發(fā)布時間：2018-07-13 17:33

【摘要】：背景:胃癌是我國的一種常見的消化道惡性腫瘤,由于我國人口眾多且經(jīng)濟(jì)社會發(fā)展的不均衡,我國早期胃癌的就診比例比較低,而進(jìn)展期胃癌的比例很高,這嚴(yán)重降低了患者的5年生存率。目前針對進(jìn)展期胃癌的治療模式主要是D2根治術(shù)聯(lián)合術(shù)后化療。由于臨床常用的化療藥物副作用較大且作用不確定,臨床急需新的治療藥物。JS-K是一種新和成的一氧化氮(NO)前體藥,研究顯示JS-K對多種腫瘤都具有良好的抗腫瘤效應(yīng),但JS-K在胃癌中的研究尚未見諸報道。方法:在本研究中,我們通過一系列的體外試驗和體內(nèi)試驗,對JS-K在胃癌中的抗腫瘤效應(yīng)進(jìn)行評估,并對其具體的作用機(jī)制進(jìn)行探討。我們通過MTT試驗、流式細(xì)胞儀檢測技術(shù)、線粒體分離技術(shù)、目的蛋白的質(zhì)粒過表達(dá)、siRNA、Western blot以及胃癌的皮下移植瘤模型等一系列的生化檢測手段對上述問題進(jìn)行研究。結(jié)果:JS-K可以通過誘導(dǎo)細(xì)胞凋亡對胃癌細(xì)胞進(jìn)行殺傷,且殺傷效果呈時間和劑量依賴性。JS-K誘導(dǎo)胃癌細(xì)胞發(fā)生的凋亡是caspase依賴性的,泛caspase抑制劑Z-VAD-FMK可以完全阻止JS-K上述的殺傷效果,而caspase3的抑制劑Z-DEVD-FMK和caspase 9抑制劑Z-LEHD-FMK只能部分地阻止JS-K的殺傷效果。JS-K通過抑制呼吸鏈復(fù)合體1 (ComplexⅠ)和復(fù)合體Ⅳ (Complex Ⅳ)使胃癌細(xì)胞活性氧(ROS)的產(chǎn)生增多,同時又降低SOD1和Catalase這兩種活性氧(ROS)清除蛋白的含量,使活性氧(ROS)在胃癌細(xì)胞內(nèi)累積。細(xì)胞內(nèi)累積的活性氧(ROS)損傷線粒體的功能,使線粒體膜電位下降(△Ψm),細(xì)胞色素C由線粒體進(jìn)入細(xì)胞質(zhì),激活caspase 9,而活化的caspase 9又激活caspase 3和剪切PARP從而導(dǎo)致凋亡的發(fā)生。JS-K通過活性氧(ROS)使胃癌細(xì)胞發(fā)生凋亡,而活性氧清除劑N-乙酰-L-半胱氨酸(NAC)可以逆轉(zhuǎn)JS-K的上述作用。JS-K可以降低胃癌細(xì)胞內(nèi)的抗凋亡蛋白Bcl-2和Bcl-xL的含量,而通過質(zhì)粒過表達(dá)Bcl-2和Bcl-xL這兩種蛋白可以保護(hù)胃癌細(xì)胞免受JS-K的殺傷。通過小干擾RNA (siRNA)技術(shù),我們發(fā)現(xiàn)凋亡誘導(dǎo)因子(AIF)和核算內(nèi)切酶G (Endo G)的核轉(zhuǎn)位并不參與JS-K誘導(dǎo)的胃癌細(xì)胞發(fā)生的凋亡過程。在體內(nèi)試驗中,JS-K可以很好抑制胃癌皮下瘤體積和質(zhì)量的增長,但對小鼠的體重并不產(chǎn)生明顯的影響,說明其生物耐受性良好。結(jié)論:JS-K可以通過使胃癌細(xì)胞累積活性氧(ROS)而損傷線粒體,通過線粒體途徑使胃癌細(xì)胞發(fā)生caspase依賴性的凋亡,從而發(fā)揮抗腫瘤效應(yīng),且其生物耐受性良好,是一種良好的潛在的可以用于治療胃癌的藥物,值得在今后的臨床應(yīng)用和試驗中進(jìn)一步的研究。
[Abstract]:Background: gastric cancer is a common malignant tumor of digestive tract in China. Due to the large population and unbalanced economic and social development, the proportion of patients with early gastric cancer in China is low, but the proportion of advanced gastric cancer is very high. This severely reduces the 5-year survival rate. At present, the main treatment mode for advanced gastric cancer is D 2 radical resection combined with postoperative chemotherapy. Because the side effects of the commonly used chemotherapeutic drugs are large and uncertain, JS-K is a new precursor of nitric oxide (no), which is in urgent need of clinical treatment. Studies show that JS-K has a good anti-tumor effect on many kinds of tumors. However, the study of JS-K in gastric cancer has not been reported. Methods: in this study, we evaluated the antitumor effect of JS-K in gastric cancer by a series of experiments in vitro and in vivo, and discussed its mechanism. We studied the above problems by a series of biochemical methods, such as MTT test, flow cytometry, mitochondrial isolation, plasmid overexpression siRNA blot and subcutaneous transplanted tumor model of gastric cancer. Results the cell apoptosis of gastric cancer cells was induced by 1: JS-K in a time-and dose-dependent manner. The apoptosis of gastric cancer cells induced by JS-K was caspase dependent. Z-VAD-FMK, a pan-caspase inhibitor, could completely prevent the killing effect of JS-K. Z-DEVD-FMK, an inhibitor of caspase3, and Z-LEHD-FMK, an inhibitor of caspase 9, could partially prevent the cytotoxicity of JS-K. JS-K inhibited the production of reactive oxygen species (Ros) in gastric cancer cells by inhibiting the production of complex I and complex 鈪，

本文編號：2120189