NKCC1促进肝癌细胞转移的分子机制及其抑制剂用于抗肿瘤药物的探索

发布时间：2018-01-03 00:33

本文关键词：NKCC1促进肝癌细胞转移的分子机制及其抑制剂用于抗肿瘤药物的探索　出处：《安徽医科大学》2015年硕士论文　论文类型：学位论文

【摘要】：癌症是一种复杂的疾病,在一个特定组织的细胞中,不再充分响应组织内的信号调节细胞分化、生存、增殖和死亡。癌细胞能够无限增殖,在适宜的条件下具有“永生”能力,而无限增殖是癌细胞的主要生物学行为之一,也是癌症患者死亡的主要原因。全球有六大常见癌症,包括肺癌、肝癌、胃癌、结直肠癌、乳腺癌和食管癌,其发病率和致死率逐年增高的趋势为我们敲响了警钟。目前通过多种干预措施可以治疗癌症,比如外科手术、化学治疗和放射治疗,以及适当的心理辅助。化学治疗在癌症治疗中一直发挥着重要作用,但其治疗效果却受到剂量、毒性等因素的影响,而靶向小分子抑制剂的治疗则以癌症发生发展相关的分子为作用靶点,将小分子类化合物、抗体等有效成分靶向肿瘤细胞,具有选择性的杀伤作用,能够减少对正常组织的损伤,提高治疗效果,减少毒副作用,从而达到治疗或改善病人生活质量的目的。近年来,为了提高医疗卫生水平,关于癌症治疗工作国内外研究学者都为此付出不少心血,并取得了卓越成效,但仍极其缺乏根治手段。因此,从源头了解癌症发生发展的分子机制,也是今后工作的重中之重。所以,抗癌工作任重而道远。本实验室前期研究内容是备受关注的肝癌转移,体外以不同转移能力的肝癌细胞系、体内裸鼠荷瘤模型为研究对象验证了NKCC1蛋白与肝癌的转移能力正相关,但是,NKCC1通过何种机制促进肝癌细胞转移尚未有人研究过。本研究重点选取遗传背景相同、转移能力不同的高转移能力肝癌细胞系MHCC97H和低转移能力肝癌细胞系MHCC97L,无转移能力的肝癌细胞系Huh7,正常肝细胞系L02为研究对象,分别检测了激酶WNK1/OSR1对NKCC1总蛋白、磷酸化蛋白表达的影响及其活性变化,验证了WNK1-OSR1/SPAK-NKCC1通路中涉及的关键分子的总蛋白和磷酸化蛋白质的表达情况,结果在高转移肝癌细胞系、低转移肝癌细胞系和无转移肝癌细胞系中,WNK1-OSR1/SPAK-NKCC1通路中涉及的关键蛋白的总蛋白和磷酸化蛋白呈明显的依次降低的趋势；高转移肝癌细胞系、低转移肝癌细胞系和无转移肝癌细胞系的胞内NKCC 1活性也呈依次降低的趋势。刺激剂替莫唑胺,能够显著提高该通路中WNK1、OSR1、NKCC1蛋白的磷酸化水平；siRNA干扰WNK1/OSR1蛋白的表达,则降低了胞内钠离子浓度,最终降低了NKCC1的活性。实验证明,WNK1/OSR1通过磷酸化调控NKCC1的活性,进而促进了肝癌细胞的转移。NKCC1抑制剂布美他尼和RNAi干扰VNK1或OSRl都能显著降低肝癌细胞的转移能力。其次,利用布美他尼具有抑制肝癌细胞增殖的作用,我们扩大了其应用范围,选取肝癌以外的其他七株肿瘤细胞系进行研究。结合前期研究结果,本文首先采用Western Blot验证了JKC C1蛋白在肝癌以外的7种肿瘤细胞,即肺癌细胞系(A549),结直肠癌细胞系(HCT116),慢性髓源白血病细胞系(K562),食管癌细胞系(Eca109),宫颈癌细胞系(Hela),T淋巴细胞白血病细胞系(Jurkat),乳腺癌细胞系(MCF7)中的表达情况,接着用CCK-8法测定NKCC1抑制剂布美他尼对上述七种不同组织来源的肿瘤细胞系的抑制率,并进行敏感度对比。其中布美他尼对肺癌细胞系(A549)和结直肠癌细胞系(HCT116)抑制效果相对较敏感,即实验中所测IC50值最低。这些为未来临床上寻找布美他尼联合肝癌化疗药物奠定了有力基础。
[Abstract]:Cancer is a complex disease in a specific tissue cells, not fully response signal within the organization to regulate cell differentiation, survival, proliferation and death. Cancer cells can proliferate, with "eternal life" ability under suitable conditions, and unlimited proliferation is one of the main biological behavior of the cancer cells. The main reason is the death of cancer patients. There are six of the world's most common types of cancer, including lung cancer, liver cancer, gastric cancer, colorectal cancer, breast cancer and esophageal cancer, the incidence rate and mortality rate increased year by year trend is a wake-up call. The treatment of cancer through a variety of interventions, such as surgery, chemotherapy and radiation treatment and appropriate psychological support. Chemical treatment has played an important role in cancer treatment, but the treatment effect was dose, toxic effects and other factors, and targeted small molecule inhibitors treatment The cancer therapy target of the development of related molecules, small molecular compounds, antibodies and other effective components of targeting tumor cells, with a selective killing effect, can reduce the damage of normal tissue, improve the therapeutic effect, reduce the toxic and side effects, so as to cure or improve the quality of life of patients in purpose. Here, in order to improve the health level of cancer treatment research by domestic and foreign scholars have paid a lot of effort, and achieved remarkable results, but still lack of radical means. Therefore, understanding the molecular mechanisms of cancer development from the source, is the priority among priorities for future work. Therefore, the