E蛋白与乳腺癌耐药的相关性研究

发布时间：2018-06-25 23:07

本文选题：E蛋白 + 乳腺癌　；参考：《天津医科大学》2016年硕士论文

【摘要】：目的:E蛋白是一个新型膜蛋白,本实验室前期通过蛋白组学分析,发现E蛋白与耐药蛋白P-gp存在相互作用。本课题将在乳腺癌组织中评价E蛋白及耐药蛋白P-gp的表达,分析E蛋白与耐药蛋白P-gp以及临床病理指标的相关性。同时,构建原代乳腺癌细胞培养方法,分析原代乳腺癌细胞中E蛋白的表达及定位与药物敏感性的关系。此外,为更好的反应原代乳腺癌细胞对药物的敏感性,我们将进一步比较二维及三维培养条件下,原代乳腺癌细胞的药物敏感性情况,并分析E蛋白的表达与三维培养环境中药物敏感性的关系。方法:1、利用免疫组化方法对87例乳腺癌患者进行指标染色,检测E蛋白及耐药蛋白P-gp的表达量,并分析E蛋白蛋白表达与耐药蛋白P-gp及临床病理之间的相关性。2、构建乳腺癌原代细胞培养方法,得到纯度较高的原代乳腺癌细胞。3、采用MTT方法检测不同患者来源的原代乳腺癌细胞对化疗药物(阿霉素和顺铂)的敏感性。4、采用Western Blot方法检测不同患者来源的原代乳腺癌细胞中E蛋白的表达量,分析原代乳腺癌细胞中E蛋白的总体表达量与药物敏感性的关系。5、采用免疫荧光方法,观察对药物敏感性不同的原代乳腺癌细胞中E蛋白与耐药蛋白P-gp的表达及定位情况。6、构建Matrigel三维细胞培养方法,研究E蛋白表达水平与三维环境下药物敏感性的关系。结果:1、乳腺癌组织中,E蛋白可以表达于胞膜、胞质、胞核中,E蛋白在膜浆的表达量与耐药蛋白P-gp呈更明显的相关性。且在E蛋白膜浆高表达组中,luminal型乳腺癌所占比例较高。2、通过胶原酶消化乳腺癌组织,梯度离心法及差异消化法进一步纯化,建立原代乳腺癌细胞培养方法,成功培养原代乳腺癌细胞标本28例,其中20例获得足够数量的状态良好的细胞进行后续实验。3、对20例患者来源的原代乳腺癌细胞的总蛋白通过Western Blot方法,检测了原代乳腺癌细胞内总体E蛋白表达水平,并计算了E蛋白/GAPDH值将患者分为E蛋白高表达组和E蛋白低表达组。4、20例患者中,对16例患者来源的原代乳腺癌细胞进行了对阿霉素的药物敏感性检测,12例进行了对顺铂的药物敏感性检测,并计算出IC50值。E蛋白表达水平与药物敏感性数据结合分析显示,在对DDP的药物敏感性中,E蛋白总体水平高表达组,对顺铂的敏感性减低,耐受性增强;在对ADR的药物敏感性中,两组中无明显差异。5、免疫荧光实验发现,在相对耐药的患者中,E蛋白主要定位于胞膜上,同耐药蛋白P-gp存在明显胞膜共定位关系;在对药物相对敏感的患者中,E蛋白在胞膜表达明显减少,同耐药蛋白P-gp无明显共定位。6、三维环境中原代乳腺癌细胞对药物的敏感性降低,在对药物相对耐受的病例中,E蛋白主要表达于胞膜上,且E蛋白及耐药蛋白P-gp均高表达,反之亦然。结论:1、乳腺癌组织中,E蛋白可以表达于胞膜、胞质、胞核中,E蛋白胞膜胞浆表达量与耐药蛋白P-gp呈更明显相关性。且在膜浆高表达组中,luminal型乳腺癌所占比例较高。2、原代乳腺癌细胞中,E蛋白总体表达水平与药物敏感性无明显相关性,表达于胞膜上的E蛋白调控耐药蛋白P-gp的表达,影响细胞对药物的敏感性。3、三维培养方法更能反应原代肿瘤细胞对药物的敏感性,在三维环境中也表现出胞膜E蛋白高表达,细胞耐药性增强,进一步验证了二维培养条件下的结果。
[Abstract]:Objective: E protein is a new membrane protein. In the early stage of the laboratory, the interaction between E protein and drug resistant protein P-gp was found by proteomics analysis. This topic will evaluate the expression of E protein and drug resistant protein P-gp in breast cancer tissue, and analyze the correlation between E protein and drug resistant protein P-gp and the clinicopathological index. Meanwhile, the primary milk is constructed. In addition, in order to better respond to the sensitivity of the primary breast cancer cells to the drug sensitivity, we will further compare the drug sensitivity of the primary breast cancer cells in the two-dimensional and three-dimensional culture conditions and analyze the table of the E protein, in addition to the relationship between the expression of E protein in the primary breast cancer cells and the relationship between the localization of the protein and the drug sensitivity. The relationship between the drug sensitivity in the three-dimensional culture environment. Methods: 1, 87 cases of breast cancer were stained by immunohistochemical method, the expression of E protein and drug resistance protein P-gp were detected, and the correlation between the expression of E protein protein and drug resistant protein P-gp and the clinicopathology was analyzed, and the primary cell culture method of breast cancer was constructed. The MTT method was used to detect the sensitivity.4 of the primary breast cancer cells of different patients to chemotherapy drugs (adriamycin and cisplatin), and the Western Blot method was used to detect the expression of E protein in the primary breast cancer cells from different patients, and the overall table of E protein in the primary breast cancer cells was analyzed by using the Western Blot method to detect the.3 of the primary breast cancer cell with high purity. The relationship between dose and drug sensitivity.5, the expression and localization of E protein and drug resistant protein P-gp in primary breast cancer cells with different drug sensitivity were observed