本文關(guān)鍵詞：NKCC1促進(jìn)肝癌細(xì)胞轉(zhuǎn)移的分子機(jī)制及其抑制劑用于抗腫瘤藥物的探索　出處：《安徽醫(yī)科大學(xué)》2015年碩士論文　論文類(lèi)型：學(xué)位論文

  更多相關(guān)文章： NKCC1 布美他尼 肝癌轉(zhuǎn)移

【摘要】：癌癥是一種復(fù)雜的疾病,在一個(gè)特定組織的細(xì)胞中,不再充分響應(yīng)組織內(nèi)的信號(hào)調(diào)節(jié)細(xì)胞分化、生存、增殖和死亡。癌細(xì)胞能夠無(wú)限增殖,在適宜的條件下具有“永生”能力,而無(wú)限增殖是癌細(xì)胞的主要生物學(xué)行為之一,也是癌癥患者死亡的主要原因。全球有六大常見(jiàn)癌癥,包括肺癌、肝癌、胃癌、結(jié)直腸癌、乳腺癌和食管癌,其發(fā)病率和致死率逐年增高的趨勢(shì)為我們敲響了警鐘。目前通過(guò)多種干預(yù)措施可以治療癌癥,比如外科手術(shù)、化學(xué)治療和放射治療,以及適當(dāng)?shù)男睦磔o助�；瘜W(xué)治療在癌癥治療中一直發(fā)揮著重要作用,但其治療效果卻受到劑量、毒性等因素的影響,而靶向小分子抑制劑的治療則以癌癥發(fā)生發(fā)展相關(guān)的分子為作用靶點(diǎn),將小分子類(lèi)化合物、抗體等有效成分靶向腫瘤細(xì)胞,具有選擇性的殺傷作用,能夠減少對(duì)正常組織的損傷,提高治療效果,減少毒副作用,從而達(dá)到治療或改善病人生活質(zhì)量的目的。近年來(lái),為了提高醫(yī)療衛(wèi)生水平,關(guān)于癌癥治療工作國(guó)內(nèi)外研究學(xué)者都為此付出不少心血,并取得了卓越成效,但仍極其缺乏根治手段。因此,從源頭了解癌癥發(fā)生發(fā)展的分子機(jī)制,也是今后工作的重中之重。所以,抗癌工作任重而道遠(yuǎn)。本實(shí)驗(yàn)室前期研究?jī)?nèi)容是備受關(guān)注的肝癌轉(zhuǎn)移,體外以不同轉(zhuǎn)移能力的肝癌細(xì)胞系、體內(nèi)裸鼠荷瘤模型為研究對(duì)象驗(yàn)證了NKCC1蛋白與肝癌的轉(zhuǎn)移能力正相關(guān),但是,NKCC1通過(guò)何種機(jī)制促進(jìn)肝癌細(xì)胞轉(zhuǎn)移尚未有人研究過(guò)。本研究重點(diǎn)選取遺傳背景相同、轉(zhuǎn)移能力不同的高轉(zhuǎn)移能力肝癌細(xì)胞系MHCC97H和低轉(zhuǎn)移能力肝癌細(xì)胞系MHCC97L,無(wú)轉(zhuǎn)移能力的肝癌細(xì)胞系Huh7,正常肝細(xì)胞系L02為研究對(duì)象,分別檢測(cè)了激酶WNK1/OSR1對(duì)NKCC1總蛋白、磷酸化蛋白表達(dá)的影響及其活性變化,驗(yàn)證了WNK1-OSR1/SPAK-NKCC1通路中涉及的關(guān)鍵分子的總蛋白和磷酸化蛋白質(zhì)的表達(dá)情況,結(jié)果在高轉(zhuǎn)移肝癌細(xì)胞系、低轉(zhuǎn)移肝癌細(xì)胞系和無(wú)轉(zhuǎn)移肝癌細(xì)胞系中,WNK1-OSR1/SPAK-NKCC1通路中涉及的關(guān)鍵蛋白的總蛋白和磷酸化蛋白呈明顯的依次降低的趨勢(shì)；高轉(zhuǎn)移肝癌細(xì)胞系、低轉(zhuǎn)移肝癌細(xì)胞系和無(wú)轉(zhuǎn)移肝癌細(xì)胞系的胞內(nèi)NKCC 1活性也呈依次降低的趨勢(shì)。刺激劑替莫唑胺,能夠顯著提高該通路中WNK1、OSR1、NKCC1蛋白的磷酸化水平；siRNA干擾WNK1/OSR1蛋白的表達(dá),則降低了胞內(nèi)鈉離子濃度,最終降低了NKCC1的活性。實(shí)驗(yàn)證明,WNK1/OSR1通過(guò)磷酸化調(diào)控NKCC1的活性,進(jìn)而促進(jìn)了肝癌細(xì)胞的轉(zhuǎn)移。NKCC1抑制劑布美他尼和RNAi干擾VNK1或OSRl都能顯著降低肝癌細(xì)胞的轉(zhuǎn)移能力。其次,利用布美他尼具有抑制肝癌細(xì)胞增殖的作用,我們擴(kuò)大了其應(yīng)用范圍,選取肝癌以外的其他七株腫瘤細(xì)胞系進(jìn)行研究。結(jié)合前期研究結(jié)果,本文首先采用Western Blot驗(yàn)證了JKC C1蛋白在肝癌以外的7種腫瘤細(xì)胞,即肺癌細(xì)胞系(A549),結(jié)直腸癌細(xì)胞系(HCT116),慢性髓源白血病細(xì)胞系(K562),食管癌細(xì)胞系(Eca109),宮頸癌細(xì)胞系(Hela),T淋巴細(xì)胞白血病細(xì)胞系(Jurkat),乳腺癌細(xì)胞系(MCF7)中的表達(dá)情況,接著用CCK-8法測(cè)定NKCC1抑制劑布美他尼對(duì)上述七種不同組織來(lái)源的腫瘤細(xì)胞系的抑制率,并進(jìn)行敏感度對(duì)比。其中布美他尼對(duì)肺癌細(xì)胞系(A549)和結(jié)直腸癌細(xì)胞系(HCT116)抑制效果相對(duì)較敏感,即實(shí)驗(yàn)中所測(cè)IC50值最低。這些為未來(lái)臨床上尋找布美他尼聯(lián)合肝癌化療藥物奠定了有力基礎(chǔ)。
[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.

【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：R735.7

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 Alfredo Guglielmi;rea Ruzzenente;Simone Conci;Alessro Valdegamberi;Marco Vitali;Francesca Bertuzzo;Michela De Angelis;Guido Mantovani;Calogero Iacono;;Hepatocellular carcinoma:Surgical perspectives beyond the barcelona clinic liver cancer recommendations[J];World Journal of Gastroenterology;2014年24期

，

本文編號(hào)：1371615