顺铂、5-氟尿嘧啶与塞来昔布联合应用对胃癌细胞株SGC-7901生物学行为的影响

发布时间：2018-01-28 03:48

本文关键词： 胃癌 COX-2 塞来昔布化疗药物细胞增殖细胞凋亡　出处：《广西医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的探讨常用化疗药物顺铂、5-氟尿嘧啶与COX-2抑制剂塞来昔布联合应用对胃癌细胞株SGC-7901生物学行为的影响及可能涉及的分子机制。方法采用免疫组化法(IHC)检测胃癌及相应癌旁组织中COX-2的表达情况;用MTT法检测顺铂(DDP)、5-氟尿嘧啶(5-Fu)与塞来昔布(Ce)单用或联合应用对胃癌细胞增殖的影响;用AnnexinV-PE和7-AAD双染进行流式细胞技术检测各组胃癌细胞的凋亡情况;qRT-PCR检测各组胃癌细胞用药前后COX-2、Caspase-3、Survivin、Mcl-1、MDR1的mRNA的表达差异。结果1.胃癌及相应癌旁组织中COX-2的表达情况:免疫组化结果显示,在46例胃癌及相应癌旁组织中,胃癌组织COX-2强阳性表达者占10.87%(5/46),阳性表达者占21.74%(10/46),弱阳性表达者占63.04%(29/46),阴性表达者占4.35%(2/46),而相应癌旁组织未见强阳性及阳性表达者,弱阳性表达者占21.74%(10/46),阴性表达者占78.26%(36/46)。与相应癌旁组织相比,胃癌组织中COX-2的表达明显增高,差异具有显著性(P0.05)2.COX-2表达差异与胃癌临床病理特征的关系:进一步分析上述胃癌组织中COX-2的表达情况发现,COX-2表达与胃癌的分化程度、淋巴结转移、TNM病理分期以及肿瘤直径大小有关;但与胃癌患者的性别、年龄、肿瘤位置、是否伴有脉管浸润及肿瘤浸润深度等因素无关。3.药物作用前后各组胃癌细胞的增殖情况:mtt检测结果显示,不同浓度的ce、ddp和5-fu均可抑制明显胃癌细胞的增殖,且其抑制作用随着药物浓度的升高而增强。计算ce、ddp和5-fu的半数抑制浓度(ic50)分别为:92.04±4.93umol/l、10.43±0.27mg/l和156.56±2.07mg/l。进一步研究中,我们以先前计算出的半数抑制浓度的平均数为药物剂量处理各组胃癌细胞,结果显示ddp+ce组与ddp组相比,24h、48h和72h的细胞增殖抑制率均增加,分别为59.66%vs46.88%、67.05%vs50.02%和63.77%vs52.17%;5-fu+ce组与5-fu组相比,在上述不同时间点的抑制率也明显增加,分别为55.58%vs39.23%、62.84%vs47.62%和59.00%vs51.19%。联合ddp+ce组和5-fu+ce组与相应单药组相比,差异具有显著性(p0.05)。4.药物作用前后各组胃癌细胞凋亡情况:流式细胞技术检测发现,以半数抑制浓度的药物剂量作用胃癌细胞48h后,空白对照组、ddp组、5-fu组、ddp+ce组、5-fu+ce组的细胞凋亡率分别为7.10%±0.30%、46.53%±1.50%、39.17%±1.10%、66.93%±0.70%、62.13%±1.60%,各药物作用组细胞凋亡率均明显高于空白对照组,差别有统计学显著性差异(p0.05),联合ddp+ce组和5-fu+ce组与相应单药组相比,细胞凋亡率明显增加,差异具有显著性(p0.05)。5.药物作用前后各组胃癌细胞中cox-2、caspase-3、survivin、mcl-1和mdr1mrna表达情况:qrt-pcr结果显示,半数抑制浓度的药物剂量作用48h后,ddp组、5-fu组、ce+ddp组和ce+5-fu组与空白对照组相比,cox-2、survivin、mcl-1和mdr1mrna表达明显降低,而caspase-3则明显升高,差异具有显著性(p0.05),且ddp+ce组和5-fu+ce组与相应单药组(ddp组和5-fu组)相比,cox-2、survivin、mcl-1和mdr1mrna表达也明显降低,caspase-3表达升高,差别具有统计学显著性差异(p0.05)。结论1.COX-2在胃癌组织中呈高表达,且其表达情况与胃癌的分化程度、淋巴结转移情况、TNM病理分期以及肿瘤直径大小有关。2.与单用化疗药相比,顺铂或5-氟尿嘧啶联合塞来昔布可进一步促进胃癌细胞凋亡,抑制细胞增殖,增强药物敏感性,提高对胃癌细胞的化疗效果。这提示顺铂或5-氟尿嘧啶与塞来昔布联合应用具有协同抗胃癌的作用。3.顺铂或5-氟尿嘧啶联合塞来昔布后其凋亡增加机制可能与COX-2下调后促进Caspase-3表达与下调Survivin和Mcl-1表达有关,而药物敏感性增加则可能与COX-2下调后抑制MDR1表达有关。
[Abstract]:Objective to investigate the molecular mechanism of cisplatin chemotherapy drugs, 5- fluorouracil and COX-2 Inhibitor Celecoxib on the biological behaviors of gastric cancer cell line SGC-7901 and may be involved. By immunohistochemical method (IHC method) to detect expression of COX-2 in gastric cancer and paracancerous tissues was detected by MTT; cisplatin (DDP). 5- fluorouracil (5-Fu) and celecoxib (Ce) effects of single or combination on the proliferation of gastric cancer cells; apoptosis by flow cytometry to detect the gastric cancer cells by AnnexinV-PE and 7-AAD double staining; qRT-PCR test before and after the treatment of gastric cancer cells COX-2, Caspase-3, Survivin, Mcl-1, expression of MDR1 mRNA the expression of COX-2. Results of 1. gastric cancer and paracancerous tissues: immunohistochemistry showed that in 46 cases of gastric cancer tissues and corresponding para carcinoma, the positive expression of COX-2 strong gastric cancer accounted for 10.87% (5/46), The positive expression was 21.74% (10/46), weakly positive expression was 63.04% (29/46), negative expression accounted for 4.35% (2/46), and the corresponding paracancerous tissues and there were no positive and positive, weak positive expression was 21.74% (10/46), negative expression accounted for 78.26% (36/46) and the corresponding cancer. Compared with the adjacent tissues, the expression of COX-2 in gastric cancer tissues were significantly increased, the difference was significant (P0.05) between 2.COX-2 expression and clinicopathological features of gastric cancer: further analysis of the expression of COX-2 in gastric carcinoma, and gastric cancer differentiation degree of expression of COX-2, lymph node metastasis, TNM