Src蛋白激酶抑制剂对5-氟尿嘧啶诱导的结肠癌细胞凋亡的影响及意义

发布时间：2018-07-08 15:36

本文选题：结肠癌 + src　；参考：《郑州大学》2016年硕士论文

【摘要】：背景与目的据调查表明:结肠癌是世界第三常见的恶性肿瘤,其中一半以上发生在发达国家;近年来,发展中国家结肠癌的发病率也正逐步上升。在我国,结肠癌发病率和死亡率均较前有较大升高,其中发病率已居我国恶性肿瘤的第四位,死亡率居第五位,已严重危害我国人民的生命健康。目前,结肠癌的基本治疗方法是早期手术切除,中晚期患者在手术切除的基本上,术后辅以化疗的综合性治疗方案。然而,我国结肠癌患者发现时大多已处于中晚期,肿瘤细胞局部浸润和转移,已破坏了结肠有效的粘膜屏障功能及肿瘤范围不明显,单纯的手术治疗无法切除所有肿瘤,容易复发和转移。而在手术的基础上,术前术后行化疗治疗,不但可以降低肿瘤分期分级,为手术切除创造可行性,还降低了术后复发和转移的风险,对患者无病生存期的延长有着重要意义。但临床数据表明,对于该类患者手术切除癌肿后有很高的复发率,且对化疗有较高的耐药性,特别是5-氟尿嘧啶,许多患者最终因耐药而导致死亡。然而,5-氟尿嘧啶是结肠癌化疗中的基本药物。因此,减少患者对5-氟尿嘧啶耐药的发生,降低复发和转移,对中晚期结肠癌的预后治疗效果的提高有着重要意义,是当前结肠癌化疗研究的重要环节之一。Src蛋白是由原癌基因src编码,是位于细胞膜的非受体酪氨酸蛋白激酶(nr PTKs)家族中的一员,与肿瘤的发生发展有着密切关系。据报道,Src蛋白在多种肿瘤中呈现出高表达,而在结肠癌患者中高表达率为80%。因此,近年来Src激酶抑制剂的研究已成为各种肿瘤治疗的热点。达沙替尼是一种二代Src激酶抑制剂,体外实验及动物实验研究发现其与奥沙利铂联用于结肠癌细胞具有协同作用。目前有多项一期及二期临床试验测试达沙替尼与紫杉醇,卡培他滨或奥沙利铂联用于乳腺癌,结肠癌等恶性肿瘤的治疗研究。Araujo,J.C.等人的一项最新三期临床研究结果表明:达沙替尼和紫杉醇联用于前列腺癌治疗,其疗效不改善转移性去势疗法抵抗的患者的生存期。但是,现在对于src激酶抑制剂普遍的观点是:与传统的化疗药物联用能改善患者的治疗效果。然而,Carrato,A.等人做的另外一项类似的三期临床试验却得出了相反的结果:受体酪氨酸激酶抑制剂舒尼替尼联用FOLFIRI方案不改善转移性结肠癌患者的生存期,且联用导致更严重的安全问题。De Bouard,S.等人对于舒尼替尼的报道同样可在细胞水平抑制Src激酶的活性。因此,结肠癌患者术后及化疗过程中Src激酶抑制剂与传统化疗药物联用的疗效尚不确定,有待进一步研究。研究表明:多种化疗耐药的发生与细胞凋亡的降低有关。本文基于此,通过细胞实验、MTS、Western blot等方法检测达沙替尼和5-氟尿嘧啶对结肠癌细胞内Src蛋白的活性的影响;流式细胞仪、Western blot等方法研究5-氟尿嘧啶与达沙替尼联用对比氟尿嘧啶单用对结肠癌细胞凋亡的影响;来进一步研究src激酶抑制剂与传统化疗药物联用的疗效及其影响。一、达沙替尼和5-氟尿嘧啶对结肠癌细胞内Src蛋白表达活性的影响方法1、MTS法检测达沙替尼、5-氟尿嘧啶对结肠癌细胞存活率的影响。2、Western blotting检测5-氟尿嘧啶对结肠癌细胞内Src蛋白活性的影响。3、Western blotting检测达沙替尼对结肠癌细胞内Src蛋白活性的影响。4、Western blotting检测达沙替尼与5-氟尿嘧啶联用对结肠癌细胞内Src蛋白活性的影响。结果1、达沙替尼、5-氟尿嘧啶对结肠癌细胞均能抑制细胞的存活MTS结果显示:SW480和HT29细胞用达沙替尼20n M处理48小时后,用酶标仪检测的结果得出,细胞的存活率为80%;SW480和HT29细胞用5-氟尿嘧啶50u M处理48小时后,用酶标仪检测的结果得出,细胞的存活率为50%(即SW480和HT29细胞用5-氟尿嘧啶处理的IC50的条件为50u M处理48小时)。2、5-氟尿嘧啶增强结肠癌细胞内src蛋白的活性Western blotting结果显示:用50u M浓度的5-氟尿嘧啶处理SW480和HT29细胞不同时间,代表src激酶活性的p-src的表达明显增加,且呈现出时间梯度依耐性。3、达沙替尼抑制结肠癌细胞内src蛋白的活性Western blotting结果显示:用达沙替尼作用于SW480和HT29细胞24小时,代表src激酶活性的p-src的表达明显降低,且呈现出浓度梯度依耐性;用20n M浓度的达沙替尼处理SW480和HT29细胞不同时间,代表src激酶活性的p-src的表达明显增加,且呈现出时间梯度依耐性。4、达沙替尼与5-氟尿嘧啶诱导联用抑制结肠癌细胞内src的激活Western blotting结果显示:达沙替尼与氟尿嘧啶联用降低结肠癌细胞p-src的表达。二、达沙替尼与5-氟尿嘧啶联用对结肠癌细胞的凋亡效率的影响方法1、流式细胞学方法测达沙替尼与5-氟尿嘧啶联用对结肠癌细胞凋亡的影响。2、Western blotting检测达沙替尼与5-氟尿嘧啶联用对结肠癌细胞内凋亡信号cleaved-caspase-3、cleaved-caspase-7、PARP表达的影响。结果1、达沙替尼与5-氟尿嘧啶联用降低的结肠癌细胞的凋亡流式细胞仪结果表明:用达沙替尼与5-氟尿嘧啶联用(达沙替尼20n M、5-氟尿嘧啶50u M)作用于结肠癌细胞HT29细胞,HT29细胞早期的凋亡率为7.8%;单用5-氟尿嘧啶(50u M),HT29细胞早期凋亡率为14.3%;两组相比较:联用组比5-氟尿嘧啶单用组的凋亡率明显降低,具有统计学意义(p0.05)。2、达沙替尼与5-氟尿嘧啶联用降低结肠癌细胞凋亡信号的表达Western blotting结果显示:用达沙替尼与5-氟尿嘧啶联用(达沙替尼20n M、5-氟尿嘧啶50u M)联用作用于结肠癌细胞SW480和HT29细胞48小时后,细胞内凋亡信号PARP、cleaved-caspase-3、cleaved-caspase-7的表达比单用5-氟尿嘧啶明显降低,结果提示达沙替尼与5-氟尿嘧啶联降低结肠癌细胞凋亡率。三、结肠癌患者原发癌与复发转移癌组织标本中Src蛋白的表达方法Envision法检测src蛋白在结肠癌组织中的表达结果结肠癌复发组织比原发结肠癌中src蛋白表达较低免疫组化结果显示:经免疫组化染色发现同一患者原发癌的Src蛋白表达总体高于复发癌或转移癌。提示癌症复发可能来源于Src低表达癌细胞对化疗不敏感。Src的存在亦可能在肿瘤化疗过程中起到积极作用。结论1、5-氟尿嘧啶与达沙替尼联用降低src蛋白在结肠癌细胞中的表达;2、达沙替尼与5-氟尿嘧啶联用降低结肠癌细胞的凋亡;3、Src作为原癌基因,其表达的src蛋白,在结肠癌化疗过程中起到积极作用。
