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目的观察选择性环氧合酶-2(COX-2)抑制剂对COX-2高表达的结肠癌细胞株HT-29增殖和凋亡的影响,明确以COX-2为靶点治疗结肠癌的作用途径以及与COX-2活性、表达水平的相关关系。方法将选择性COX-2抑制剂NS-398作用于结肠癌细胞系HT-29,运用MTT法检测细胞增殖状态。流式细胞仪观察NS-398对细胞凋亡的影响。进一步用逆转录聚合酶链式反应(RT-PCR)检测药物作用前后HT-29中COX-2mRNA表达。ELISA法测定前列腺素E2(PGE2)水平。Westernblot检测药物作用前后细胞周期素D1、Bcl-2的表达。结果结肠癌细胞系HT-29中COX-2mRNA高表达,NS-398呈时间和剂量依赖性抑制HT-29细胞增殖,促进其凋亡。加入NS-398的HT-29细胞中COX-2mRNA表达水平无明显变化(P>0.05),PGE2却显著下降(P<0.01)。72h时空白组与NS-398(75μmol/L)处理组细胞周期素D1、Bcl-2表达水平比值分别为2.21和3.25(P<0.01),两者表达水平随作用时间延长而下降。结论选择性COX-2抑制剂NS-398不影响结肠癌细胞COX-2mRNA表达水平,而与其活性相关(PGE2水平),可能通过细胞周期素D1、Bcl-2影响结肠癌细胞系HT-29的增殖与凋亡,揭示了COX-2为靶点治疗结肠癌的分子机制。
Objective To investigate the effects of cyclooxygenase-2 (COX-2) inhibitor on the proliferation and apoptosis of colorectal cancer cell line HT-29 with high expression of COX-2. To investigate the effect of cyclooxygenase-2 Pathways and their relationship with COX-2 activity and expression levels. Methods NS-398, a selective COX-2 inhibitor, was administered to human colon cancer cell line HT-29. The cell proliferation was detected by MTT assay. The effect of NS-398 on apoptosis was observed by flow cytometry. The expression of COX-2 mRNA in HT-29 was detected by reverse transcription-polymerase chain reaction (RT-PCR). The level of prostaglandin E2 (PGE2) was measured by ELISA. Western blot was used to detect the expression of cyclin D1 and Bcl-2 before and after treatment. Results COX-2 mRNA was highly expressed in colon cancer cell line HT-29. NS-398 could inhibit the proliferation and promote the apoptosis of HT-29 cells in a time and dose-dependent manner. The expression of COX-2 mRNA in HT-29 cells treated with NS-398 had no significant change (P> 0.05), PGE2 decreased significantly (P <0.01). The ratio of cyclin D1 and Bcl-2 expression in blank group and NS-398 (75μmol / L) group at 72h was 2.21 and 3.25 respectively (P <0.01), and the expression of both decreased with the extension of time. Conclusion NS-398, a selective COX-2 inhibitor, does not affect the COX-2 mRNA expression in colon cancer cells, but its activity (PGE2 level) may affect the expression of COX-2 mRNA in colon cancer cell line HT-29 by cyclin D1 and Bcl- Proliferation and apoptosis, revealing the molecular mechanism of COX-2 as a target for the treatment of colon cancer.