胰腺癌MR分子探针多靶点的筛

发布时间：2018-06-16 00:01

  本文选题：胰腺癌 + 分子影像学　； 参考：《第四军医大学》2015年硕士论文

【摘要】：目的:1.验证MMP14、Survivin及Kras在胰腺癌高表达,且与胰腺炎及正常胰腺组织表达情况进行比较,差异有统计学意义。2.构建MMP14基因序列及质粒载体p ET-30a-m MMP14,将其导入大肠杆菌中构成原核表达体系,通过IPTG诱导表达并纯化出MMP14蛋白。方法:1.收集43例胰腺癌、13例胰腺炎及21例正常胰腺组织标本进行免疫组织化学染色,分析MMP14、Survivin及Kras蛋白在三种组织内表达的程度、差异及与胰腺癌患者临床病理参数间的关系。2.合成并优化MMP14基因序列,构建原核质粒载体p ET-30a-m MMP14,对其进行双酶切鉴定,确定该质粒载体构建成功。再将p ET-30a-m MMP14转化至Rosetta(DE3)细胞中,以不同浓度IPTG进行诱导表达,摸索适宜条件,对表达产物进行镍柱层析纯化。结果:1.显示MMP14、Survivin及Kras蛋白在胰腺癌组织中高表达,与非癌组织比较,差异有统计学意义。2.MMP14及Survivin蛋白的表达情况与胰腺癌患者临床病理参数无关,Kras蛋白的表达与年龄呈正相关,年龄越大的胰腺癌患者越容易出现Kras蛋白的高表达。3.MMP14、Survivin及Kras蛋白在胰腺炎及正常胰腺组织内表达差异无统计学意义。4.通过对截短后的MMP14基因序列进行稀有密码子优化,得到了m MMP14序列,将其与原始MMP14序列进行基因比对,发现相符率约为74%,达到实验要求,遂即全基因合成原核表达载体p ET-30a-m MMP14。5.将p ET-30a-m MMP14转化至Rosetta(DE3)细胞后,在0.4 mm IPTG浓度,37℃条件下诱导4 h后可获得高度表达的MMP14蛋白,最终经过多次镍柱层析纯化得到了纯度在80%以上的MMP14蛋白。结论:1.MMP14、Survivin及Kras蛋白在胰腺癌明显高表达,可以作为胰腺癌分子诊断的靶点。2.成功构建了原核质粒载体p ET-30a-m MMP14,将该载体转化入感受态细胞Rosetta(DE3)后,可以正常表达MMP14蛋白,再经镍柱层析纯化,最终得到了纯度在80%以上的MMP14蛋白,可以用于后续的细胞SELEX筛选实验。
[Abstract]:Purpose 1. To verify the high expression of survivin and K ras in pancreatic carcinoma, and compared with pancreatitis and normal pancreatic tissue, the difference was statistically significant. 2. MMP14 gene sequence and plasmid vector pET-30a-m MMP14 were constructed and introduced into E. coli to form a prokaryotic expression system. MMP14 protein was induced and purified by IPTG. Method 1: 1. Immunohistochemical staining was performed in 43 cases of pancreatic carcinoma with pancreatitis and 21 cases of normal pancreatic tissues. The expression of MMP14, survivin and K ras protein in the three tissues was analyzed. The relationship between MMP14, survivin and the clinicopathological parameters of pancreatic cancer was analyzed. The sequence of MMP14 gene was synthesized and optimized, and the prokaryotic plasmid vector pET-30a-m MMP14 was constructed. Then pET-30a-m MMP14 was transformed into Rosetta-DE3) cells. The expression was induced by IPTG at different concentrations. The optimal conditions were explored and the expressed product was purified by nickel column chromatography. The result is 1: 1. The results showed that the expression of survivin and Kras protein in pancreatic carcinoma was significantly higher than that in non-cancerous tissue. 2. The expression of MMP14 and survivin protein was not correlated with the clinicopathologic parameters of pancreatic cancer and the expression of Kras protein was positively correlated with age. The higher the expression of Kras protein, the higher the expression of Kras protein. 3. There was no significant difference in the expression of Kras protein and survivin protein between pancreatitis and normal pancreatic tissues. 4. By optimizing the truncated MMP14 gene sequence with rare codon, the mMMP14 sequence was obtained and compared with the original MMP14 sequence. It was found that the coincidence rate was about 74%, which met the experimental requirements. Therefore, the whole gene synthesis prokaryotic expression vector pET-30a-m MMP14.5. After transforming pET-30a-m MMP14 into Rosetta-DE3) cells, the highly expressed MMP14 protein was obtained after induction at 0.4 mm IPTG concentration at 37 鈩，

本文编号：2024242