硒是人体内一种必需的微量元素，对身体健康和预防肿瘤具有重要作用。大量的流行病学及临床数据表明，超营养剂量的硒可以有效诱导包括结肠癌、甲状腺癌、食管癌、肝癌、前列腺癌、膀胱癌、肺癌及白血病等在内的多种肿瘤细胞发生凋亡，探究硒的抗癌分子机制成为目前研究的热点。结直肠癌是一种常见的胃肠道恶性肿瘤，在我国发病率逐年增加，治愈率低，而目前的治疗方案都存在着一定程度的局限性。因此，研究硒诱导结直肠癌细胞凋亡的分子机制可以为含硒药物的研发以及结直肠癌的治疗提供新思路和理论依据，具有重要意义。

 本实验室前期研究结果表明:超营养剂量（10μmol/L）的亚硒酸钠可以抑制多株结直肠癌细胞的增殖并诱导细胞凋亡，在移植瘤小鼠模型亚硒酸钠能够抑制肿瘤的生长，并具有诱导肿瘤组织凋亡的作用。但是关于亚硒酸钠诱导结直肠癌细胞及组织凋亡所涉及到的信号通路种类与蛋白分子机制还不完全清楚。

本实验选用结直肠癌SW480细胞和亚硒酸钠为研究对象，对亚硒酸诱导SW480细胞凋亡过程中产生变化的细胞内信号分子与调控蛋白进行了研究。发现随着硒处理时间的延长，细胞总蛋白的bcl-2表达量下降，caspase-3和cleaved  parp切割水平上升。采用DCFH-DA荧光探针法对细胞内ROS水平进行连续实时监测，发现ROS含量随着亚硒酸钠处理时间的增加而上升。结果表明亚硒酸钠确实诱导了SW480细胞的凋亡,同时也诱导了细胞内活性氧的产生，由此推测亚硒酸钠处理SW480细胞后，其凋亡可能与细胞内ROS含量有着一定的关联。

为了验证上述猜测，随后采用活性氧的清除剂MnTMPyP预处理SW480细胞，通过western blot检测发现由亚硒酸钠引起的抑凋亡蛋白bcl-2表达量下调，cleaved parp的切割水平上升均被逆转，同时通过流式细胞术检测发现由亚硒酸钠引起的细胞凋亡同样被逆转，MnTMPyP预处理的SW480细胞再加入亚硒酸钠处理后，凋亡率与双阴对照组相差无几。实验结果说明诱导SW480细胞凋亡的直接因素是ROS，是由亚硒酸钠处理SW480细胞后产生ROS而引起细胞凋亡。为了进一步探讨哪些蛋白分子参与了ROS对结直肠癌SW480细胞凋亡的调控，采用了10μmol亚硒酸钠处理SW480细胞不同时间点的总蛋白样品，进行western blot检测发现，JNK/P-ATF-2通路被抑制，三种核转录因子c-myc,c-jun,c-fos的蛋白表达量都相应降低，重要周期蛋白cyclinA2和cyclinD1表达量降低，导致了细胞凋亡增加。为了验证是否是ROS所引起的作用，随后采用活性氧的清除剂MnTMPyP预处理SW480细胞，再用亚硒酸钠处理，通过western blot检测，发现JNK/P-ATF-2的通路抑制，三种核转录因子c-myc,c-jun,c-fos的蛋白表达量降低，重要周期蛋白cyclinA2和cyclinD1表达量降低均被逆转。这些结果从细胞学和分子内蛋白水平两方面证明了ROS在亚硒酸钠诱导结直肠癌SW480细胞凋亡过程中的核心作用，但是其详细的分子机制还有待进一步深入的研究。

以上发现为筛选亚硒酸钠特异性杀伤结直肠癌等肿瘤细胞的分子靶标提供了重要的依据，也为硒对肿瘤的预防和治疗作用提供了重要的理论和实验基础。

The essential trace element, selenium, is of fundamental importance to human health and the prevention of tumors. Mounting epidemiological and clinical evidence indicate that ultra doses of selenium could effectively induce tumor cells apoptosis ,including prostate cancer, bladder cancer, thyroid cancer, esophageal cancer, liver cancer, colon cancer, lung cancer and colorectal cancer. Exploring the molecular mechanisms underlying the anti-cancer effect of selenium became a hot spot of current research. A growing number of people are diagnosed with colorectal cancer in China and the cure rate is very low. But there are all some limitations in the current treatment plan. Therefore, making a thorough inquiry into the molecular mechanisms of selenium induced colorectal cancer cell apoptosis would be of great significance for the development of drugs containing selenium and the treatment of colorectal cancer with new ideas and theoretical basis.

Our previous data show that supranutritional doses of selenite can inhibit proliferation and induce apoptosis in multiple colorectal cancer cell lines, and in xenograft mouse model, sodium selenite can inhibit tumor growth and induce apoptosis of tumor tissue. But the types of signaling pathways involved and the detailed mechanism is unclear.

In this experiment, we used the colorectal cancer cell SW480 and sodium selenite as the research object, to studied the intracellular signaling molecules and regulatory proteins which have played a role in the process of colorectal cancer cell apoptosis that induced by sodium selenite. SW480 cells were treated with supranutritional dose of selenite (10μmol/L) for different times, the expression of bcl-2 and cleaved parp ,cleaved caspase-3 were detected by western blot, we found decreased protein expression on bcl-2, the cutting level of cleaved parp and cleaved caspase-3 had risen, showed that sodium selenite did induced the apoptosis of SW480 cells, and incubated SW480 cells with ROS detection probe and continuous real-time monitoring, the results showed that the ROS content increased over time. It indicated that in the SW480 cells treated with sodium selenite, there was a certain relevance  between the apoptosis and the intracellular ROS that we unknown.

In order to verified the above speculation, SW480 cells were pretreated with MnTMPyP for 1.5 h, followed by selenite or PBS solution treatment for 24 h, total lysates were prepared and subjected to immunoblot analysis,apoptosis related proteins including cleaved parp and bcl-2 were detectd, we found the change that caused by sodium selenite about bcl-2 and cleaved parp , cleaved caspase-3 were all reversed, the result of flow cytometry assay did the same. There were not much difference between the cells pretreated with MnTMPyP and the double negative control group, it showed that the direct factor induced apoptosis in SW480 cells was ROS, rather than sodium selenite. In order to further explore which proteins have involved in the regulation of ROS on the apoptosis of colorectal cancer , SW480 cells were treated with supranutritional dose of selenite (10μmol/L) for different times, blot western detection found some signal transduction proteins involved in this process, including P-JNK, P-ATF-2, c-myc, c-jun, c-fos, cyclinD1 and cyclinA2 were all inhibitied, these changes lead to apoptosis. After this experiment, we pretreated SW480 Cells with MnTMPyP for 1.5 h, followed by selenite or PBS solution treatment for 24 h, total lysates were prepared and subjected to immunoblot analysis. The signal transduction proteins which have changed in the previous experiment were detectd. We also found the change that caused by sodium selenite about P-JNK, P-ATF-2, c-myc, c-jun, c-fos, cyclinD1 and cyclinA2 were all reversed. These results have proved the core role of ROS in the process of sodium selenite induced colon cancer cell apoptosis from two levels, cytology and protein molecules.

The above findings have provided an important theoretical and experimental basis for the screening of molecular targets about sodium selenite specific killing of colorectal cancer cells, also for the prevention and treatment of selenium on the role of cancer.