本文選題：E蛋白 + 乳腺癌��； 參考：《天津醫(yī)科大學(xué)》2016年碩士論文

【摘要】：目的:E蛋白是一個(gè)新型膜蛋白,本實(shí)驗(yàn)室前期通過蛋白組學(xué)分析,發(fā)現(xiàn)E蛋白與耐藥蛋白P-gp存在相互作用。本課題將在乳腺癌組織中評(píng)價(jià)E蛋白及耐藥蛋白P-gp的表達(dá),分析E蛋白與耐藥蛋白P-gp以及臨床病理指標(biāo)的相關(guān)性。同時(shí),構(gòu)建原代乳腺癌細(xì)胞培養(yǎng)方法,分析原代乳腺癌細(xì)胞中E蛋白的表達(dá)及定位與藥物敏感性的關(guān)系。此外,為更好的反應(yīng)原代乳腺癌細(xì)胞對(duì)藥物的敏感性,我們將進(jìn)一步比較二維及三維培養(yǎng)條件下,原代乳腺癌細(xì)胞的藥物敏感性情況,并分析E蛋白的表達(dá)與三維培養(yǎng)環(huán)境中藥物敏感性的關(guān)系。方法:1、利用免疫組化方法對(duì)87例乳腺癌患者進(jìn)行指標(biāo)染色,檢測(cè)E蛋白及耐藥蛋白P-gp的表達(dá)量,并分析E蛋白蛋白表達(dá)與耐藥蛋白P-gp及臨床病理之間的相關(guān)性。2、構(gòu)建乳腺癌原代細(xì)胞培養(yǎng)方法,得到純度較高的原代乳腺癌細(xì)胞。3、采用MTT方法檢測(cè)不同患者來源的原代乳腺癌細(xì)胞對(duì)化療藥物(阿霉素和順鉑)的敏感性。4、采用Western Blot方法檢測(cè)不同患者來源的原代乳腺癌細(xì)胞中E蛋白的表達(dá)量,分析原代乳腺癌細(xì)胞中E蛋白的總體表達(dá)量與藥物敏感性的關(guān)系。5、采用免疫熒光方法,觀察對(duì)藥物敏感性不同的原代乳腺癌細(xì)胞中E蛋白與耐藥蛋白P-gp的表達(dá)及定位情況。6、構(gòu)建Matrigel三維細(xì)胞培養(yǎng)方法,研究E蛋白表達(dá)水平與三維環(huán)境下藥物敏感性的關(guān)系。結(jié)果:1、乳腺癌組織中,E蛋白可以表達(dá)于胞膜、胞質(zhì)、胞核中,E蛋白在膜漿的表達(dá)量與耐藥蛋白P-gp呈更明顯的相關(guān)性。且在E蛋白膜漿高表達(dá)組中,luminal型乳腺癌所占比例較高。2、通過膠原酶消化乳腺癌組織,梯度離心法及差異消化法進(jìn)一步純化,建立原代乳腺癌細(xì)胞培養(yǎng)方法,成功培養(yǎng)原代乳腺癌細(xì)胞標(biāo)本28例,其中20例獲得足夠數(shù)量的狀態(tài)良好的細(xì)胞進(jìn)行后續(xù)實(shí)驗(yàn)。3、對(duì)20例患者來源的原代乳腺癌細(xì)胞的總蛋白通過Western Blot方法,檢測(cè)了原代乳腺癌細(xì)胞內(nèi)總體E蛋白表達(dá)水平,并計(jì)算了E蛋白/GAPDH值將患者分為E蛋白高表達(dá)組和E蛋白低表達(dá)組。4、20例患者中,對(duì)16例患者來源的原代乳腺癌細(xì)胞進(jìn)行了對(duì)阿霉素的藥物敏感性檢測(cè),12例進(jìn)行了對(duì)順鉑的藥物敏感性檢測(cè),并計(jì)算出IC50值。E蛋白表達(dá)水平與藥物敏感性數(shù)據(jù)結(jié)合分析顯示,在對(duì)DDP的藥物敏感性中,E蛋白總體水平高表達(dá)組,對(duì)順鉑的敏感性減低,耐受性增強(qiáng);在對(duì)ADR的藥物敏感性中,兩組中無明顯差異。5、免疫熒光實(shí)驗(yàn)發(fā)現(xiàn),在相對(duì)耐藥的患者中,E蛋白主要定位于胞膜上,同耐藥蛋白P-gp存在明顯胞膜共定位關(guān)系;在對(duì)藥物相對(duì)敏感的患者中,E蛋白在胞膜表達(dá)明顯減少,同耐藥蛋白P-gp無明顯共定位。6、三維環(huán)境中原代乳腺癌細(xì)胞對(duì)藥物的敏感性降低,在對(duì)藥物相對(duì)耐受的病例中,E蛋白主要表達(dá)于胞膜上,且E蛋白及耐藥蛋白P-gp均高表達(dá),反之亦然。結(jié)論:1、乳腺癌組織中,E蛋白可以表達(dá)于胞膜、胞質(zhì)、胞核中,E蛋白胞膜胞漿表達(dá)量與耐藥蛋白P-gp呈更明顯相關(guān)性。且在膜漿高表達(dá)組中,luminal型乳腺癌所占比例較高。2、原代乳腺癌細(xì)胞中,E蛋白總體表達(dá)水平與藥物敏感性無明顯相關(guān)性,表達(dá)于胞膜上的E蛋白調(diào)控耐藥蛋白P-gp的表達(dá),影響細(xì)胞對(duì)藥物的敏感性。3、三維培養(yǎng)方法更能反應(yīng)原代腫瘤細(xì)胞對(duì)藥物的敏感性,在三維環(huán)境中也表現(xiàn)出胞膜E蛋白高表達(dá),細(xì)胞耐藥性增強(qiáng),進(jìn)一步驗(yàn)證了二維培養(yǎng)條件下的結(jié)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.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R737.9

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