staging and tumor diameter size but in patients with gastric cancer; gender, age, tumor location, whether associated with vascular invasion and tumor invasion depth and other factors unrelated to proliferation of gastric cancer cells in each group before and after.3. drugs: MTT test results showed that different concentrations of CE, DDP and 5-FU Can inhibit the proliferation of gastric cancer cells, and the inhibition effect increases with the increase of drug concentration. The calculation of CE, DDP and 5-FU half inhibitory concentration (IC50) respectively: 92.04 + 4.93umol/l, further study of 10.43 + 0.27mg/l and 156.56 + 2.07mg/l., the average number of our half inhibitory concentration to previously calculated for the dose of drug treatment of gastric cancer cells in each group, the results show that the ddp+ce group compared with DDP group, 24h, 48h and 72h cell proliferation inhibition rate were increased, respectively 59.66%vs46.88%, 67.05%vs50.02% and 63.77%vs52.17%; 5-fu+ce group and 5-FU group than in the different time points of the inhibition rate also increased significantly, respectively 55.58%vs39.23%, 62.84%vs47.62% and 59.00%vs51.19%. combined with ddp+ce group and 5-fu+ce group compared with the corresponding single drug group, with significant difference (P0.05) before and after apoptosis of gastric cancer cell.4. drug situation: flow cytometry. Test found that half inhibition dose effect to gastric cancer cell 48h concentration, the blank control group, DDP group, 5-FU group, ddp+ce group, the apoptosis rate of 5-fu+ce group was 7.10% + 0.30%, 46.53% + 1.50%, 39.17% + 1.10%, 66.93% + 0.70%, 62.13% + 1.60%, the drug group the rate of cell apoptosis were significantly higher than control group, the difference was statistically significant difference (P0.05), ddp+ce group and 5-fu+ce group compared with the corresponding single drug group, the apoptosis rate was significantly increased, the difference was significant (P0.05) before and after.5. drugs COX-2, gastric cancer cells in each group Caspase-3, survivin, Mcl-1 and mdr1mrna expression: qRT-PCR the results showed that half inhibition dose effect of 48h concentration, DDP group, 5-FU group, ce+ddp group and ce+5-fu group compared with the control group, COX-2, survivin, Mcl-1 and mdr1mrna expression was significantly reduced, while caspase-3 increased significantly, the difference is significant A (P0.05), and ddp+ce group and 5-fu+ce group and single drug group (DDP group and 5-FU group) compared to COX-2, survivin, Mcl-1 and mdr1mrna expression was significantly decreased, the expression of Caspase-3 increased, the difference was statistically significant difference (P0.05). Conclusion the high expression of 1.COX-2 in gastric cancer tissues, and the the expression and degree of differentiation of gastric cancer, lymph node metastasis, TNM staging and tumor diameter of about.2. compared with chemotherapy, cisplatin and 5- fluorouracil combined with celecoxib can further promote gastric cancer cell apoptosis, inhibit cell proliferation, increase drug sensitivity, improve the effect of chemotherapy on gastric cancer cells. This suggests that cisplatin or 5- fluorouracil and celecoxib have synergistic anti-cancer effect of.3. cisplatin and 5- fluorouracil combined with celecoxib after its apoptosis mechanism may be related to the down-regulation of COX-2 expression and down-regulation of Caspase-3 after the promotion The expression of Survivin is related to the expression of Mcl-1, and the increase of drug sensitivity may be related to the inhibition of the expression of MDR1 after the downregulation of COX-2.

【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.2

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