本文選題：腫瘤耐藥 + 5-氟尿嘧啶��； 參考：《鄭州大學(xué)》2017年碩士論文

【摘要】：首先,我們采用濃度遞增法刺激人胃癌細(xì)胞MGC803,建立了人胃癌體外耐藥細(xì)胞模型MGC803/5-FU,并初步探討了MGC803和MGC803/5-FU的生物學(xué)性質(zhì)及耐藥株MGC803/5-FU的耐藥機(jī)制。在這部分的研究中,我們以人胃癌細(xì)胞MGC803為親本細(xì)胞株,以化療藥物5-氟尿嘧啶低濃度持續(xù)刺激,并逐漸加大刺激濃度,建立人胃癌體外耐藥細(xì)胞模型MGC803/5-FU。耐藥細(xì)胞株建立后,我們采用CCK8法檢測(cè)了親本細(xì)胞株及耐藥細(xì)胞株對(duì)5-氟尿嘧啶、順鉑、紫杉醇、阿霉素的耐藥性及耐藥細(xì)胞株撤藥6個(gè)月內(nèi)的耐藥穩(wěn)定性。同時(shí),5-氟尿嘧啶作用親本細(xì)胞與耐藥細(xì)胞后,經(jīng)DAPI染色和Annexin V/PI染色進(jìn)一步驗(yàn)證耐藥株MGC803/5-FU對(duì)5-FU的耐藥性;然后,我們通過PI染色、平板克隆和繪制生長(zhǎng)曲線等檢測(cè)了MGC803和MGC803/5-FU生物學(xué)性質(zhì)的差異。最后,通過Western blot檢測(cè)親本細(xì)胞和耐藥細(xì)胞P-gp、TS、β-catenin等蛋白表達(dá)量的差異,分析可能的耐藥機(jī)制。經(jīng)過8個(gè)月的誘導(dǎo),成功建立了人胃癌體外耐藥細(xì)胞模型MGC803/5-FU,耐藥指數(shù)為13.02,但交叉耐藥現(xiàn)象不明顯;同時(shí),耐藥細(xì)胞株撤藥六個(gè)月內(nèi)耐藥指數(shù)能夠保持在9到13之間,耐藥穩(wěn)定性較好。5-氟尿嘧啶作用后,相同濃度下,耐藥細(xì)胞的凋亡率明顯低于親本細(xì)胞。與親本細(xì)胞相比,耐藥細(xì)胞G0/G1期比例增加,S期比例減少;耐藥細(xì)胞生長(zhǎng)速度減慢,群體倍增時(shí)間增大,克隆形成率降低。耐藥細(xì)胞中TS蛋白表達(dá)量增加,同時(shí)Wnt/β-catenin通路相關(guān)蛋白發(fā)生改變。在耐藥模型的基礎(chǔ)上,我們接著研究了新型甾體聯(lián)苯類化合物ZYL-263對(duì)胃癌細(xì)胞MGC803及其耐藥細(xì)胞MGC803/5-FU的抗腫瘤活性及其機(jī)制。我們采用CCK8法和平板克隆法檢測(cè)化合物ZYL-263對(duì)MGC803及MGC803/5-FU細(xì)胞生長(zhǎng)增殖的影響。DAPI染色和Annexin V/PI染色檢測(cè)ZYL-263對(duì)兩株細(xì)胞細(xì)胞凋亡的影響;同時(shí),DCFH-DA染色檢測(cè)ZYL-263對(duì)兩株細(xì)胞活性氧的影響。最后,ZYL-263作用兩株細(xì)胞后,Western blot檢測(cè)Bcl-2、Bax、Total-Caspase-7、Cleaved-Caspase-7、Total-PARP-1、Cleaved-PARP-1、TS等蛋白表達(dá)量的變化。結(jié)果顯示,ZYL-263對(duì)MGC803及MGC803/5-FU有較好的生長(zhǎng)抑制作用,同時(shí)MGC803/5-FU的IC50低于其對(duì)MGC803的的IC50;同時(shí),ZYL-263能夠抑制兩株細(xì)胞的克隆形成。ZYL-263能夠使兩株細(xì)胞的細(xì)胞核發(fā)生皺縮和明顯的凋亡,且MGC803/5-FU的凋亡率高于MGC803的凋亡率。ZYL-263作用兩株細(xì)胞后,胞內(nèi)活性氧水平明顯增高。ZYL-263能夠降低兩株細(xì)胞內(nèi)的Bcl-2、Total-Caspase-7、Total-PARP-1蛋白表達(dá)量,同時(shí)增加兩株細(xì)胞內(nèi)的Bax、Cleaved-Caspase-7、Cleaved-PARP-1蛋白表達(dá)量;另外,ZYL-263顯著下調(diào)耐藥株MGC803/5-FU的TS蛋白表達(dá)量,而對(duì)MGC803的TS蛋白表達(dá)量沒有明顯影響。綜上所述,我們成功建立了穩(wěn)定的人胃癌體外耐藥模型MGC803/5-FU,檢測(cè)了其相關(guān)生物學(xué)特性及其可能的耐藥機(jī)制,為進(jìn)一步研究耐藥相關(guān)原因及尋找新型抗腫瘤化合物提供了穩(wěn)定的體外模型。新型化合物ZYL-263能夠明顯促進(jìn)親本細(xì)胞MGC803及其耐藥細(xì)胞MGC803/5-FU的凋亡,且對(duì)MGC803/5-FU的促凋亡作用更明顯,為尋找新型抗腫瘤化合物研究提供一定的思路。
[Abstract]:First, we used the concentration increase method to stimulate human gastric cancer cell MGC803, and established the human gastric cancer cell model MGC803/5-FU, and preliminarily discussed the biological properties of MGC803 and MGC803/5-FU and the resistance mechanism of the drug resistant strain MGC803/5-FU. In this part, we use the human gastric cancer cell MGC803 as the parent cell line and the chemotherapeutic drug. After the low concentration of 5- fluorouracil was sustained and the concentration was gradually increased, the drug resistant cell line of human gastric cancer was established, and the drug resistance of MGC803/5-FU., cisplatin, paclitaxel and amamycin and the withdrawal of drug resistant cell lines were detected by CCK8 method within 6 months. Resistance stability. At the same time, after 5- fluorouracil acted on the parent and drug-resistant cells, the resistance of MGC803/5-FU to 5-FU was further verified by DAPI staining and Annexin V/PI staining. Then, we detected the differences in the biological properties of MGC803 and MGC803/5-FU by PI staining, flat clones and plotting growth curves. Finally, we passed Weste. RN blot detected the