本文關(guān)鍵詞： 胃癌 COX-2 塞來昔布 化療藥物 細胞增殖 細胞凋亡　出處：《廣西醫(yī)科大學》2017年碩士論文　論文類型：學位論文

【摘要】：目的探討常用化療藥物順鉑、5-氟尿嘧啶與COX-2抑制劑塞來昔布聯(lián)合應(yīng)用對胃癌細胞株SGC-7901生物學行為的影響及可能涉及的分子機制。方法采用免疫組化法(IHC)檢測胃癌及相應(yīng)癌旁組織中COX-2的表達情況;用MTT法檢測順鉑(DDP)、5-氟尿嘧啶(5-Fu)與塞來昔布(Ce)單用或聯(lián)合應(yīng)用對胃癌細胞增殖的影響;用AnnexinV-PE和7-AAD雙染進行流式細胞技術(shù)檢測各組胃癌細胞的凋亡情況;qRT-PCR檢測各組胃癌細胞用藥前后COX-2、Caspase-3、Survivin、Mcl-1、MDR1的mRNA的表達差異。結(jié)果1.胃癌及相應(yīng)癌旁組織中COX-2的表達情況:免疫組化結(jié)果顯示,在46例胃癌及相應(yīng)癌旁組織中,胃癌組織COX-2強陽性表達者占10.87%(5/46),陽性表達者占21.74%(10/46),弱陽性表達者占63.04%(29/46),陰性表達者占4.35%(2/46),而相應(yīng)癌旁組織未見強陽性及陽性表達者,弱陽性表達者占21.74%(10/46),陰性表達者占78.26%(36/46)。與相應(yīng)癌旁組織相比,胃癌組織中COX-2的表達明顯增高,差異具有顯著性(P0.05)2.COX-2表達差異與胃癌臨床病理特征的關(guān)系:進一步分析上述胃癌組織中COX-2的表達情況發(fā)現(xiàn),COX-2表達與胃癌的分化程度、淋巴結(jié)轉(zhuǎn)移、TNM病理分期以及腫瘤直徑大小有關(guān);但與胃癌患者的性別、年齡、腫瘤位置、是否伴有脈管浸潤及腫瘤浸潤深度等因素無關(guān)。3.藥物作用前后各組胃癌細胞的增殖情況:mtt檢測結(jié)果顯示,不同濃度的ce、ddp和5-fu均可抑制明顯胃癌細胞的增殖,且其抑制作用隨著藥物濃度的升高而增強。計算ce、ddp和5-fu的半數(shù)抑制濃度(ic50)分別為:92.04±4.93umol/l、10.43±0.27mg/l和156.56±2.07mg/l。進一步研究中,我們以先前計算出的半數(shù)抑制濃度的平均數(shù)為藥物劑量處理各組胃癌細胞,結(jié)果顯示ddp+ce組與ddp組相比,24h、48h和72h的細胞增殖抑制率均增加,分別為59.66%vs46.88%、67.05%vs50.02%和63.77%vs52.17%;5-fu+ce組與5-fu組相比,在上述不同時間點的抑制率也明顯增加,分別為55.58%vs39.23%、62.84%vs47.62%和59.00%vs51.19%。聯(lián)合ddp+ce組和5-fu+ce組與相應(yīng)單藥組相比,差異具有顯著性(p0.05)。4.藥物作用前后各組胃癌細胞凋亡情況:流式細胞技術(shù)檢測發(fā)現(xiàn),以半數(shù)抑制濃度的藥物劑量作用胃癌細胞48h后,空白對照組、ddp組、5-fu組、ddp+ce組、5-fu+ce組的細胞凋亡率分別為7.10%±0.30%、46.53%±1.50%、39.17%±1.10%、66.93%±0.70%、62.13%±1.60%,各藥物作用組細胞凋亡率均明顯高于空白對照組,差別有統(tǒng)計學顯著性差異(p0.05),聯(lián)合ddp+ce組和5-fu+ce組與相應(yīng)單藥組相比,細胞凋亡率明顯增加,差異具有顯著性(p0.05)。5.藥物作用前后各組胃癌細胞中cox-2、caspase-3、survivin、mcl-1和mdr1mrna表達情況:qrt-pcr結(jié)果顯示,半數(shù)抑制濃度的藥物劑量作用48h后,ddp組、5-fu組、ce+ddp組和ce+5-fu組與空白對照組相比,cox-2、survivin、mcl-1和mdr1mrna表達明顯降低,而caspase-3則明顯升高,差異具有顯著性(p0.05),且ddp+ce組和5-fu+ce組與相應(yīng)單藥組(ddp組和5-fu組)相比,cox-2、survivin、mcl-1和mdr1mrna表達也明顯降低,caspase-3表達升高,差別具有統(tǒng)計學顯著性差異(p0.05)。結(jié)論1.COX-2在胃癌組織中呈高表達,且其表達情況與胃癌的分化程度、淋巴結(jié)轉(zhuǎn)移情況、TNM病理分期以及腫瘤直徑大小有關(guān)。2.與單用化療藥相比,順鉑或5-氟尿嘧啶聯(lián)合塞來昔布可進一步促進胃癌細胞凋亡,抑制細胞增殖,增強藥物敏感性,提高對胃癌細胞的化療效果。這提示順鉑或5-氟尿嘧啶與塞來昔布聯(lián)合應(yīng)用具有協(xié)同抗胃癌的作用。3.順鉑或5-氟尿嘧啶聯(lián)合塞來昔布后其凋亡增加機制可能與COX-2下調(diào)后促進Caspase-3表達與下調(diào)Survivin和Mcl-1表達有關(guān),而藥物敏感性增加則可能與COX-2下調(diào)后抑制MDR1表達有關(guān)。
[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.

【學位授予單位】：廣西醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R735.2

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