激活素A在人大肠癌组织中的表达及其作用研究

发布时间：2018-06-22 13:53

本文选题：激活素A大肠癌 + 肿瘤标记物　；参考：《吉林大学》2016年博士论文

【摘要】：大肠癌是临床上常见的恶性肿瘤之一,其发病率逐年上升。但目前对大肠癌的早期诊断及治疗效果依然不甚理想。临床上,急需更为有效的方法提高大肠癌的诊断效率及治疗效果,使大肠癌患者从中获益。激活素A具有多种生物学活性,包括参与多种肿瘤的发生、发展;尽管已有部分研究表明激活素A在大肠癌组织表达,但关于大肠癌患者血清激活素A水平、激活素A对大肠癌细胞作用的信号转导机制等仍未明确。因此,本研究检测了健康对照者、大肠良性息肉及大肠癌患者血清激活素A水平,结果发现大肠癌患者血清激活素A水平较健康对照者及大肠良性息肉患者血清激活素A水平明显升高,并且激活素A水平与大肠癌分期存在一定关联。RT-PCR、Western blotting及免疫组织化学染色等结果确认了激活素A是由大肠癌细胞分泌,提示激活素A可能成为辅助诊断大肠癌的新的肿瘤标记物。本研究又通过吉姆萨染色及流式细胞术等方法检测激活素A对大肠癌细胞系SW480细胞的作用,发现激活素A可以诱导SW480细胞凋亡。另外,本研究还通过基因敲低及过表达等方法证明激活素A通过Smads信号传导途径诱导SW480细胞凋亡。一、大肠癌患者血清及组织中激活素A水平变化1.健康对照者、大肠良性息肉患者及大肠癌患者血清激活素A水平应用ELISA试剂盒检测健康对照者、大肠良性息肉患者及大肠癌患者血清激活素A水平,结果发现:大肠癌患者血清激活素A水平较健康对照者及大肠良性息肉患者血清激活素A水平明显升高;大肠癌术后患者血清激活素A水平较术前明显下降,提示大肠癌患者血清激活素A来源于大肠癌组织。2.激活素A在大肠癌组织中的表达应用RT-PCR及Western blotting检测激活素A m RNA及蛋白在大肠癌组织中的表达。结果显示,大肠癌组织可高表达激活素A。免疫组织化学染色进一步表明大肠癌细胞高表达激活素A。3.血清激活素A水平与大肠癌分期的关系大肠癌患者血清激活素A水平与其分期相关,即I期大肠癌患者血清激活素A水平明显低于II期、III期大肠癌患者,提示血清激活素A水平与大肠癌的术前分期有关。4.血清激活素A与癌胚抗原的比较与常用的大肠癌肿瘤标记物癌胚抗原(CEA)相比,激活素A具有更高的特异性,故在临床上,联合应用CEA及血清激活素A可能提高大肠癌诊断的准确性。通过以上结果确认了激活素A在大肠癌患者血清及组织中的表达,并提示激活素A可能成为辅助诊断大肠癌的肿瘤标记物。二、激活素A诱导大肠癌细胞系SW480发生凋亡的作用1.激活素A在大肠癌细胞系SW480中的表达应用RT-PCR及Western blotting检测大肠癌细胞系SW480细胞激活素A表达,结果发现SW480细胞可表达激活素A。与前述实验结果相符。2.激活素A诱导SW480细胞凋亡实验采用MTT法测定细胞活力,结果提示:在外源性激活素A作用下,大肠癌细胞系SW480细胞活力减弱。应用吉姆萨染色检测激活素A对SW480细胞的作用。结果发现:SW480细胞发生核固缩、凋亡小体等。提示激活素A可诱导SW480大肠癌细胞发生凋亡。进一步应用流式细胞术检测激活素A对SW480的凋亡作用。结果显示:激活素A可诱导SW480大肠癌细胞发生凋亡,且呈时间及浓度依赖性。3.激活素受体及Smad3在SW480细胞中的表达Smads途径为经典的激活素A信号转导通路,本实应用RT-PCR及Western blotting检测了激活素A受体及Smad3在SW480细胞中的表达。结果发现:激活素A处理的SW480细胞Smads通路的关键蛋白Smad3及Act RIIA与Act RIIB均升高,且呈浓度依赖性。提示激活素A可能通过Smads信号传导途径诱导SW480大肠癌细胞凋亡。三、Smad3基因敲低减弱激活素A诱导的SW480细胞凋亡作用SW480细胞转染Smad3 sh RNA表达质粒敲低Smad3基因表达,再应用流式细胞术检测激活素A诱导SW480大肠癌细胞的凋亡作用。结果显示,Smad3基因敲低后激活素A诱导SW480细胞凋亡明显减少。该结果进一步表明Smad3信号介导了激活素A诱导SW480大肠癌细胞凋亡作用。四、Smad3基因过表达对激活素A诱导SW480细胞凋亡作用的增强SW480细胞转染Smad3表达质粒,再应用流式细胞术检测激活素A诱导SW480大肠癌细胞的凋亡作用。结果显示,Smad3基因过表达后激活素A诱导SW480细胞凋亡明显增加。该结果进一步确定Smad3信号介导了激活素A诱导SW480大肠癌细胞凋亡作用。结论:1.研究发现大肠癌患者血清激活素A水平升高,表明其可以作为大肠癌肿瘤标记物用于临床辅助诊断。2.研究表明激活素A用于大肠癌辅助诊断其特异性明显高于CEA,更具有早期临床样本筛查价值。3.研究证实激活素A可诱导大肠癌SW480细胞凋亡,其作用与Smads信号传导途径有关,提示激活素A也具有治疗大肠癌的潜能。
[Abstract]:Colorectal cancer is one of the most common malignant tumors in the clinic, its incidence is increasing year by year. But the early diagnosis and treatment effect of colorectal cancer is still not ideal. In clinical, it is urgent to improve the diagnosis efficiency and treatment effect of colorectal cancer and make the patients with large intestine cancer benefit from it. The activin A has many biological activities. It is involved in the occurrence and development of a variety of tumors. Although some studies have shown that activin A is expressed in colorectal carcinoma, the level of serum activin A in colorectal cancer patients and the signal transduction mechanism of activin A on colorectal cancer cells are still not clear. Therefore, this study has been used to detect healthy controls, colorectal polyps and colorectal cancer patients. Serum activin A levels showed that serum activin A levels in patients with colorectal cancer were significantly higher than those of healthy controls and patients with benign colorectal polyps, and the levels of activin A were associated with.RT-PCR, Western blotting, and immunohistochemical staining, which confirmed that activin A is a large amount of A. The secretion of colon cancer cells suggests that activin A may be a new tumor marker to assist in the diagnosis of colorectal cancer. This study also detected the effect of activin A on SW480 cells in colorectal cancer cell lines by means of Giemsa staining and flow cytometry, and found that activin A can induce apoptosis of SW480 cells. The expression of activin A induced SW480 cell apoptosis through Smads signal transduction pathway. 1. Changes in serum and tissue activin A level in colorectal cancer patients 1. healthy controls, benign colorectal polyps and colorectal cancer patients serum activin A level using ELISA kit to detect healthy controls, patients with colorectal polyps and The serum activin A level in colorectal cancer patients showed that the serum activin A level of the patients with colorectal cancer was significantly higher than that of the healthy controls and the patients with benign colorectal polyps. The serum activin A level of the patients with colorectal cancer was significantly lower than that before the operation, suggesting that the serum activin A of the patients with colorectal cancer was derived from