本文選題：胰腺癌 + 分子影像學(xué)��； 參考：《第四軍醫(yī)大學(xué)》2015年碩士論文

【摘要】：目的:1.驗(yàn)證MMP14、Survivin及Kras在胰腺癌高表達(dá),且與胰腺炎及正常胰腺組織表達(dá)情況進(jìn)行比較,差異有統(tǒng)計(jì)學(xué)意義。2.構(gòu)建MMP14基因序列及質(zhì)粒載體p ET-30a-m MMP14,將其導(dǎo)入大腸桿菌中構(gòu)成原核表達(dá)體系,通過IPTG誘導(dǎo)表達(dá)并純化出MMP14蛋白。方法:1.收集43例胰腺癌、13例胰腺炎及21例正常胰腺組織標(biāo)本進(jìn)行免疫組織化學(xué)染色,分析MMP14、Survivin及Kras蛋白在三種組織內(nèi)表達(dá)的程度、差異及與胰腺癌患者臨床病理參數(shù)間的關(guān)系。2.合成并優(yōu)化MMP14基因序列,構(gòu)建原核質(zhì)粒載體p ET-30a-m MMP14,對(duì)其進(jìn)行雙酶切鑒定,確定該質(zhì)粒載體構(gòu)建成功。再將p ET-30a-m MMP14轉(zhuǎn)化至Rosetta(DE3)細(xì)胞中,以不同濃度IPTG進(jìn)行誘導(dǎo)表達(dá),摸索適宜條件,對(duì)表達(dá)產(chǎn)物進(jìn)行鎳柱層析純化。結(jié)果:1.顯示MMP14、Survivin及Kras蛋白在胰腺癌組織中高表達(dá),與非癌組織比較,差異有統(tǒng)計(jì)學(xué)意義。2.MMP14及Survivin蛋白的表達(dá)情況與胰腺癌患者臨床病理參數(shù)無關(guān),Kras蛋白的表達(dá)與年齡呈正相關(guān),年齡越大的胰腺癌患者越容易出現(xiàn)Kras蛋白的高表達(dá)。3.MMP14、Survivin及Kras蛋白在胰腺炎及正常胰腺組織內(nèi)表達(dá)差異無統(tǒng)計(jì)學(xué)意義。4.通過對(duì)截短后的MMP14基因序列進(jìn)行稀有密碼子優(yōu)化,得到了m MMP14序列,將其與原始MMP14序列進(jìn)行基因比對(duì),發(fā)現(xiàn)相符率約為74%,達(dá)到實(shí)驗(yàn)要求,遂即全基因合成原核表達(dá)載體p ET-30a-m MMP14。5.將p ET-30a-m MMP14轉(zhuǎn)化至Rosetta(DE3)細(xì)胞后,在0.4 mm IPTG濃度,37℃條件下誘導(dǎo)4 h后可獲得高度表達(dá)的MMP14蛋白,最終經(jīng)過多次鎳柱層析純化得到了純度在80%以上的MMP14蛋白。結(jié)論:1.MMP14、Survivin及Kras蛋白在胰腺癌明顯高表達(dá),可以作為胰腺癌分子診斷的靶點(diǎn)。2.成功構(gòu)建了原核質(zhì)粒載體p ET-30a-m MMP14,將該載體轉(zhuǎn)化入感受態(tài)細(xì)胞Rosetta(DE3)后,可以正常表達(dá)MMP14蛋白,再經(jīng)鎳柱層析純化,最終得到了純度在80%以上的MMP14蛋白,可以用于后續(xù)的細(xì)胞SELEX篩選實(shí)驗(yàn)。
[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，

本文編號(hào)：2024242