本文選題：膀胱癌 + 多藥耐藥�。� 參考：《福建醫(yī)科大學(xué)》2014年碩士論文

【摘要】：目的探討細(xì)胞間P 糖蛋白轉(zhuǎn)移現(xiàn)象在形成并維持膀胱癌BIU 87細(xì)胞多藥耐藥中的作用及機(jī)制。 方法用Transwell共培養(yǎng)膀胱癌耐藥株細(xì)胞BIU 87/ADM與敏感株細(xì)胞BIU 87，在共聚焦顯微鏡下觀察共培養(yǎng)至24h、48h、72h、96h Transwell下室BIU 87細(xì)胞（即獲得性耐藥細(xì)胞AqMDR）P 糖蛋白含量，細(xì)胞計數(shù)法繪制BIU 87/ADM、BIU 87、共培養(yǎng)至96h AqMDR三種在加與不加1μg/ml阿霉素（adriamycin,ADM）培養(yǎng)液中的生長曲線并比較其倍增時間，分離出共培養(yǎng)至96h AqMDR繼續(xù)分2組傳代培養(yǎng)，一組培養(yǎng)基中加入1μg/ml阿霉素，另一組不加。分別運用CCK 8、Western Blot、RT PCR方法、流式細(xì)胞儀檢測0、4、8、16、20代及第20代凍存1個月后復(fù)蘇的AqMDR的耐藥指數(shù)、P gp表達(dá)量、MDR1mRNA表達(dá)水平及細(xì)胞內(nèi)羅丹明 123熒光強(qiáng)度。 結(jié)果共聚焦顯微鏡結(jié)果顯示P 糖蛋白轉(zhuǎn)移量隨著共培養(yǎng)時間延長逐漸增多。細(xì)胞生長曲線提示：在不加ADM組，BIU 87倍增時間（25.30+0.04h）較BIU 87/ADM（31.58+0.37h）短（P0.001），AqMDR介于兩者之間（28.39+0.33h，P0.001）；在加入1μg/ml ADM組，BIU 87培養(yǎng)5天后全部死亡，而AqMDR與BIU 87/ADM細(xì)胞生長并不受限制。CCK 8、Western Blot、RT PCR及羅丹明 123外排實驗證實：AqMDR細(xì)胞隨著傳代增加，在不加阿霉素組，耐藥指數(shù)和P gp表達(dá)量逐漸下降，細(xì)胞內(nèi)羅丹明 123熒光強(qiáng)度逐漸升高，最終回到敏感株BIU 87水平，，MDR1mRNA的表達(dá)水平未見明顯改變。在加阿霉素組，耐藥指數(shù)和P gp表達(dá)量略呈增高趨勢，細(xì)胞內(nèi)羅丹明 123熒光強(qiáng)度略呈下降趨勢，MDR1mRNA的表達(dá)水平逐漸增高到耐藥株細(xì)胞BIU 87/ADM水平。 結(jié)論細(xì)胞間P 糖蛋白轉(zhuǎn)移量隨著耐藥株與敏感株共培養(yǎng)時間延長而逐漸增多，AqMDR細(xì)胞能夠在略大于敏感株IC50的藥物濃度（此濃度會導(dǎo)致敏感株死亡）下穩(wěn)定生長，細(xì)胞間P gp轉(zhuǎn)移有利于膀胱癌敏感細(xì)胞逃避化療藥物的作用并由暫時性耐藥發(fā)展為獲得性永久耐藥。
[Abstract]:Objective to investigate the role and mechanism of intercellular P glycoprotein transfer in the formation and maintenance of multidrug resistance (MDR) in bladder cancer cells. Methods Transwell co-cultured bladder cancer cell line BIU / 87 / ADM and sensitive cell line BIU / 87 / ADM were used to observe the content of P-glucoprotein (P) in BIU _ (87) cells (namely, acquired drug resistant cells AqMDR) under confocal microscope to 24 h ~ (48) h ~ (72) h ~ 96 h. The cell count method was used to draw the growth curve of three kinds of AMDR in medium supplemented or without 1 渭 g/ml adriamycin (ADM) for 96 h, and the doubling time was compared. The co-cultured cells were isolated and cultured to 96 h, AqMDR was further subcultured in two groups, and the results were as follows: 1 渭 g/ml adriamycin (adriamycin) adriamycin was added to the culture medium, and the time of multiplication was compared. In one group, 1 渭 g/ml adriamycin was added to the medium, while in the other group, no adriamycin was added. The expression level of MDR1 mRNA and the fluorescence intensity of Neronerol 123 were detected by flow cytometry and flow cytometry, respectively. The drug resistance index (MDR) and the expression level of MDR1 mRNA were detected by flow cytometry after cryopreservation for 1 month or 20 months after cryopreservation for 1 month. The expression of MDR1 mRNA and the fluorescence intensity of Nerosin 123 in cells were detected by flow cytometry (FCM). The expression level of MDR1 mRNA and the expression level of MDR1 mRNA were measured. Results the results of confocal microscopy showed that the amount of P glycoprotein transfer increased with the prolongation of co culture time. The cell growth curve indicated that the doubling time of BIU87 (25.300.04 h) was shorter than that of BIU-87 / ADM (31.58 0.37 h) in the group without ADM (P0.001). However, the growth of AqMDR and Biu 87 / ADM cells was not restricted. The results of Western Blot RT PCR and Rhodamine 123 efflux assay showed that the drug resistance index and the expression of P P in the group without adriamycin decreased gradually with the increase of passage. The fluorescence intensity of Nero-danmycin 123 increased gradually, and the expression level of MDR1 mRNA in the sensitive strain BIU-87 did not change significantly. In the adriamycin group, the drug resistance index and the expression of P ~ + GP increased slightly, and the fluorescence intensity of Nero-danmycin 123 decreased slightly. The expression level of MDR1 mRNA increased gradually to 87% ADM level in the resistant cell line. Conclusion with the increase of co-culture time between resistant strain and sensitive strain, the amount of P-glucoprotein transfer between cells increased gradually, and the cells of AqMDR could grow stably at a concentration slightly larger than that of sensitive strain IC50 (which would lead to the death of sensitive strain). Intercellular P P GP metastasis was beneficial to the escape of chemotherapeutic agents from transient resistance to acquired permanent resistance in bladder cancer sensitive cells.
【學(xué)位授予單位】：福建醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R737.14

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