乌贼墨寡肽EPSI-1的制备与抗前列腺癌机制研究

Prostate cancer is one of the most common malignant tumors all over the world. Most prostate cancer are low growing. However, there are cases of aggressive prostate cancers. Toxicity, drug resistance and limited transient benefits in patients are the main problems associated with standard chemotherapeutic regimens, and new drugs are therefore needed to fight this devastating disease. In recent five years, we get EPSI of five different molecular weights defined as EPSI-1, EPSI-2, EPSI-3, EPSI-4 and EPSI-5,respectively. EPSI-1 whose molecular weight is under 1KDa possessed high anti-cancer properties including cell apoptosis and regulated the gen expression. EPSI-1 is isolated by ultrafiltration, purified on G-25 gel filtration and HPLC and demonstrated the purity of EPSI-1 by HPLC.Its peptide sequence analysis is detected. What's more, the structure of EPSI-1 is identified by NMR, GC and GE. The anti-tumor effect and mechanism of purified EPSI-1 in vitro and in vivo are investigated. The hormone-independent DU-145 and PC-3 are used to study the anti-tumor effect and mechanism of purified EPSI-1 in vitro. Concentration-dependent responses of the effect of EPSI-1on cell proliferation is examined using CCK-8 assay. AO/EB staining and FCM are employed for observation of apoptosis on DU-145 and PC-3 cells after treating with EPSI-1.WB, FQ-PCR and ISH are used to analyze the expression of Bcl-2,Caspase-3, Caspase-9,p53,mm23H1and VEGF-C in PC-3 and DU-145 cells treated with EPSI-1.The nude mouse model of human prostate cancer is established and its expression of p53,mm23H1,VEGF-C and MVD and the number of MLC are studied. From the above experiment ,the key technology of marine drug selection and preparation can be cleared and defined .This study will prepared a new anti-prostate cancer predecessor of marine drug which can lay a foundation for the development of peptide drug with independent intellectual property. Therefor ,this study possesses important academic values and a good perspective of application.

前列腺癌（PCa）是全球最常见恶性肿瘤之一，一旦发生或转移，治疗效果较差，且肿瘤长期化疗损害重要脏器，也会产生肿瘤耐药。因此，寻找低毒、高效新型抗癌药物势在必行。在研究PCa发生、转移及相关基因表达基础上，近5年来，又从乌贼墨中经酶解法首次得到5种不同分子量乌贼墨多肽（EPSI），其中分子量小于1KDa的EPSI-1具有显著诱导PCa细胞凋亡和基因调控作用。采用GE、GC、HPLC、CD和NMR等对EPSI-1进行分离纯化和结构表征。对其纯化物进行抗肿瘤机制研究，采用PCa细胞株和荷瘤小鼠模型进行体内外试验，阐明PCa的p53、nm23H1、VEGF-C表达以及MVD、MLC变化，明确海洋药物筛选和制备的关键技术，评价其抗PCa作用机制。本研究将制备一种新型抗PCa海洋药物前身物，为一种具有自主知识产权的抗肿瘤海洋肽类药物的研发奠定基础。因此，具有重要的学术价值和较好的应用前景。

本项目以新鲜的乌贼墨为原料，进行乌贼墨寡肽和乌贼墨肽聚糖进行分离纯化。乌贼墨寡肽主要是通过蛋白酶酶解获得乌贼墨酶解物并进行分离纯化，抗癌活性筛选，然后将收集的峰进行进一步分离纯化。分离纯化得到的寡肽进行氨基酸序列检测。乌贼墨肽聚糖粗提物经过分离纯化，并用高效液相色谱和聚丙烯酰胺凝胶电泳检测纯度，得到单一组分，最后将该样品通过紫外和红外扫描进行结构表征。. 研究结果表明乌贼墨寡肽的N端氨基酸序列为：Gln-Pro-Lys，分子量为343.4。CCK-8法结果显示，不同浓度的乌贼墨寡肽和乌贼墨肽聚糖对DU-145和PC-3细胞均有增殖抑制作用，呈剂量和时间依赖。AO/EB双染显示，对照组无明显凋亡细胞，5mg/mL乌贼墨寡肽和肽聚糖组开始出现早期凋亡细胞，随着乌贼墨寡肽和乌贼墨肽聚糖组浓度增加，晚期凋亡细胞逐渐增多，核浓聚、偏位、被染成桔红色，可见明显的凋亡小体。应用Annexin V-FITC/ PI 双标记流式细胞术检测后发现，不同浓度乌贼墨寡肽和墨肽聚糖作用24h后DU-145和PC-3早期凋亡率随着作用药物浓度的增加而增加。乌贼墨寡肽作用后，三种细胞中Bcl-2蛋白随着作用浓度的增加。2种细胞中Caspase-3和Caspase-9随着作用浓度的增加，阳性表达增加。乌贼墨寡肽以10mg/mL作用于DU-145和PC-3细胞24h后，两种细胞中p53mRNA表达量增加；两种细胞中BRCA1mRNA表达量增加。细胞周期结果显示寡肽将DU-145细胞阻滞在S期和G2/M期;；PC-3细胞被阻滞在G0/G1期和SubG1期。. 免疫组化法检测肽聚糖作用前后结果表明，乌贼墨肽聚糖对DU-145和PC-3细胞作用24h后，COX-2和VEGF蛋白表达急剧下降，阳性表达减弱。原位杂交方法结果表明，以15mg/mL肽聚糖作用于DU-145和PC-3细胞24h后，两种细胞中Bcl-2mRNA表达量下降；Caspase-3 mRNA表达量增加。. 建立小鼠PC-3细胞移植瘤模型，结果表明乌贼墨肽聚糖低、中和高剂量组对PC-3移植瘤均有抑制作用，抑瘤率分别为7.81%、23.43%和35.36%。急性毒理试验结果发现，乌贼墨肽聚糖实验组小鼠最大给药量为21.00g/kg.d，且试验过程中没有小鼠死亡。