[Abstract]:Background and objective investigation shows that colon cancer is the third common malignant tumor in the world, and more than half of them occur in developed countries. In recent years, the incidence of colon cancer in developing countries is increasing gradually. In China, the incidence and mortality of colon cancer are higher than before, and the incidence of colorectal cancer is fourth in China. The death rate is fifth, which has seriously jeopardize the life and health of the people of our country. At present, the basic treatment method of colon cancer is early surgical resection, the middle and late patients are basically surgical excision and combined with chemotherapy after operation. However, most of the colon cancer patients in our country have been found in the middle and late stages, and the tumor cells are locally infiltrated. Metastasis has destroyed the effective mucosal barrier function of the colon and the tumor's range is not obvious. Simple surgical treatment can not remove all the tumors and easily relapse and metastases. On the basis of surgery, chemotherapy is performed before and after operation. It can not only reduce the classification of tumor, create the feasibility for hand resection, but also reduce the recurrence and metastasis of the operation. The risk is of great significance for the prolongation of the patient's disease-free life. But clinical data show that there is a high recurrence rate and high resistance to chemotherapy for this type of cancer, especially 5- fluorouracil, and many patients eventually cause death due to resistance. However, 5- fluorouracil is the basis of chemotherapy for colon cancer. Therefore, it is of great significance to reduce the incidence of 5- fluorouracil resistance and reduce the recurrence and metastasis of the patients with intermediate and advanced colon cancer. It is one of the important links in the chemotherapy of colon cancer. The.Src protein is encoded by the proto oncogene SRC and is a non receptor tyrosine kinase (NR PTKs) located in the cell membrane. A member of the family is closely related to the development of the tumor. It is reported that Src protein is highly expressed in a variety of tumors, and the high expression rate is 80%. in colon cancer patients. Therefore, the study of Src kinase inhibitors has become a hot spot in all kinds of cancer treatment in recent years. Laboratory and animal studies have found a synergistic effect of the combination of oxaliplatin and oxaliplatin in colon cancer cells. There are a number of phase one and two clinical trials to test the treatment of dabatinib and paclitaxel, capecitabine or oxaliplatin in the treatment of breast cancer, colon cancer and other malignant tumors,.Araujo, J.C. et al. A latest phase of clinical research The results show that the combination of dasatinib and paclitaxel in the treatment of prostate cancer does not improve the survival time of