β-tubulinⅢ在宣威非小细胞肺癌中的表达与紫杉醇敏感性研究
发布时间：2018-08-05 12:44
【摘要】：[研究背景和目的]肺癌的发病率在我国恶性肿瘤中占首位,非小细胞肺癌(NSCLC)占到肺癌80%左右,近年来其发病率有增长趋势。化疗是治疗中晚期NSCLC主要手段,但化疗客观有效率仅21%左右,甚至更低。已有研究发现β-tubulinⅢ基因定位在6p21. 3,含有4个外显子,而第4个外显子是最容易发生突变的结合位点,其基因突变可改变微管稳定性使紫杉醇、长春瑞滨等药物与结合位点作用降低,导致其疗效降低甚至无效。因此,探索用于确定云南宣威敏感人群、选择适宜治疗对象、减少紫杉醇类药物耐药的发生是目前肺癌化学治疗亟待解决的一大问题。目前国内外不同研究机构报道的“肿瘤组织β-tubulinⅢ的表达与紫杉醇敏感性的关系”结果差异较大,而这种差异是否与研究目标人群的地域性差异有关目前尚不清楚。虽然我国北京、上海、广州等个别地区进行了部分化疗药物疗效相关基因的检测研究,而这些研究机构大多局限于为当地肺癌患者的检测,且其与肺癌临床疗效的关系报道结果差异较大。本课题主要通过免疫组化法和Western blot检测β-tubulinⅢ蛋白在宣威肺腺癌的表达,同时通过MTT分析β-tubulinⅢ蛋白的表达对紫杉醇类药物敏感性的影响,探索紫杉醇类药物耐药的靶点,从而为临床合理使用紫杉醇类药物提供参考依据,提出符合云南宣威地区肺癌的“个体化”综合治疗方案,对提高云南宣威地区肺癌的临床疗效无疑具有重要的社会价值和科学意义。[研究方法]1.分析β-tubulinⅢ在宣威肺腺癌细胞株与肺腺癌A549细胞株中的表达差异(1)采用免疫组织化学法检测宣威肺腺癌与肺腺癌A549中β-tubulinⅢ的表达。(2)在200倍光学显微镜下选取5个不同区域计算β-tubulinⅢ在宣威肺腺癌与肺腺癌A549中表达的阳性细胞数。(3)通过Western blot检测宣威腺肺癌与肺腺癌A549中β-tubulinⅢ蛋白的差异性表达2.分析β-tubulinⅢ在宣威肺腺癌细胞株与肺腺癌A549细胞株中的表达对紫杉醇敏感性的影响通过MTT法检测宣威肺腺癌与肺腺癌A549对紫杉醇不同浓度随时间迁移其细胞生物活性的影响。3.统计学方法采用SPSS 17.0统计软件进行数据分析,进行两组间的均数比较采用单因素方差分析及独立样本t检验,P0.05为差异有统计学意义。[结果]1.在免疫组化实验中:β-tubulinⅢ微管蛋白在宣威肺腺癌细胞株中的平均表达率为95%,而β-tubulinⅢ微管蛋白在肺腺癌A549细胞株中的表达率为86%。2.在Western blot实验中:通过Image J分析β-tubulinⅢ微管蛋白在宣威肺腺癌细胞株中表达的平均灰度值为1.196,β-tubulinⅢ微管蛋白在肺腺癌A549细胞株中表达的平均灰度值为0.912。3.在MTT试验中,肺腺癌A549细胞株抑制率随着紫杉醇浓度的增加而增加,当紫杉醇浓度达25ug/mL时其平均抑制率为93%,紫杉醇浓度为50ug/mL时平均抑制率为95%。在同浓度条件下,在24h、48h、72h之间相比较随时间的推移其抑制率逐渐增加(如图2-1所示)。宣威肺腺癌细胞株抑制率随着紫杉醇浓度的增加而增加,当紫杉醇浓度达25ug/mL时其平均抑制率为52%,紫杉醇浓度为50ug/mL时平均抑制率为90%。在同浓度条件下,在24h、48h、72h之间相比较随时间的推移其抑制率逐渐增加(如图2-2所示)。在同时间同浓度调件下,紫杉醇对肺腺癌A549细胞株的平均抑制率高于宣威肺腺癌细胞株(如图2-3至图2-5所示)。[结论]1.β-tubulinⅢ微管蛋白在宣威肺腺癌中高表达,可能存在地域性差异。2.在β-tubulinⅢ微管蛋白高表达的宣威肺腺癌中,对紫杉醇表现出了耐药性。3.β-tubulinⅢ微管蛋白表达水平与宣威肺腺癌对紫杉醇敏感性密切相关,可作为一种以紫杉醇为基础化疗药物的疗效预测因子。
[Abstract]:[background and objective] the incidence of lung cancer is the first in China's malignant tumor. Non small cell lung cancer (NSCLC) accounts for about 80% of lung cancer. In recent years, the incidence of lung cancer has been increasing. Chemotherapy is the main method for the treatment of middle and late NSCLC, but the objective and effective rate of chemotherapy is only about 21% and even lower. The study found that the gene of beta -tubulin III is located in 6p 21.3, there are 4 exons, and fourth exons are the most likely mutation binding sites. The mutation of the gene can change the stability of microtubules to reduce the effect of taxol and Changchun Rui litbin and other drugs and binding sites, leading to the reduction of the efficacy and even ineffectiveness. Therefore, the exploration is used to determine the sensitive population of Yunnan Xuanwei, and to select the suitable treatment target. The occurrence of drug resistance of fewer paclitaxel is a major problem to be solved urgently at present. The results of the relationship between the expression of beta -tubulin III in tumor tissue and the sensitivity of taxol in different research institutions at home and abroad differ greatly, and whether this difference is related to the regional difference of the target population is still concerned. It is not clear. Although some regions of China, such as Beijing, Shanghai and Guangzhou, have carried out the detection and study of some genes related to the therapeutic effect of chemotherapeutic drugs, most of these research institutions are limited to the detection of local lung cancer patients, and their relationship with the clinical efficacy of lung cancer is very different. This subject is mainly through immunohistochemical method and Western blot The expression of beta -tubulin III protein in the lung adenocarcinoma of Xuanwei was detected, and the effect of the expression of beta -tubulin III protein on the sensitivity of taxol drugs was analyzed by MTT, and the target of taxol drug resistance