环氧合酶2反义寡核苷酸对胰腺癌新生血管生成的抑制作用

目的 研究环氧合酶-2反义寡核苷酸（COX-2 AS-ODN）对胰腺癌新生血管生成的抑制作用,探讨前列腺素E2（PGE2）在胰腺癌新生血管生成中的调节作用。方法 设计、合成特异性靶向COX-2 AS-ODN。人胰腺癌PC-3细胞进行体外培养,转染COX-2 AS-ODN后0、12、24、40和72h以荧光显微镜观察细胞情况。第二批PC-3细胞用于研究量-效关系,分为5组：对照组、Lipo组（接种Lopofectin脂质体）、C1组（1μg COX-2 AS-ODN Lipo/孔）。第三批PC-3细胞用于研究时-效关系,接种3μg COX-2 AS-ODN Lipo/孔后观察0、12、24、48和72h。用RT-PCR观察COX-2 mRNA的表达。以Western印迹观察分别加入3μg和9μg COX-2 AS-ODN/瓶后PC-3细胞COX-2蛋白质的表达。18只鸡胚分3组,其绒毛尿囊（CAM）分别接种PC-3细胞以及Lipo、Lipo COX-2 AS-ODN、Lipo COX-2 AS-ODN 前列腺素E2（PGE2）,另6只鸡胚仅接种PC3细胞用作对照。用Leica体视显微镜观察新生血管生成情况。结果 RT-PCR示随转染COX-2 AS-ODN浓度的增加,PC3细胞的COX-2 mRNA表达逐渐下调（直到COX-2 AS-ODN浓度为0.2μmol/L时）。COX-2 AS-ODN的作用于12h后最强,以后渐弱,48h后基本消失。Western印迹表明COX-2 AS-ODN转染组的COX-2蛋白质表达受抑制,9μg COX-2 AS-ODN/瓶组的抑制强于3μg COX-2 AS-ODN/瓶组。Lipo COX-2 AS-ODN组鸡胚CAM移植肿瘤中的新生血管生成密度明显低于Lipo组;Lipo COX-2 AS-ODN PGE2组的新生血管密度介于以上两组之间。结论 COX-2 AS-ODN显著抑制胰腺癌新生血管的生成。内源性COX-2参与对胰腺癌新生血管生成的调节,PGE2在该过程中起重要的介导作用。

Inhibition of angiogenesis in pancreatic carcinoma by cyclooxygenase-2 antisense oligodeoxynucleotides

OBJECTIVE: To investigate the effect of cyclooxygenase-2 antisense oligodeoxynucleotides (COX-2 AS-ODNs) on the angiogenesis in pancreatic carcinoma and to evaluate the intermediary effect of prostaglandin 2 in this process. METHODS: Specific targeting COX-2 AS-ODNs were designed and synthesized, and transfected into the PC3 human pancreatic carcinoma cells cultured in vitro. Fluorescence microscopy was used to observe the PC3 cells 0.12. 24, 40, and 72 hours after the transfection. the second cultured PC3 cells were divided into 5 groups: control group, Lipo group (transfected with Lipofectin only), C1 group (transfected with 1 micro g COX-2 AS-ODN + Lipo/well), C2 group (transfected with 2 micro g COX-2 AS-ODN + Lipo/well), and C3 group (transfected with 3 micro g COX-2 AS-ODN + Lipo/well). RT-PCR was used to observe the expression of COX-2 mRNA in the PC3 cells. The third batch of PC3 cells were transfected with 3 micro g COX-2 AS-ODN + Lipo/well, and the expression of COX-2 mRNA was observed 0, 12, 24, 48, and 72 hours later by RT-PCR. 3 micro g COX-2 AS-ODN + Lipo/bottle and 9 micro g COX-2 AS-ODN + Lipo/bottle were added into the cultured PC3 cells and Western blotting was used to observe the expression of COX-2 protein 24 hours later. 24 chicken eggs were inoculated with PC3 cells into the chorio-allantoic membrane and then divided equally into 5 groups; control group, Lipo group, COX-2 AS-ODN + Lipo group, and COX-2 AS-ODN + Lipo + PGE2 group. Leica microscopy was used to observe the angiogenesis in the transplanted carcinoma. RESULTS: RT-PCR showed that the downregulation of expression of COX-2 mRNA in the PC3 cells with the increase of the COX-2 AS-ODN concentration, peaking at the concentration of 0.2 micro mol/L. The effect of COX-2 AS-ODN was strongest by the 12th hour after transfection and then began to decrease and basically disappeared 48 hours after. Western blotting showed that COX-2 AS-ODN, especially that of the concentration of the expression of 9 micro g/bottle, inhibited the expression of COX-2 AS-ODN. The angiogenesis of the transplanted carcinoma in the eggs was significantly inhibited in the Lipo + COX-2 AS-ODN group, the density of newly generated vessels in the Lipo + COX-2 AS-ODN + PGE2 group was between those of the other 2 groups. CONCLUSION: COX-2 AS-ODN significantly inhibits the angiogenesis in the pancreatic carcinoma. Endogenous COX-2 AS-ODN may play an important role in such a process and PGE2 may play an intermediate role therein.