本文選題：激活素A大腸癌 + 腫瘤標記物��； 參考：《吉林大學》2016年博士論文

【摘要】：大腸癌是臨床上常見的惡性腫瘤之一,其發(fā)病率逐年上升。但目前對大腸癌的早期診斷及治療效果依然不甚理想。臨床上,急需更為有效的方法提高大腸癌的診斷效率及治療效果,使大腸癌患者從中獲益。激活素A具有多種生物學活性,包括參與多種腫瘤的發(fā)生、發(fā)展;盡管已有部分研究表明激活素A在大腸癌組織表達,但關(guān)于大腸癌患者血清激活素A水平、激活素A對大腸癌細胞作用的信號轉(zhuǎn)導機制等仍未明確。因此,本研究檢測了健康對照者、大腸良性息肉及大腸癌患者血清激活素A水平,結(jié)果發(fā)現(xiàn)大腸癌患者血清激活素A水平較健康對照者及大腸良性息肉患者血清激活素A水平明顯升高,并且激活素A水平與大腸癌分期存在一定關(guān)聯(lián)。RT-PCR、Western blotting及免疫組織化學染色等結(jié)果確認了激活素A是由大腸癌細胞分泌,提示激活素A可能成為輔助診斷大腸癌的新的腫瘤標記物。本研究又通過吉姆薩染色及流式細胞術(shù)等方法檢測激活素A對大腸癌細胞系SW480細胞的作用,發(fā)現(xiàn)激活素A可以誘導SW480細胞凋亡。另外,本研究還通過基因敲低及過表達等方法證明激活素A通過Smads信號傳導途徑誘導SW480細胞凋亡。一、大腸癌患者血清及組織中激活素A水平變化1.健康對照者、大腸良性息肉患者及大腸癌患者血清激活素A水平應(yīng)用ELISA試劑盒檢測健康對照者、大腸良性息肉患者及大腸癌患者血清激活素A水平,結(jié)果發(fā)現(xiàn):大腸癌患者血清激活素A水平較健康對照者及大腸良性息肉患者血清激活素A水平明顯升高;大腸癌術(shù)后患者血清激活素A水平較術(shù)前明顯下降,提示大腸癌患者血清激活素A來源于大腸癌組織。2.激活素A在大腸癌組織中的表達應(yīng)用RT-PCR及Western blotting檢測激活素A m RNA及蛋白在大腸癌組織中的表達。結(jié)果顯示,大腸癌組織可高表達激活素A。免疫組織化學染色進一步表明大腸癌細胞高表達激活素A。3.血清激活素A水平與大腸癌分期的關(guān)系大腸癌患者血清激活素A水平與其分期相關(guān),即I期大腸癌患者血清激活素A水平明顯低于II期、III期大腸癌患者,提示血清激活素A水平與大腸癌的術(shù)前分期有關(guān)。4.血清激活素A與癌胚抗原的比較與常用的大腸癌腫瘤標記物癌胚抗原(CEA)相比,激活素A具有更高的特異性,故在臨床上,聯(lián)合應(yīng)用CEA及血清激活素A可能提高大腸癌診斷的準確性。通過以上結(jié)果確認了激活素A在大腸癌患者血清及組織中的表達,并提示激活素A可能成為輔助診斷大腸癌的腫瘤標記物。二、激活素A誘導大腸癌細胞系SW480發(fā)生凋亡的作用1.激活素A在大腸癌細胞系SW480中的表達應(yīng)用RT-PCR及Western blotting檢測大腸癌細胞系SW480細胞激活素A表達,結(jié)果發(fā)現(xiàn)SW480細胞可表達激活素A。與前述實驗結(jié)果相符。2.激活素A誘導SW480細胞凋亡實驗采用MTT法測定細胞活力,結(jié)果提示:在外源性激活素A作用下,大腸癌細胞系SW480細胞活力減弱。應(yīng)用吉姆薩染色檢測激活素A對SW480細胞的作用。結(jié)果發(fā)現(xiàn):SW480細胞發(fā)生核固縮、凋亡小體等。提示激活素A可誘導SW480大腸癌細胞發(fā)生凋亡。進一步應(yīng)用流式細胞術(shù)檢測激活素A對SW480的凋亡作用。結(jié)果顯示:激活素A可誘導SW480大腸癌細胞發(fā)生凋亡,且呈時間及濃度依賴性。3.激活素受體及Smad3在SW480細胞中的表達Smads途徑為經(jīng)典的激活素A信號轉(zhuǎn)導通路,本實應(yīng)用RT-PCR及Western blotting檢測了激活素A受體及Smad3在SW480細胞中的表達。結(jié)果發(fā)現(xiàn):激活素A處理的SW480細胞Smads通路的關(guān)鍵蛋白Smad3及Act RIIA與Act RIIB均升高,且呈濃度依賴性。提示激活素A可能通過Smads信號傳導途徑誘導SW480大腸癌細胞凋亡。三、Smad3基因敲低減弱激活素A誘導的SW480細胞凋亡作用SW480細胞轉(zhuǎn)染Smad3 sh RNA表達質(zhì)粒敲低Smad3基因表達,再應(yīng)用流式細胞術(shù)檢測激活素A誘導SW480大腸癌細胞的凋亡作用。結(jié)果顯示,Smad3基因敲低后激活素A誘導SW480細胞凋亡明顯減少。該結(jié)果進一步表明Smad3信號介導了激活素A誘導SW480大腸癌細胞凋亡作用。四、Smad3基因過表達對激活素A誘導SW480細胞凋亡作用的增強SW480細胞轉(zhuǎn)染Smad3表達質(zhì)粒,再應(yīng)用流式細胞術(shù)檢測激活素A誘導SW480大腸癌細胞的凋亡作用。結(jié)果顯示,Smad3基因過表達后激活素A誘導SW480細胞凋亡明顯增加。該結(jié)果進一步確定Smad3信號介導了激活素A誘導SW480大腸癌細胞凋亡作用。結(jié)論:1.研究發(fā)現(xiàn)大腸癌患者血清激活素A水平升高,表明其可以作為大腸癌腫瘤標記物用于臨床輔助診斷。2.研究表明激活素A用于大腸癌輔助診斷其特異性明顯高于CEA,更具有早期臨床樣本篩查價值。3.研究證實激活素A可誘導大腸癌SW480細胞凋亡,其作用與Smads信號傳導途徑有關(guān),提示激活素A也具有治療大腸癌的潛能。
[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.
【學位授予單位】：吉林大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R735.34

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