was explored, thus providing a reference for the rational use of taxol drugs in clinical practice, and the individualization of lung cancer in Xuanwei area of Yunnan was proposed. "The comprehensive treatment scheme is of great social and scientific significance to improve the clinical efficacy of lung cancer in Xuanwei area of Yunnan. [research method]1. analyses the difference between the expression of beta -tubulin III in the lung adenocarcinoma cell lines of Xuanwei and the A549 cell line of lung adenocarcinoma (1) the detection of beta -tu in Xuanwei lung adenocarcinoma and lung adenocarcinoma A549 by immunohistochemistry Expression of Bulin III. (2) the number of positive cells expressed by beta -tubulin III in Xuanwei lung adenocarcinoma and lung adenocarcinoma A549 was calculated under 200 times optical microscope. (3) the difference of 2. analysis of beta -tubulin III protein in Xuanwei adenocarcinoma and lung adenocarcinoma A549 by Western blot was reached to 2. analysis of beta -tubulin III in Xuanwei lung adenocarcinoma cell line The effect of expression in A549 cell line with lung adenocarcinoma cell line on Taxol sensitivity and the effect of MTT method to detect the effect of different concentrations of paclitaxel on the cell bioactivity of lung adenocarcinoma and lung adenocarcinoma A549,.3. statistical method was used to analyze the data of SPSS 17 statistical software, and the average number of the two groups was compared by single factor. The difference was statistically significant in the difference analysis and independent sample t test. [results]1. in immunohistochemical experiment: the average expression rate of beta -tubulin III microtubulin in Xuanwei lung adenocarcinoma cell line was 95%, while the expression rate of beta -tubulin III microtubulin in A549 cell line of lung adenocarcinoma was 86%.2. in Western blot experiment: Image J score. The average gray value of the expression of beta -tubulin III microtubule in lung adenocarcinoma cell lines was 1.196. The average gray value of the expression of beta -tubulin III microtubulin in A549 cell lines of lung adenocarcinoma was 0.912.3. in the MTT test. The inhibition rate of A549 cell lines in lung adenocarcinoma increased with the increase of paclitaxel concentration. When paclitaxel concentration reached 25ug/mL, the concentration of paclitaxel was 25ug/mL The average inhibitory rate was 93% and the average inhibitory rate of paclitaxel was 50ug/mL at the same concentration of 95%. at the same concentration. The inhibition rate of 24h, 48h and 72h increased gradually with time (as shown in Figure 2-1). The inhibitory rate of the lung adenocarcinoma cell line of Xuanwei increased with the increase of paclitaxel concentration, and the average of paclitaxel concentration reached 25ug/mL. The inhibition rate was 52% and the average inhibitory rate of paclitaxel concentration was 50ug/mL was 90%. at the same concentration. The inhibition rate of 24h, 48h, 72h was gradually increased with time (as shown in Figure 2-2). The average inhibition rate of paclitaxel to lung adenocarcinoma cell line in the same time same concentration was higher than that of Xuanwei lung adenocarcinoma cell line (G. 2- (3 to 2-5) [Conclusion] [conclusion]1. beta -tubulin III microtubulin is highly expressed in Xuanwei lung adenocarcinoma, and there may be regional difference in.2. in the high expression of beta -tubulin III microtubulin in lung adenocarcinoma. The expression of.3. beta -tubulin III microtubulin resistance to paclitaxel is closely related to the sensitivity of Xuanwei lung adenocarcinoma to Taxol sensitivity. It can be used as a predictive factor for the efficacy of paclitaxel based chemotherapy drugs.