patients with metastatic castration therapy. However, the common view of Src kinase inhibitors is that the combination of traditional chemotherapy drugs can improve the treatment effect of patients. However, another similar of Carrato, A. and others is similar. The three phase clinical trial yielded the opposite results: the receptor tyrosine kinase inhibitor suneinib combined with the FOLFIRI scheme did not improve the survival of patients with metastatic colon cancer, and associated with a more serious safety problem.De Bouard. S. et al. Reports on suloninib also inhibit the activity of Src kinase at the cell level. The effect of combination of Src kinase inhibitor and traditional chemotherapeutic drugs on postoperative and chemotherapy of colon cancer patients is still uncertain. Further study is needed. The study shows that the occurrence of multiple chemotherapeutic drug resistance is related to the decrease of cell apoptosis. Based on this, dasatinib and 5- fluorouracil were detected by cell experiments, MTS, Western blot and so on. The effect of Src protein activity in colon cancer cells; flow cytometry, Western blot and other methods to study the effect of 5- fluorouracil combined with dasatinib on the apoptosis of colon cancer cells in single use of fluorouracil; to further study the effect and influence of the combination of Src kinase inhibitor and traditional chemotherapeutic drugs. Methods 1, MTS assay of dasitinib, the effect of 5- fluorouracil on the survival rate of colon cancer cells.2, Western blotting detection of the effect of 5- fluorouracil on the activity of Src protein in colon cancer cells by Western blotting, and Western blotting to detect the activity of Src in colon cancer cells. The effect of.4, Western blotting on the combined use of dasatinib and 5- fluorouracil on the activity of Src protein in colon cancer cells. Results 1, dasatinib, 5- fluorouracil can inhibit the survival of colon cancer cells and the survival MTS results show: SW480 and HT29 cells with dasitinib 20n M treatment for 48 hours, the results of enzyme labeling results, The survival rate of cells was 80%. After 48 hours of treatment with 5- fluorouracil 50U M, the survival rate of SW480 and HT29 cells was 50% (i.e., SW480 and HT29 cells treated with 5- fluorouracil for 50U M 48 hours). G results showed that the expression of p-src, which represents the activity of Src kinase, increased significantly at different times of SW480 and HT29 cells treated with 5- fluorouracil at the concentration of 5- M, and showed a time gradient dependent.3. Dasitinib inhibited the activity of Src protein in colon cancer cells Western blotting results showed that dasitinib was used for 24 small cells. The expression of p-src representing the activity of Src kinase significantly decreased and showed a concentration gradient dependent manner. The expression of p-src, which represented Src kinase