發(fā)布時(shí)間：2018-07-12 21:09

  本文選題：多藥耐藥 + ABCB1抑制劑　； 參考：《鄭州大學(xué)》2017年碩士論文

【摘要】：多藥耐藥性的產(chǎn)生,是導(dǎo)致腫瘤化療失敗的主要因素。研究報(bào)道ABC轉(zhuǎn)運(yùn)體在腫瘤耐藥中發(fā)揮著重要作用,ABC轉(zhuǎn)運(yùn)體家族成員之一的ABCB1,是最早發(fā)現(xiàn)的ABC轉(zhuǎn)運(yùn)體,ABCB1的過(guò)表達(dá)是導(dǎo)致腫瘤耐藥性產(chǎn)生的關(guān)鍵因素之一。尋找高效低毒的ABCB1選擇性抑制劑已成為科研工作者的研究熱點(diǎn)。本課題利用ABCB1過(guò)表達(dá)的腫瘤耐藥細(xì)胞株,對(duì)一系列甾體衍生物進(jìn)行了逆轉(zhuǎn)耐藥活性研究,旨在篩選出高效低毒的ABCB1選擇性抑制劑,并研究其逆轉(zhuǎn)耐藥的作用機(jī)制,為解決腫瘤耐藥性問(wèn)題提供研究模型。具體研究?jī)?nèi)容如下:1.以ABCB1過(guò)表達(dá)的結(jié)腸癌耐阿霉素SW620/Ad300細(xì)胞株為基礎(chǔ),建立逆轉(zhuǎn)劑篩選平臺(tái),采用MTT法對(duì)一系列甾體衍生物進(jìn)行篩選,旨在尋找高效低毒的耐藥逆轉(zhuǎn)劑。結(jié)果表明,新型甾體化合物ASC26在濃度為5μM時(shí)能顯著逆轉(zhuǎn)ABCB1調(diào)控的耐藥,并且對(duì)細(xì)胞的抑制率僅為7.39%。2.利用MTT法評(píng)價(jià)ASC26對(duì)七對(duì)腫瘤耐藥及其親本細(xì)胞的增殖抑制作用,結(jié)果發(fā)現(xiàn)ASC26在8μM時(shí),對(duì)這七對(duì)腫瘤耐藥及親本細(xì)胞的抑制率均小于15%,即沒(méi)有細(xì)胞毒性。3.進(jìn)一步研究ASC26在濃度為8μM時(shí)的體外逆轉(zhuǎn)耐藥活性。結(jié)果表明,ASC26能使高表達(dá)ABCB1的腫瘤耐藥細(xì)胞對(duì)紫杉醇(ABCB1的底物)的敏感性顯著增加,而對(duì)順鉑(非ABCB1的底物)的抗腫瘤活性則無(wú)明顯影響。此外,對(duì)于ABCG2過(guò)表達(dá)的耐藥細(xì)胞,ASC26未表現(xiàn)出逆轉(zhuǎn)耐藥的作用。4.利用高效液相法檢測(cè)ASC26對(duì)ABCB1高表達(dá)的腫瘤耐藥細(xì)胞及其親本細(xì)胞內(nèi)紫杉醇蓄積和外排的影響。結(jié)果發(fā)現(xiàn),ASC26能顯著增加紫杉醇在ABCB1高表達(dá)的腫瘤耐藥細(xì)胞中的蓄積,抑制ABCB1高表達(dá)的腫瘤耐藥細(xì)胞對(duì)紫杉醇的外排,提高細(xì)胞內(nèi)紫杉醇含量,而對(duì)親本腫瘤細(xì)胞內(nèi)紫杉醇的蓄積和外排無(wú)明顯作用。5.流式細(xì)胞術(shù)檢測(cè)ASC26對(duì)ABCB1高表達(dá)的SW620/Ad300細(xì)胞內(nèi)羅丹明(Rho123)蓄積的影響。結(jié)果表明,ASC26能顯著增加SW620/Ad300細(xì)胞內(nèi)Rho123的滯留率,但對(duì)親本SW620細(xì)胞內(nèi)Rho123的蓄積無(wú)明顯影響。6.免疫熒光檢測(cè)ASC26對(duì)SW620/Ad300細(xì)胞內(nèi)ABCB1轉(zhuǎn)運(yùn)蛋白表達(dá)位點(diǎn)的影響。ASC26作用于SW620/Ad300細(xì)胞72 h后,免疫熒光法檢測(cè)ASC26對(duì)ABCB1表達(dá)位點(diǎn)的作用。結(jié)果表明,ASC26不改變細(xì)胞內(nèi)ABCB1的蛋白表達(dá)位點(diǎn)。8.利用Western blot檢測(cè)ASC26對(duì)ABCB1蛋白表達(dá)水平的影響。由Western blot結(jié)果可知,ASC26分別作用于SW620/Ad300耐藥細(xì)胞0 h,24 h,48 h,72h后,并不改變細(xì)胞內(nèi)ABCB1的蛋白表達(dá)水平。9.分子對(duì)接實(shí)驗(yàn)研究ASC26與人同源ABCB1分子模型的相互作用關(guān)系。分子對(duì)接實(shí)驗(yàn)表明,化合物ASC26能結(jié)合在ABCB1轉(zhuǎn)運(yùn)蛋白的跨膜結(jié)構(gòu)域的藥物結(jié)合口袋上,從而抑制ABCB1對(duì)化療藥物的外排轉(zhuǎn)運(yùn)。本課題發(fā)現(xiàn)了一個(gè)選擇性ABCB1抑制劑,新型甾體化合物ASC26,在無(wú)毒濃度下,能顯著逆轉(zhuǎn)ABCB1介導(dǎo)的腫瘤耐藥。進(jìn)一步的機(jī)制研究表明,ASC26的逆轉(zhuǎn)耐藥機(jī)制是通過(guò)抑制ABCB1轉(zhuǎn)運(yùn)泵的外排轉(zhuǎn)運(yùn)功能,而不是通過(guò)影響ABCB1的蛋白表達(dá)來(lái)發(fā)揮逆轉(zhuǎn)耐藥作用。ASC26能與ABCB1轉(zhuǎn)運(yùn)蛋白的跨膜結(jié)構(gòu)域的藥物結(jié)合口袋結(jié)合,抑制ABCB1對(duì)抗腫瘤藥物的外排作用,增加細(xì)胞內(nèi)抗腫瘤藥物的濃度,從而提高腫瘤細(xì)胞對(duì)化療藥物的敏感性,達(dá)到逆轉(zhuǎn)腫瘤耐藥的效果。這一研究成果將為解決腫瘤耐藥性問(wèn)題提供新的研究方案和研究模型。
[Abstract]:The production of multidrug resistance is the main factor leading to the failure of tumor chemotherapy. The study reports that the ABC transporter plays an important role in the drug resistance of the tumor. ABCB1, one of the members of the ABC transporter family, is the earliest found ABC transporter. The overexpression of ABCB1 is one of the key factors leading to the production of tumor resistance. The search for high efficiency and low toxicity ABCB1 Selective inhibitors have become the research hotspots of researchers. This topic uses ABCB1 overexpressed tumor resistant cell lines to reverse the drug resistance of a series of steroid derivatives. The purpose of this study is to screen out highly toxic and low toxic ABCB1 selective inhibitors, and to study the mechanism of its reversal of drug resistance, in order to solve the problem of drug resistance. The specific research contents are as follows: 1. based on the ABCB1 overexpressed adriamycin resistant SW620/Ad300 cell line of colon cancer, the reverse agent screening platform was established and a series of steroid derivatives were screened by the MTT method. The