by immunofluorescence. The relationship between the expression of Matrigel and the relationship between the level of E protein expression and drug sensitivity in the three dimensional environment was studied. Results: 1, breast cancer group. In the fabric, E protein can be expressed in the membrane, cytoplasm and nucleus. The expression of E protein in the membrane pulp is more closely related to the drug resistant protein P-gp. And in the high expression group of E protein membrane pulp, the proportion of luminal type breast cancer is higher than.2, and further purified by collagenase digestion of breast cancer tissue, ladder centrifugation and differential digestion method, the original generation is established. In breast cancer cell culture, 28 cases of primary breast cancer cells were successfully cultured. 20 of them obtained sufficient numbers of good cells for follow-up experiment.3. The total protein of primary mammary cancer cells from 20 patients was detected by Western Blot method, and the expression of total E protein in the primary mammary gland cancer cells was measured. The /GAPDH value of E protein was divided into the high expression group of E protein and the low expression group of E protein in the.4,20 patients. The sensitivity of the drug sensitivity to adriamycin was detected in 16 cases of the primary breast cancer cells derived from the patients. The drug sensitivity of cisplatin was detected in 12 cases, and the expression of IC50 value.E protein was combined with the drug sensitivity data. The analysis showed that in the drug sensitivity to DDP, the high expression group of E protein was lower in sensitivity to cisplatin, and the tolerance was enhanced. In the sensitivity to ADR, there was no significant difference between the two groups. The immunofluorescence test found that in the relatively resistant patients, the E egg white was mainly located on the membrane, and there was a distinct cell with the drug resistant protein P-gp. In the patients with relatively sensitive drug, the expression of E protein decreased significantly in the cell membrane, and there was no obvious co localization of.6 with the drug resistant protein P-gp. The sensitivity of the breast cancer cells to the drug was reduced in the three-dimensional environment. In the cases of relative tolerance to the drug, the E protein was expressed on the cell membrane, and the E protein and the drug resistance protein P-gp were both high. Conclusion: 1, in breast cancer tissue, E protein can be expressed in the cell membrane, cytoplasm and nucleus. The cytoplasmic expression of E protein is more closely related to the drug resistant protein P-gp. In the high expression group, the proportion of luminal type breast cancer is higher.2. In the primary breast cancer cells, the overall expression level of E protein and drug sensitivity are in the primary breast cancer cells. No obvious correlation, the expression of E protein expressed on the cell membrane regulates the expression of drug resistant protein P-gp and affects the sensitivity of cell to drug sensitivity.3. The three-dimensional culture method is more responsive to the sensitivity of the original tumor cells to the drug. In the three-dimensional environment, the expression of E protein in the cell membrane is highly expressed and the cell resistance is enhanced. Further verification of the two-dimensional culture conditions The result.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R737.9

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