光动力疗法对人胆管癌细胞QBC939凋亡的影响

The influence of photodynamic therapy on the apoptosis of bile duct cancer cells QBC939

目的研究血卟啉衍生物（hematoporphyrin derivative，HPD）介导的光动力作用（pho－todynamic therapy，PDT）对人胆管癌细胞QBC939凋亡的影响及其作用机制。方法体外培养人胆管癌QBC939细胞株，用不同浓度HPD处理，并用半导体激光治疗仪不同强度光照后，应用CCK8法检测PDT对QBC939细胞生长的相对抑制率。采用流式细胞术检测PDT作用前后QBC939细胞凋亡情况。应用RT-PCR检测PDT作用前后QBC939细胞中血管内皮细胞生长因子-C（VEGF-C）、环氧合酶-2（COX-2）基因表达情况。用SP免疫细胞化学法测定PDT作用前后QBC939细胞质中VEGF-C、COX-2两种蛋白表达情况。用ELISA法测定PDT作用前后QBC939细胞上清中VEGF-C、COX-2两种蛋白分泌情况。结果HPD-PDT在体外能够抑制QBC939细胞生长，HPD10mg／L经5J／cm2光照强度时，实验组与空白组A值差异有统计学意义（P〈0．05），细胞生长抑制率达到70％。继续增加药物浓度或光照剂量，差异无统计学意义，细胞存活率降低不明显。流式细胞术显示HPD-PDT明显促进QBC939细胞早期凋亡。RT_PCR显示HPD-PDT能明显抑制QBC939细胞中VEGF-C、COX-2基因表达。SP免疫细胞化学法显示HPD-PDT能抑制QBC939细胞质中VEGF-C、COX-2两种蛋白表达。ELISA法显示HPD-PDT能抑制QBC939细胞VEGF-C、COX-2两种蛋白细胞外分泌。结论HPD-PDT能抑制胆管癌QBC939细胞生长，促进胆管癌细胞早期凋亡。HPD-PDT对胆管癌QBC939细胞生长的抑制作用可能是通过促进早期凋亡实现的，而VEGF-C、COX-2从基因到蛋白水平低表达可能是促进胆管癌QBC939细胞早期凋亡的途径。

Objective To investigate the apoptotic effect and mechanism of photodynamic therapy, mediated by hematoporphyrin derivative, on the cholangiocellular carcinoma QBC939 cell line. Methods Cultured cholangiocellular carcinoma QBC939 cells in vitro were given different concentrations of hematoporphyrin derivatives with different light intensities. The relative growth inhibition rate of QBC939 cells was detected by the CCK8 method, and flow cytometry assay was used to detect apoptosis. RT-PCR examination was used to detect the transcriptional changes ot the mRNA ot vascular endothelial growth factor-C （VEGF-C） and cyclooxygenase-2 （COX-2）. Immunocytochemistry was used to measure the changes of proteins VEGF-C and COX-2, and enzyme-linked immunosorbent assay （ELISA） was used to examine the secretion of VEGF-C and COX-2 in QBC939 cells. Results HPD- PDT inhibited QBC939 cell line growth in vitro, and a significant difference of the A value between the PDT group and control group was observed （P^0. 05）. When the concentration of HPD was 10 mg/L and the light radiation was 5 J/cm2, the relative growth inhibition rate of the QBC939 cell line was 70%. If the drug concentration or light dose was increased, the result showed no statistically significant differences. Flow cytometry assay showed that HPD-PDT could promote apoptosis of QBC939 cells in an early stage. The RT-PCR examination showed that HPD-PDT could reduce the transcriptional changes of the mRNA of VEGF-C and COX-2 in QBC939 cells. Immunocytochemistry examination revealed that HPD-PDT could restrain the proteins of VEGF-C and COX-2 in QBC939 cells, and ELISA demonstrated that HPD-PDT could decrease the secretion of VEGF-C and COX-2 in QBC939 cells. Conelusions HPD-PDT could inhibit QBC939 cell growth and promote QBC939 cell apoptosis in an early stage. The growth inhibition effect may be achieved by the promotion of early apoptosis, which may result from the lower expression of VEGF-C and COX-2 from mRNA to protein level.