食源性功能肽抑制胰腺癌细胞迁移作用和机制研究

发布时间：2018-07-25 21:11

【摘要】：胰腺癌是比较常见的消化道恶性肿瘤,近年的发病率和死亡率呈明显上升趋势,其诊断与治疗都十分的困难,且手术治疗的死亡率较高,五年生存率不足5%,是预后最差的恶性肿瘤之一,极大多数病人死于癌细胞的转移。我们拟利用农副产品牛血及大豆残渣,通过计算机模拟胃蛋白酶和胰蛋白酶的消化,筛选出抗胰腺癌细胞转移的功能性多肽。我们用计算机模拟胰蛋白酶/胃蛋白酶将牛血红蛋白进行水解,产生一系列的多肽片段,再依据与Hsp90对接消耗的自由能的大小排序,得到消耗自由能最低的6个牛血肽序列。经过划痕愈伤实验的筛选,四肽Asn-Phe-Gly-Lye(NFGK)对胰腺癌细胞MIAPaCa-2迁移的抑制有着比较可观的潜力。Transwell实验结果进一步证实了NFGK的抑制效果,同时,我们通过MTS实验得知,NFGK对细胞的生长增殖没有影响,即没有细胞毒性。我们将NFGK的N端与荧光素FITC连接,用荧光示踪的方法,查看NFGK在细胞中的分布情况,发现NFGK只出现细胞膜上。此外,利用Western-Blot检测蛋白水平的变化,证明NFGK以浓度依赖的方式抑制细胞外分泌型Hsp90α及其下游的MMP-9,而对细胞内的Hsp90及其客户蛋白CDK4均无抑制作用。同样地,我们利用划痕愈伤实验筛选出了效果相对最理想的大豆肽YPTSK,然后通过tranwell实验证明了其确有抑制MIAPaCa-2细胞迁移和侵袭的作用,并且,经MTS实验确认其也无细胞毒性。与NFGK不同的是,荧光共聚焦定位显示YPTSK进入了细胞,但不入核。而ATP酶活检测实验则证明YPTSK不是通过抑制Hsp90的N端ATP酶活来抑制胰腺癌细胞转移的,相应的,我们发现YPTSK也并不抑制细胞内外Hsp90及其客户蛋白的表达水平。反而,AKT和MMP家族蛋白的表达水平有所下降,且进一步的RT-PCR实验中,我们证实YPTSK的确抑制了一些MMP家族蛋白的转录水平,如MMP-2、MMP-9。在本研究中,我们首次证明胰腺癌细胞MIAPaCa-2也会分泌Hsp90α到细胞外基质,从而行使其分子伴侣功能,影响其下游如MMP-9等蛋白的功能。我们还首次从牛血及大豆残渣中提取出具有抗癌细胞转移作用的四肽NFGK及五肽YPTSK,既解决了农副产品重新利用的问题,减少了浪费,又为辅助治疗胰腺癌的方案提供了新的策略。
[Abstract]:Pancreatic cancer is a common malignant tumor of digestive tract. In recent years, the morbidity and mortality of pancreatic cancer are increasing obviously. The diagnosis and treatment of pancreatic cancer are very difficult, and the mortality rate of surgical treatment is high. The five-year survival rate is less than 5%, and is one of the worst malignant tumors. Most patients die of cancer cell metastasis. Using bovine blood and soybean residue as agricultural by-product, the functional peptides against pancreatic cancer cell metastasis were screened by computer simulation of pepsin and trypsin digestion. The bovine hemoglobin was hydrolyzed by computer simulated trypsin / pepsin to produce a series of polypeptide fragments. According to the order of the free energy consumed by docking with Hsp90, six bovine blood peptide sequences with the lowest consumption of free energy were obtained. The inhibitory effect of tetrapeptide Asn-Phe-Gly-Lye (NFGK) on MIAPaCa-2 migration of pancreatic cancer cells was confirmed by the screening of scratch callus assay. The results of Transwell experiment further confirmed the inhibitory effect of NFGK. We know by MTS experiment that NFGK has no effect on cell growth and proliferation, that is, no cytotoxicity. The N-terminal of NFGK was connected with fluorescein FITC, and the distribution of NFGK in cells was examined by fluorescence tracer. It was found that NFGK only appeared on the cell membrane. In addition, the change of protein level detected by Western-Blot showed that NFGK inhibited exocrine Hsp90 伪 and its downstream MMP-9 in a concentration-dependent manner, but had no inhibitory effect on intracellular Hsp90 and its client protein CDK4. In the same way, the soybean peptide YPTSKK, which is the most effective soybean peptide, was screened by scratch callus test, and its inhibitory effect on the migration and invasion of MIAPaCa-2 cells was proved by tranwell assay. Moreover, the MTS assay confirmed that YPTSKK had no cytotoxicity. Unlike NFGK, fluorescent confocal localization showed that YPTSK entered the cell but not into the nucleus. ATP activity assay showed that YPTSK did not inhibit the metastasis of pancreatic cancer cells by inhibiting the N-terminal ATP activity of Hsp90. Accordingly, we found that YPTSK did not inhibit the expression of Hsp90 and its client proteins in and out of the cells. On the contrary, the expression of AKT and MMP family proteins decreased, and in further RT-PCR experiments, we confirmed that YPTSK did inhibit the transcription level of some MMP family proteins, such as MMP-2, MMP-9. In this study, we demonstrated for the first time that pancreatic cancer cells MIAPaCa-2 also secreted Hsp90 伪 into extracellular matrix, thus exercising its molecular chaperone function and affecting the function of downstream proteins such as MMP-9. We also extracted tetrapeptide NFGK and pentapeptide YPTSKfrom bovine blood and soybean residue for the first time, which not only solved the problem of reuse of agricultural and sideline products, but also provided a new strategy for adjuvant treatment of pancreatic cancer.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.9

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