOL content of high dose group was significantly higher than the control group; high dose group and middle dose, low dose group, high levels of CHOL, P=0.015,0.001; compared with the control group, the content of CHOL is high. P=0.003; between middle dose group and low dose group, low dose group and control group, the difference was not statistically significant. Conclusion: CMC-Zn+-P composite material not only has no cytotoxicity of HPDLCs in vitro, but also can promote cell proliferation and no acute toxicity in rats, skin irritation and no acute toxicity, no oral mucosal irritation. No long-term toxicity.

【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R783.1

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