血卟啉光动力对人胆管癌细胞转移潜能影响实验研究

Inbibitional effects of photodynamic therapy of hematoporphyrin derivative on proliferation and invasion of human cholangiocarcinoma cells

目的:探讨血卟啉衍生物介导的光动力疗法(hematoporphyrin derivative,HPD-PDT)对人胆管癌细胞增殖和侵袭的影响。方法:体外培养人胆管癌细胞系QBC939,经系列浓度的HPD处理,并应用半导体激光治疗仪给予不同强度的光照,CCK-8试剂盒检测HPD-PDT对人胆管癌细胞增殖的影响;Transwell小室(Matrigel侵袭实验)检测HPD-PDT对人胆管癌细胞体外侵袭能力的影响;酶联免疫吸附试验(enzyme linked immunosorbent assay,ELISA)检测细胞培养上清液中分泌的VEGF-C浓度变化;RT-PCR检测细胞中VEGF-C mRNA的表达变化;SP免疫组化染色观察细胞中VEGF-C蛋白的表达变化。结果:CCK-8检测结果显示,HPD-PDT在体外能够抑制QBC939细胞生长,当HPD质量浓度4μg/mL和光照剂量5J/cm2时,实验组A450值为1.541 7±0.098 3,明显低于空白对照组的2.027 8±0.198 6,P<0.01;细胞生长抑制率达到23.97%,且随着激光能量密度及光敏剂浓度的增加,其抑制作用也越明显。体外侵袭实验结果显示,实验组穿过Matrigel胶的细胞数为13.33±1.155,明显少于空白对照组的31.67±2.517,P<0.01;ELISA结果显示,实验组上清液中分泌的VEGF-C浓度为(1 558.241 7±97.114 3)ng/L,低于空白对照组的(1 716.375 0±45.047 2)ng/L,P<0.05;RT-PCR结果显示,实验组VEGF-C mRNA/hGAPDH的比值为0.7009±0.1872,明显低于空白对照组的1.542 5±0.107 8,P<0.01;SP免疫组化染色结果显示,实验组VEGF-C蛋白表达阳性的细胞百分率为24.52%±2.22%,明显低于空白对照组的56.94%±3.38%,P<0.01。结论:HPD-PDT能够抑制人胆管癌细胞QBC939的增殖活性及体外侵袭能力,并有可能通过下调VEGF-C因子的表达进一步抑制胆管癌的生长和转移。

OBJECTIVE： To discuss the effects of photodynamic therapy of hematoporphyrin derivative （HPD-PDT） on proliferation and invasion of human cholangiocarcinoma cell line QBC939. METHODS： Human cholangiocareinoma cells were cultured with serial concentrations of hematoporphyrin derivatives （HPD） followed by irradiation of different dosage of laser light,then Cell Counting Kit-8 （CCK-8） was applied to measure the relative inhibitory rate of HPD-PDT for the cells. The invasive ability of HPD-PDT on QBC939 cells was detected by Transwell assay. The effects of HPD-PDT on the secretion of VEGF-C in QBC939 cells were measured by enzyme linked immunosorbent assay （ELISA）;RT-PCR tech- nique was used to investigate the transcriptional changes of gene in VEGF-C Furthermore, immunocytochemistry was used to examine the changes of its protein expression. RESULTS： HPD-PDT inhibited QBC939 cell line growth in vitro, and a significant difference of the A450 value between the experimental group and control group was observed （1. 541 7 ± 0. 098 3 vs 2. 027 8±0. 198 6,P〈0.01）. When the concentration of HPD was 4/lg/mL and light irradiation was 5 J/cm2 , the relative growth inhibition rate of QBC939 cells was 23.97% ,and with the laser energy density and the concentration of photosensitizer increased, the inhibitory effect was more obviously. In vitro invasion, cells passed Matrigel in experimental group （ 13.33 ± 1.155） were less than that in control group （31.67 ± 2.517）, with P〈 0.01. In ELISA test, the secretion of VEGF-C in experimental group （1 558. 241 7±97. 114 3） ng/L was less than that of control group （1 716. 375 0±45. 047 2） ng/L,with P〈0.05. In RT-PCR test,the expression of mRNA of VEGF-C（0. 700 9-t-0. 187 2） in experimen- tal group was significantly less than that in control group （i. 542 5± 0. 107 8）, with P〈0.01. In IHC test, the positive rate of VEGF-C protein （24.52±2.22）% was also significantly less than that in control group （56.94± 3.38）%, with P%0.01. CONCLUSIONS.. HPD-PDT inhibits the growth proliferation and invasion ability of human cholangiocarcinoma cells. HPD-PDT is likely to inhibit the growth and metastasis of cholangiocarcinoma further by reducing the translational level of VEGF-C gene.