the.2. of the colorectal carcinoma tissue. The expression of activin A in colorectal carcinoma tissue using RT-PCR and Western blotting to detect the expression of activin A m RNA and protein in colorectal cancer tissue. The results show that the high expression of activin A. in colorectal cancer tissues is a further indication of the high expression activin A.3. serum activin A level in colorectal cancer cells and the score of colorectal cancer. Serum activin A level in patients with colorectal cancer is related to its stages, that is, serum activin A level in patients with stage I colorectal cancer is significantly lower than phase II, III stage colorectal cancer patients, suggesting that serum activin A level is related to the preoperative staging of colorectal cancer, which is related to the comparison of.4. serum activin A and carcinoembryonic antigen, and the common cancer markers for colorectal cancer. Compared with the antigen (CEA), activin A has a higher specificity. Therefore, the combination of CEA and serum activin A may improve the accuracy of the diagnosis of colorectal cancer. The expression of activin A in the serum and tissue of patients with colorectal cancer is confirmed by the above results, and the activin A may be a marker for the diagnosis of colorectal cancer. Two, activin A induced the apoptosis of colorectal cancer cell line SW480, 1. activin A expression in colorectal cancer cell line SW480, RT-PCR and Western blotting were used to detect the A expression of SW480 cell activin in colorectal cancer cell lines. The results showed that SW480 cells expressed activin A. consistent with the experimental results. MTT assay was used to determine cell viability. The results showed that the activity of SW480 cells in the colorectal cancer cell line was weakened under the effect of exogenous activin A. The effect of activin A on SW480 cells was detected by Giemsa staining. The results showed that SW480 cells had nuclear condensation and apoptotic bodies. It suggested that activin A can induce SW480 colorectal cancer cells to occur. Apoptosis. Further application of flow cytometry to detect the apoptotic effect of activin A on SW480. The results show that activin A can induce apoptosis in SW480 colorectal cancer cells, and the time and concentration dependent.3. activin receptor and the expression of Smad3 in SW480 cells are the classical activin A signal transduction pathway. This is a real application of RT-PCR and We. Stern blotting detected the expression of activin A receptor and Smad3 in SW480 cells. The results showed that the key protein Smad3 and Act RIIA and Act Act increased in the SW480 cell Smads pathway of activin A, and showed a concentration dependence. Smad3 sh RNA expression plasmid was transfected with Smad3 sh RNA expression plasmid to knock down the expression of Smad3 gene by knocking down the apoptosis induced by activin A, and then using flow cytometry to detect the apoptosis effect of activin A induced SW480 colorectal cancer cells. The results showed that the apoptosis of A inducement SW480 cells after the Smad3 gene knockdown was significantly reduced. The step indicated that the Smad3 signal mediated the apoptosis of SW480 colorectal cancer cells induced by activin A. Four, Smad3 gene overexpressed the apoptosis of SW480 cells induced by activin A, SW480 cells transfected with Smad3 expression plasmid, and then using flow cytometry to detect the apoptosis effect of activin A inducing SW480 colon cancer cells. The results showed that Smad3 gene was over. The expression of activin A induces a significant increase in apoptosis of SW480 cells. The results further confirm that the Smad3 signal mediates the apoptosis of SW480 colorectal cancer cells induced by activin A. Conclusion: 1. the increase of serum activin A level in colorectal cancer patients shows that it can be used as a tumor marker for colorectal cancer to be used in clinical auxiliary diagnosis of.2. research. Activin A is used in the diagnosis of colorectal cancer, its specificity is significantly higher than that of CEA, and it has a more early clinical sample screening value.3. study confirmed that activin A can induce apoptosis of SW480 cells in colorectal cancer, which is related to the Smads signal transduction pathway, suggesting that activin A also has the potential for the treatment of colorectal cancer.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.34

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