work of anticancer go15. Our previous research content is concerned with in vitro liver cancer metastasis, hepatocellular carcinoma cell lines with different metastatic ability of tumor bearing nude mice model, validation of the NKCC1 protein and as the research object Metastatic hepatocellular carcinoma is related, however, NKCC1 promotes the metastasis of hepatocellular carcinoma has not been studied by what mechanism. This study focuses on the same genetic background, different metastatic ability of high metastatic hepatocellular carcinoma cell line MHCC97H and low metastatic hepatocellular carcinoma cell line MHCC97L, hepatocellular carcinoma cell line Huh7 without metastasis and normal liver cell line L02 as the research object, were detected on NKCC1 protein kinase WNK1/OSR1 activity, change and influence the expression of phosphorylated protein, verified the expression of total protein and protein phosphorylation of key molecules involved in WNK1-OSR1/SPAK-NKCC1 pathway, results in highly metastatic hepatocellular carcinoma cell lines, low metastatic hepatocellular carcinoma cell lines and metastasis of hepatocellular carcinoma cell line in the key proteins involved in WNK1-OSR1/SPAK-NKCC1 pathway protein and phosphorylated protein showed a significant decreasing trend; fine high metastatic liver cancer 1 cell lines, NKCC activity of low metastatic hepatocellular carcinoma cell lines and no metastasis of hepatocellular carcinoma cell lines also showed a decreasing trend. Stimulating agent temozolomide, can significantly improve the WNK1, the OSR1 pathway, the phosphorylation level of NKCC1 protein; siRNA interference WNK1/OSR1 expression, while reducing the sodium ion the concentration of intracellular, and ultimately reduce the activity of NKCC1. The experimental results show that WNK1/OSR1 phosphorylation by regulating the activity of NKCC1, thus promoting HCC cell metastasis.NKCC1 inhibitor VNK1 or OSRl bumetanide and RNAi interference can significantly reduce the metastasis of hepatocellular carcinoma cells. Secondly, the use of bumetanide inhibits the proliferation of hepatocellular carcinoma cells has the effect, we expand the the scope of application, the other seven strains of hepatocellular carcinoma cell lines. Combined with previous research results, this paper adopts Western Blot to verify the JKC C1 protein in hepatocellular carcinoma in 7 outside Tumor cells, lung cancer cell line (A549), colorectal cancer cell line (HCT116), chronic myeloid leukemia cell line (K562), esophageal carcinoma cell line (Eca109), cervical cancer cell line (Hela), T lymphocyte leukemia cell line (Jurkat), breast cancer cell line (MCF7) expression in then, inhibition of NKCC1 inhibitor bumetanide on the seven different tissue derived tumor cell lines was determined by CCK-8 rate, and contrast sensitivity. The bumetanide in lung cancer cell lines (A549) and colorectal cancer cell lines (HCT116) inhibitory effect is relatively sensitive to the experimental data. The lowest value of IC50 for the future clinical finding of bumetanide combined liver cancer chemotherapy drug has laid a strong foundation.

【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R735.7

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