β-tubulinⅢ在宣威非小細胞肺癌中的表達與紫杉醇敏感性研究
發(fā)布時間：2018-08-05 12:44
【摘要】：[研究背景和目的]肺癌的發(fā)病率在我國惡性腫瘤中占首位,非小細胞肺癌(NSCLC)占到肺癌80%左右,近年來其發(fā)病率有增長趨勢�；熓侵委熤型砥贜SCLC主要手段,但化療客觀有效率僅21%左右,甚至更低。已有研究發(fā)現(xiàn)β-tubulinⅢ基因定位在6p21. 3,含有4個外顯子,而第4個外顯子是最容易發(fā)生突變的結合位點,其基因突變可改變微管穩(wěn)定性使紫杉醇、長春瑞濱等藥物與結合位點作用降低,導致其療效降低甚至無效。因此,探索用于確定云南宣威敏感人群、選擇適宜治療對象、減少紫杉醇類藥物耐藥的發(fā)生是目前肺癌化學治療亟待解決的一大問題。目前國內外不同研究機構報道的“腫瘤組織β-tubulinⅢ的表達與紫杉醇敏感性的關系”結果差異較大,而這種差異是否與研究目標人群的地域性差異有關目前尚不清楚。雖然我國北京、上海、廣州等個別地區(qū)進行了部分化療藥物療效相關基因的檢測研究,而這些研究機構大多局限于為當地肺癌患者的檢測,且其與肺癌臨床療效的關系報道結果差異較大。本課題主要通過免疫組化法和Western blot檢測β-tubulinⅢ蛋白在宣威肺腺癌的表達,同時通過MTT分析β-tubulinⅢ蛋白的表達對紫杉醇類藥物敏感性的影響,探索紫杉醇類藥物耐藥的靶點,從而為臨床合理使用紫杉醇類藥物提供參考依據,提出符合云南宣威地區(qū)肺癌的“個體化”綜合治療方案,對提高云南宣威地區(qū)肺癌的臨床療效無疑具有重要的社會價值和科學意義。[研究方法]1.分析β-tubulinⅢ在宣威肺腺癌細胞株與肺腺癌A549細胞株中的表達差異(1)采用免疫組織化學法檢測宣威肺腺癌與肺腺癌A549中β-tubulinⅢ的表達。(2)在200倍光學顯微鏡下選取5個不同區(qū)域計算β-tubulinⅢ在宣威肺腺癌與肺腺癌A549中表達的陽性細胞數。(3)通過Western blot檢測宣威腺肺癌與肺腺癌A549中β-tubulinⅢ蛋白的差異性表達2.分析β-tubulinⅢ在宣威肺腺癌細胞株與肺腺癌A549細胞株中的表達對紫杉醇敏感性的影響通過MTT法檢測宣威肺腺癌與肺腺癌A549對紫杉醇不同濃度隨時間遷移其細胞生物活性的影響。3.統(tǒng)計學方法采用SPSS 17.0統(tǒng)計軟件進行數據分析,進行兩組間的均數比較采用單因素方差分析及獨立樣本t檢驗,P0.05為差異有統(tǒng)計學意義。[結果]1.在免疫組化實驗中:β-tubulinⅢ微管蛋白在宣威肺腺癌細胞株中的平均表達率為95%,而β-tubulinⅢ微管蛋白在肺腺癌A549細胞株中的表達率為86%。2.在Western blot實驗中:通過Image J分析β-tubulinⅢ微管蛋白在宣威肺腺癌細胞株中表達的平均灰度值為1.196,β-tubulinⅢ微管蛋白在肺腺癌A549細胞株中表達的平均灰度值為0.912。3.在MTT試驗中,肺腺癌A549細胞株抑制率隨著紫杉醇濃度的增加而增加,當紫杉醇濃度達25ug/mL時其平均抑制率為93%,紫杉醇濃度為50ug/mL時平均抑制率為95%。在同濃度條件下,在24h、48h、72h之間相比較隨時間的推移其抑制率逐漸增加(如圖2-1所示)。宣威肺腺癌細胞株抑制率隨著紫杉醇濃度的增加而增加,當紫杉醇濃度達25ug/mL時其平均抑制率為52%,紫杉醇濃度為50ug/mL時平均抑制率為90%。在同濃度條件下,在24h、48h、72h之間相比較隨時間的推移其抑制率逐漸增加(如圖2-2所示)。在同時間同濃度調件下,紫杉醇對肺腺癌A549細胞株的平均抑制率高于宣威肺腺癌細胞株(如圖2-3至圖2-5所示)。[結論]1.β-tubulinⅢ微管蛋白在宣威肺腺癌中高表達,可能存在地域性差異。2.在β-tubulinⅢ微管蛋白高表達的宣威肺腺癌中,對紫杉醇表現(xiàn)出了耐藥性。3.β-tubulinⅢ微管蛋白表達水平與宣威肺腺癌對紫杉醇敏感性密切相關,可作為一種以紫杉醇為基礎化療藥物的療效預測因子。
[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.
【學位授予單位】：昆明醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：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.
【學位授予單位】：昆明醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R734.2
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