activity at different time of SW480 and HT29 cells with 20n M concentration, increased significantly, and showed a time gradient dependent.4. Dasatinib and 5- fluorouracil induced the inhibition of colon cancer. Intracellular Src activation Western blotting results showed that dasatinib combined with fluorouracil to reduce the expression of p-src in colon cancer cells. Two, the effect of dasatinib and 5- fluorouracil on the apoptosis efficiency of colon cancer cells 1. Flow cytometry was used to determine the effect of dasatinib and 5- fluorouracil on the apoptosis of colon cancer cells. The effects of dasatinib and 5- fluorouracil on the expression of cleaved-caspase-3, cleaved-caspase-7 and PARP in colon cancer cells were detected by.2 and Western blotting. Results 1, dasatinib combined with 5- fluorouracil combined with reduced apoptosis of colon cancer cells showed that dasitinib was combined with 5- fluorouracil (DA). Satini 20n M, 5- fluorouracil 50U M) acted on HT29 cells in colon cancer cells, the apoptosis rate of early HT29 cells was 7.8%, and the apoptotic rate of HT29 cells was 14.3% with 5- fluorouracil (50U M) alone. The two groups compared: the apoptosis rate of the combined group was significantly lower than that of the 5- fluorouracil single use group. The expression of Western blotting to reduce the apoptosis signal of colon cancer cells with pyrimidine combined with dasatinib combined with 5- fluorouracil (Dashti Ni 20n M, 5- fluorouracil 50U M) combined with SW480 and HT29 cells of colon cancer cells for 48 hours, the apoptotic signal in cell was PARP, cleaved-caspase-3, expression was compared to single use. 5- fluorouracil decreased significantly. The results suggest that dasatinib and 5- fluorouracil reduce the apoptosis rate of colon cancer cells. Three, the expression of Src protein in the primary and recurrent metastatic carcinoma tissues of colon cancer patients Envision method to detect the expression of Src protein in colon cancer tissue, the recurrence of colon cancer in colon cancer is more than the SRC egg in the primary colon cancer The results of low expression of white expression showed that the expression of Src protein in the primary cancer of the same patient was higher than that of recurrent or metastatic carcinoma in the same patient. It suggested that the recurrence of cancer may originate from the presence of Src low expression cancer cells for chemotherapy insensitive.Src and may play an active role in the tumor chemotherapy process. Conclusion 1,5- fluorouracil may be found. Combined with dasatinib to reduce the expression of Src protein in colon cancer cells; 2, dasatinib combined with 5- fluorouracil to reduce the apoptosis of colon cancer cells; 3, Src as a proto oncogene, the expression of Src protein plays an active role in the chemotherapy of colon cancer.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R735.35

【相似文献】