探讨 PUMA 基因转染是否增强胰腺癌AsPC 1细胞对5 FU致凋亡的敏感性。方法: 采用脂质体转染法将PUMA pCEP4和空载体pCEP4质粒转染入胰腺癌AsPC 1细胞中，G418筛选阳性细胞。将系列浓度（0.01～100 μmol/L）的5 FU 分别作用于AsPC 1、AsPC 1/PUMA和AsPC 1/pCEP4细胞72 h，MTT法测定各组细胞的存活率并计算IC 50 ， FCM、断裂DNA琼脂糖凝胶电泳和TUNEL法检测细胞凋亡情况,Western blotting检测各组细胞PUMA蛋白表达的变化。 结果: AsPC 1 、AsPC 1/PUMA和AsPC 1/pCEP4细胞的5 FU IC 50 分别为(12±1.9)、（1.6±04）和(10.4±1.6)μmol/L，AsPC 1/PUMA细胞对5 FU作用的敏感性增加了7.5倍。5 FU以剂量依赖方式诱导AsPC 1细胞凋亡，但对AsPC 1/PUMA细胞所诱导的凋亡比AsPC 1和AsPC 1/pCEP4 更明显。低浓度5 FU(0.1 μmol/L)轻微引起AsPC 1/pCEP4\[(1.14±0.28)%\]和AsPC 1细胞凋亡\[（0.9±0.23)%\]，但能诱导AsPC 1/PUMA细胞明显凋亡\[（6.47±1.42)%\]；高浓度5 FU (1 μmol/L)诱导各组细胞凋亡，但 AsPC 1/PUMA 细胞凋亡率\[（34.54±9.36)%\]明显高于AsPC 1\[（12.8±3.74)%\]和AsPC 1/pCEP4细胞\[ （15.8±5.15)% \]，差异均有统计学意义（ P <0.01）；FCM、断裂DNA琼脂糖凝胶电泳和TUNEL方法检测显示相同的结果。PUMA蛋白在AsPC 1/ PUMA 细胞中的表达明显高于AsPC 1和AsPC 1/pCEP4细胞。结论: PUMA 基因转染明显地增强了胰腺癌AsPC 1细胞对 5 FU 致凋亡作用的敏感性。

To investigate whether PUMA gene transfection can increase sensitivity of pancreatic cancer cells (PC) to 5 FU induced apoptosis. Methods: PUMA pCEP4 containing full length PUMA cDNA or pCEP4 was transfected into human pancreatic cancer cell line AsPC 1 by lipofectamine transfection, G418 selection was used to select positive cells. AsPC 1, AsPC 1/PUMA and AsPC 1/pCEP4 cells were separately treated with serial concentrations of 5 FU（0.01 100 μmol/L）. MTT assay was used to determine the cell survival rate in each group and IC 50 of 5 FU was calculated. TUNEL，FCM and DNA ladder observation were employed to study cell apoptosis. Western blotting was performed to detect the expression of PUMA protein. Results: The 5 FU IC 50 values of AsPC 1, AsPC 1/PUMA and AsPC 1/pCEP4 cells were (12±1.9)μmol/L,（1.6±0.4）μmol/L and (10.4±1.6) μmol/L, respectively, with the sensitivity of AsPC 1/PUMA cells increased by 7.5 folds. 5 FU induced cell apoptosis of AsPC 1 cells in a dose dependent manner, with the apoptosis of AsPC 1/PUMA cells more prominent than those of AsPC 1 and AsPC 1/pCEP4 cells. Low concentration of 5 FU (0.1 μmol/L) induced few apoptosis of AsPC 1/pCEP4 cells（\[1.14±0.28\]%) and AsPC 1 cells (\[0.9±023\]%), and induced apoptosis in AsPC 1/PUMA cells（\[6.47±1.42\]%). High concentration of 5 FU (1.0 μmol/L ) induced apoptosis in all groups, with that in AsPC 1/PUMA cells（\[34.54±9.36\]%) significantly higher than those in AsPC 1/pCEP4 cells（\[15.8±5.15\]%) and AsPC 1 cells (\[12.8±3.74\]%, both P <0.01) . FCM and electrophoresis showed the same results. Expression of PUMA protein in AsPC 1/PUMA cells was significantly higher than those in AsPC 1 and AsPC 1pCEP4 cells. Conclusion: PUMA gene transfection greatly enhances the sensitivity of AsPC 1 cells to 5 FU induced apoptosis.

卫生部科学研究基金资助项目(No.WKJ 2004 2 001)