difference in the expression of P-gp, TS, and beta -catenin in the parent and drug-resistant cells, and analyzed the possible mechanism of drug resistance. After 8 months of induction, the drug resistance cell model of human gastric carcinoma was successfully established, and the resistance index was 13.02, but the cross resistance was not obvious. At the same time, drug resistant cell lines were withdrawn for six months. At the same concentration, the apoptosis rate of drug resistant cells was significantly lower than that of parental cells at the same concentration of 9 to 13, with the same resistance and stability of.5- fluorouracil. Compared with the parent cells, the proportion of G0/G1 phase increased, the proportion of S phase decreased, the growth rate of drug-resistant cells decreased, the population doubling time increased, and the clone formation rate decreased. The expression of TS protein in the cells increased and the Wnt/ beta -catenin pathway related proteins changed. On the basis of the drug resistance model, we then studied the anti-tumor activity and mechanism of the new steroidal biphenyl compound ZYL-263 on the gastric cancer cell MGC803 and its drug-resistant cell MGC803/5-FU. We used the CCK8 method and the plate cloning method to detect the antitumor activity. The effect of ZYL-263 on the growth and proliferation of MGC803 and MGC803/5-FU cells,.DAPI staining and Annexin V/PI staining were used to detect the effect of ZYL-263 on cell apoptosis of two cells. At the same time, DCFH-DA staining was used to detect the effect of ZYL-263 on the reactive oxygen species of two cells. Finally, ZYL-263 action two cells, Western blot -Caspase-7, Total-PARP-1, Cleaved-PARP-1, TS and other protein expression changes. The results showed that ZYL-263 had better growth inhibition on MGC803 and MGC803/5-FU, while MGC803/5-FU's IC50 was lower than the IC50 on MGC803. At the same time, ZYL-263 could inhibit the formation of two cells, which could cause the cells of two cells to hair wrinkle. Contraction and obvious apoptosis, and MGC803/5-FU apoptosis rate is higher than the apoptosis rate of MGC803.ZYL-263 two cells, the intracellular active oxygen level is obviously increased,.ZYL-263 can reduce the Bcl-2, Total-Caspase-7, Total-PARP-1 protein expression in two cells, and increase the Bax, Cleaved-Caspase-7, Cleaved-PARP-1 protein table in two cells. In addition, ZYL-263 significantly downregulated the TS protein expression of the drug resistant strain MGC803/5-FU, but had no significant influence on the TS protein expression of MGC803. In summary, we successfully established a stable human gastric cancer drug resistance model MGC803/5-FU, which detected its related biological characteristics and its possible mechanism of resistance to further study of drug resistance. The cause and the search for new antitumor compounds provide a stable in vitro model. The new compound, ZYL-263, can obviously promote the apoptosis of the parent cell MGC803 and its drug-resistant MGC803/5-FU cells, and the apoptosis promoting effect on MGC803/5-FU is more obvious, which provides a certain way of thinking for the study of new antitumor compounds.

【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)】：R735.2

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