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目的研究环氧合酶-2选择性抑制制塞来昔布联合放射治疗对胰腺癌的作用,并探讨其作用机制。方法克隆形成实验和裸鼠移植瘤模型观察塞来昔布、放疗和两者联合对胰腺癌细胞SW1990体内外增殖的影响。Western印迹法检测细胞增殖相关蛋白表达。原位缺口末端标记法(TUNEL)研究胰腺癌细胞凋亡。RT-PCR检测凋亡相关基因表达的变化。体外血管生成和体外侵袭力测定方法检测塞来昔布、放疗及两者联合对胰腺癌细胞新生血管生成和细胞侵袭力的影响,RT-PCR、明胶酶谱和反式酶谱方法检测基质金属蛋白酶(MMP)-2、MMP-9及其组织抑制剂(TIMP)-1、TIMP-2的表达。结果塞来昔布在体内外均有放射增敏作用。胰腺癌细胞增殖细胞核抗原(PCNA)蛋白表达水平在塞来昔布组和联合放疗组均降低,联合组较塞来昔布组有进一步降低。塞来昔布诱导胰腺癌细胞凋亡,单独放疗并不能诱导SW1990细胞发生凋亡,两者联合凋亡细胞数明显增加。塞来昔布下调bcl-2 mRNA表达,而放疗诱导bcl-2 mRNA表达上调,联合组bcl-2的表达最低。单剂量放疗时,裸鼠胰腺癌移植瘤的生长延迟时间为22 d,联合塞来昔布为38 d。单独放疗并不能抑制体外胰腺癌细胞的新生血管生成和侵袭,塞来昔布在体外抑制胰腺癌新生血管形成和侵袭,塞来昔布与放疗联合其抑制作用较单用塞来昔布无明显增强。塞来昔布抑制胰腺癌细胞合成、分泌和激活MMP-2、MMP-9,但对TIMP- 1、TIMP-2的合成、分泌和激活无明显影响。结论COX-2选择性抑制剂塞来昔布对胰腺癌放疗有增敏作用,其机制涉及诱导细胞凋亡,并通过抑制胰腺癌细胞新生血管生成和细胞侵袭,间接对胰腺癌的放疗起增敏作用。
Objective To study the effect of cyclooxygenase-2 (COX-2) selective inhibition of celecoxib combined with radiotherapy on pancreatic cancer and its mechanism of action. Methods The experimental and nude mice xenografts were established by cloning to observe the effects of celecoxib, radiotherapy and their combination on the proliferation of pancreatic cancer cell line SW1990 in vitro and in vivo. Western blotting was used to detect the expression of cell proliferation related proteins. In situ nick end labeling (TUNEL) study of pancreatic cancer cell apoptosis. The changes of apoptosis related gene expression were detected by RT-PCR. In vitro angiogenesis and in vitro invasiveness assay was used to detect the effect of celecoxib, radiotherapy and their combination on neovascularization and invasiveness of pancreatic cancer cells. RT-PCR, gelatin zymography and trans-zymogram were used to detect the effect of matrix metalloproteinases Protease (MMP) -2, MMP-9 and its tissue inhibitor of metalloproteinase (TIMP) -1, TIMP-2 expression. Results Celecoxib had radiosensitization both in vitro and in vivo. The expression of PCNA protein in pancreatic cancer cells decreased in both celecoxib group and combination radiotherapy group, and decreased in combination group compared with celecoxib group. Celecoxib induced apoptosis of pancreatic cancer cells. Radiotherapy alone did not induce apoptosis of SW1990 cells, and the number of apoptotic cells increased significantly. Celecoxib down-regulated the expression of bcl-2 mRNA, and up-regulated the expression of bcl-2 mRNA by radiotherapy. The expression of bcl-2 in the combination group was the lowest. Single-dose radiotherapy, the growth of transplanted pancreatic cancer in nude mice was 22 d, combined with celecoxib 38 d. Celecoxib inhibited the angiogenesis and invasion of pancreatic cancer in vitro, and the combination of celecoxib and radiotherapy had no significant difference compared with celecoxib Enhanced. Celecoxib inhibited pancreatic cancer cell synthesis, secretion and activation of MMP-2, MMP-9, but TIMP-1, TIMP-2 synthesis, secretion and activation had no significant effect. CONCLUSION: Celecoxib, a selective inhibitor of COX-2, has a radiosensitizing effect on radiotherapy of pancreatic cancer, and its mechanism involves the induction of apoptosis. By inhibiting the angiogenesis and cell invasion of pancreatic cancer cells, it indirectly increases the radiotherapy of pancreatic cancer Sensitive effect.