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1、 型胶原蛋白的制备、鉴定及其抗UVA氧化损伤作用的初步研究学科专业：动 物 学 研究方向：生化制药 指导教师：魏泓 教授 研 究 生：王 琳（2001345） 内 容 摘 要本文围绕型胶原蛋白（CP）的制备及其理化性质进行研究，同时在其防止长波紫外线（UVA）对细胞的要用于填充深的皱纹，皮肤损伤造成的缺损（如青春痘疤）和修补脸形的缺陷等。其效果立竿见影，但注射到皮肤内的胶元蛋白会被人体逐渐吸收，因此其功效只能维持半年至一年，而且少数人群可能会出现过敏氧化损伤方面进行了初步探索，为CP的进一步研发提供理论基础和科学依据。以猪皮为材料，比较酸溶法、酶解法、中性盐溶液法三种粗提方法，选择得率和纯度都
2、较高的酶解法为最佳粗提法，结合高效液相色谱进行CP的分离纯化。CP理化性质鉴定结果表明：样品在223 nm处有紫外最大吸收值；含有十八种氨基酸，氨基酸总量在90%以上；样品的蛋白质浓度为1.03mg/ml；SDS-PAGE测定CP由、三条链组成；色谱分析纯度约为100%。通过小鼠急性毒性试验、豚鼠致敏试验、细胞毒性试验对自制CP进行整体水平和细胞水平的生物安全性评价，观察期内，小鼠未见异常毒性反应；豚鼠注射点无红斑及水肿情况；细胞毒性小于级，结果证明本方法制备的CP无毒副作用。评价CP对UVA氧化损伤的L929细胞的保护作用。结果表明：随着UVA照射时间的延长，细胞的活性明显降低，说明UVA对
3、细胞的氧化损伤是明显的，其中表现为MDA的含量升高、GSH-px的含量降低。加入不同浓度的CP后照射同样的时间，MDA的含量有所下降、GSH-px的含量有所升高，其中50%CP组的效果极为显著（P0.01）。提示该物质有防止UVA氧化损伤的作用。综上所述，利用酶解和高效液相色谱制备相结合的方法可以从猪皮中分离纯化CP，该方法具有高效性，高选择性和易放大性；理化性质鉴定及生物安全性试验表明，该物质为纯活性胶原蛋白，无毒副作用；同时对体外细胞的UVA氧化损伤具有较好的保护作用。关键词：型胶原蛋白；高效液相色谱；制备；鉴定；氧化损伤Studies on the preparation，identif
4、ication of CPand its protective effect on cells damaged by UVAMajor : Biology Speciality : Zoology Tutor : Prof. Wei Hong Author : Wang Lin（2001345）AbstractIn this paper,how to prepare CPand identify physico-chemical property had been studied, and UVA oxidative damage L929 was perliminary discussed.
5、 These datas provided theoretic foundation and scientific proof for further research and development of this product.Pigskin was used as material. According to the comparation among the three kinds of crude extraction methods including acid dissolve,enzymolysis and neutral salt solution. The method
6、of enzymolysis combined with HPLC was chosen to prepare CP.The physico-chemical property of CPwas identified. The results showed that: CPshowed character absorption peak at 223nm; CPhad 18 kinds of amino acids, total count of amino acid was more than 90%; the concentration of protein was 1.03 mg/ml;
7、 CPwas composed of chain,chain and chain by SDS-PAGE; One pure peak with HPLC was founded and its purity was 100%. The security of CPaccording to the experiments of mice accute toxicity was evaluated, Guinea pig sensitization and cytotoxicity experiment in integer level and cell level. It was showed
8、 that : the mice had no toxic reaction; the Guinea pigs points of injection had no erythema and dropsy; the cytotoxicity was lower thanclass. The results proved that CPhad no poison side effects.CPprevented UVA from oxidative damaging L929 was studied. The activity of cells became weaker and weaker
9、with exposure time. It was conspicuous that UVA damaged the activity of cells. The contents of MDA upgraded and the contents of GSH-px degraded. When adding different concentration of CP, the contents of MDA decreased and that of GSH-px increased. The effect of 50 % concentration group was extremely
10、 remarkable (P10000048小时阴性对照组100% CP组50% CP组25% CP组12.5% CP组1.2450.4321.0570.3381.0990.3011.2550.4911.3950.356100858810010000072小时阴性对照组100% CP组50% CP组25% CP组12.5% CP组1.7450.2331.5780.3921.6140.4481.7530.3911.7940.35710090921001000004.3 CP防止长波紫外线（UVA）氧化损伤实验4.3.1 UVA对L929细胞活性的影响 见表5，随着UVA辐射时间的延长，MTT实验检测细胞的OD值由0.842下降到0.494，在照射时间为90min