results showed that the new steroid compound ASC26 was at the concentration of 5 u M. The resistance to ABCB1 regulation was significantly reversed and the inhibitory rate to the cells was only 7.39%.2. using MTT to evaluate the inhibitory effect of ASC26 on the proliferation of tumor resistance and their parent cells by ASC26. The results showed that the inhibition rate of the seven against tumor resistance and parental cells was less than 15% when ASC26 was at 8 micron, that is, no cytotoxic.3. was further studied for ASC26. The reversal of drug resistance in vitro at a concentration of 8 M showed that ASC26 could significantly increase the sensitivity of the tumor resistant cells with high expression of ABCB1 to Taxol (ABCB1 substrate), while the antitumor activity of cisplatin (non ABCB1 substrate) was not significantly affected. In addition, ASC26 did not reverse the reversal of resistance to ABCG2 overexpressed drug-resistant cells. The effect of.4. on the effect of ASC26 on the accumulation and excretion of paclitaxel in ABCB1 high expression of tumor resistant cells and their parent cells by high performance liquid phase method. The results showed that ASC26 could significantly increase the accumulation of taxol in the tumor resistant cells with high expression of ABCB1, and inhibit the outer row of paclitaxel by the high expression of ABCB1. Intracellular paclitaxel content, but no significant effect on the accumulation and outer row of paclitaxel in the parent tumor cells.5. flow cytometry was used to detect the effect of ASC26 on the accumulation of Luo Danming (Rho123) in SW620/Ad300 cells with high expression of ABCB1. The results showed that ASC26 could significantly increase the retention rate of Rho123 in SW620/Ad300 cells, but Rho12 in the parent SW620 cell Rho12. The effect of the accumulation of 3 on the expression of ABCB1 transporter protein in SW620/Ad300 cells by.6. immunofluorescence was not significantly affected by.ASC26 on SW620/Ad300 cell 72 h, and the effect of ASC26 on the ABCB1 expression site was detected by immunofluorescence. The results showed that ASC26 did not change the ABCB1 protein expression loci in the cells. The effect of C26 on the expression level of ABCB1 protein. The result of Western blot shows that ASC26 acts on SW620/Ad300 resistant cells 0 h, 24 h, 48 h, and 72h, it does not change the ABCB1 protein expression level in the cell and the interaction relationship between the.9. molecular docking experiment and the human homologous molecular model. 6 can bind to the drug binding pocket of the transmembrane domain of the ABCB1 transporter, which inhibits the translocation of ABCB1 against chemotherapeutic agents. A selective ABCB1 inhibitor, a new steroid compound, ASC26, can be found to reverse the ABCB1 mediated swelling resistance of the tumor significantly in the non toxic concentration. Further mechanism studies show the inverse of ASC26 The mechanism of trans resistance is to inhibit the efflux transport of the ABCB1 transport pump, not by affecting the protein expression of ABCB1 to reverse the resistance of.ASC26 to the drug binding pocket of the trans membrane domain of the ABCB1 transporter, inhibit the use of ABCB1 against tumor drugs, and increase the concentration of intracellular antitumor drugs. To improve the sensitivity of tumor cells to chemotherapeutic drugs and to reverse the effect of tumor resistance, this research will provide a new research scheme and model for solving the problem of tumor resistance.
【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R96

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