【学位授予单位】：昆明医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R734.2
[Abstract]:[background and objective] the incidence of lung cancer is the first in China's malignant tumor. Non small cell lung cancer (NSCLC) accounts for about 80% of lung cancer. In recent years, the incidence of lung cancer has been increasing. Chemotherapy is the main method for the treatment of middle and late NSCLC, but the objective and effective rate of chemotherapy is only about 21% and even lower. The study found that the gene of beta -tubulin III is located in 6p 21.3, there are 4 exons, and fourth exons are the most likely mutation binding sites. The mutation of the gene can change the stability of microtubules to reduce the effect of taxol and Changchun Rui litbin and other drugs and binding sites, leading to the reduction of the efficacy and even ineffectiveness. Therefore, the exploration is used to determine the sensitive population of Yunnan Xuanwei, and to select the suitable treatment target. The occurrence of drug resistance of fewer paclitaxel is a major problem to be solved urgently at present. The results of the relationship between the expression of beta -tubulin III in tumor tissue and the sensitivity of taxol in different research institutions at home and abroad differ greatly, and whether this difference is related to the regional difference of the target population is still concerned. It is not clear. Although some regions of China, such as Beijing, Shanghai and Guangzhou, have carried out the detection and study of some genes related to the therapeutic effect of chemotherapeutic drugs, most of these research institutions are limited to the detection of local lung cancer patients, and their relationship with the clinical efficacy of lung cancer is very different. This subject is mainly through immunohistochemical method and Western blot The expression of beta -tubulin III protein in the lung adenocarcinoma of Xuanwei was detected, and the effect of the expression of beta -tubulin III protein on the sensitivity of taxol drugs was analyzed by MTT, and the target of taxol drug resistance was explored, thus providing a reference for the rational use of taxol drugs in clinical practice, and the individualization of lung cancer in Xuanwei area of Yunnan was proposed. "The comprehensive treatment scheme is of great social and scientific significance to improve the clinical efficacy of lung cancer in Xuanwei area of Yunnan. [research method]1. analyses the difference between the expression of beta -tubulin III in the lung adenocarcinoma cell lines of Xuanwei and the A549 cell line of lung adenocarcinoma (1) the detection of beta -tu in Xuanwei lung adenocarcinoma and lung adenocarcinoma A549 by immunohistochemistry Expression of Bulin III. (2) the number of positive cells expressed by beta -tubulin III in Xuanwei lung adenocarcinoma and lung adenocarcinoma A549 was calculated under 200 times optical microscope. (3) the difference of 2. analysis of beta -tubulin III protein in Xuanwei adenocarcinoma and lung adenocarcinoma A549 by Western blot was reached to 2. analysis of beta -tubulin III in Xuanwei lung adenocarcinoma cell line The effect of expression in A549 cell line with lung adenocarcinoma cell line on Taxol sensitivity and the effect of MTT method to detect the effect of different concentrations of paclitaxel on the cell bioactivity of lung adenocarcinoma and lung adenocarcinoma A549,.3. statistical method was used to analyze the data of SPSS 17 statistical software, and the average number of the two groups was compared by single factor. The difference was statistically significant in the difference analysis and independent sample t test. [results]1. in immunohistochemical experiment: the average expression rate of beta -tubulin III microtubulin in Xuanwei lung adenocarcinoma cell line was 95%, while the expression rate of beta -tubulin III microtubulin in A549 cell line of lung adenocarcinoma was 86%.2. in Western blot experiment: Image J score. The average gray value of the expression of beta -tubulin III microtubule in lung adenocarcinoma cell lines was 1.196. The average gray value of the expression of beta -tubulin III microtubulin in A549 cell lines of lung adenocarcinoma was 0.912.3. in the MTT test. The inhibition rate of A549 cell lines in lung adenocarcinoma increased with the increase of paclitaxel concentration. When paclitaxel concentration reached 25ug/mL, the concentration of paclitaxel was 25ug/mL The average inhibitory rate was 93% and the average inhibitory rate of paclitaxel was 50ug/mL at the same concentration of 95%. at the same concentration. The inhibition rate of 24h, 48h and 72h increased gradually with time (as shown in Figure 2-1). The inhibitory rate of the lung adenocarcinoma cell line of Xuanwei increased with the increase of paclitaxel concentration, and the average of paclitaxel concentration reached 25ug/mL. The inhibition rate was 52% and the average inhibitory rate of paclitaxel concentration was 50ug/mL was 90%. at the same concentration. The inhibition rate of 24h, 48h, 72h was gradually increased with time (as shown in Figure 2-2). The average inhibition rate of paclitaxel to lung adenocarcinoma cell line in the same time same concentration was higher than that of Xuanwei lung adenocarcinoma cell line (G. 2- (3 to 2-5) [Conclusion] [conclusion]1. beta -tubulin III microtubulin is highly expressed in Xuanwei lung adenocarcinoma, and there may be regional difference in.2. in the high expression of beta -tubulin III microtubulin in lung adenocarcinoma. The expression of.3. beta -tubulin III microtubulin resistance to paclitaxel is closely related to the sensitivity of Xuanwei lung adenocarcinoma to Taxol sensitivity. It can be used as a predictive factor for the efficacy of paclitaxel based chemotherapy drugs.
【学位授予单位】：昆明医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R734.2
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4 李鹏，
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