纳米脂质体槲皮素对Eca-9706细胞凋亡及相关蛋白表达的影响

Effects of nanoliposomal quercetin on apoptosis and expression of related proteins in Eca-9706 cells

目的:探讨纳米脂质体槲皮素(nLQ)诱导人食管癌Eca-9706细胞逆转化及凋亡的效应及其机制。方法:采用旋转蒸发法制备以氯仿和DMSO为溶剂的脂质体槲皮素,将其超声波破碎并经80nm滤膜过滤制成nLQ,不同浓度(10、20、40、80和100μmol/L)作用于Eca-9706细胞,用MTT法检测各组Eca-9706细胞的生长抑制率。分别取Eca-9706细胞,分为nLQ组(加40μmol/LnLQ)和对照组。TUNEL法检测2组细胞凋亡率;免疫细胞化学/免疫印迹法检测2组处理48h后Eca-9706细胞CyclinD1、PTEN、c-Met、VEGF、组蛋白去乙酰化酶(HDAC)1及NF-κB表达的变化。结果:各组Eca-9706细胞生长抑制率(F分别为128.041、142.683和231.054,P<0.001)和2组细胞凋亡率(t=94.770,P<0.001)比较,差异均有统计学意义。PTEN免疫反应性和印记信号增强(t分别为5.352和4.308,P均<0.05),Cyclin D1、c-Met、VEGF、HDAC1和NF-κB的免疫反应性减弱(t分别为4.006、9.184、13.853、4.698和3.575,P均<0.05),其印迹信号亦减弱(t分别为3.827、6.399、7.868、4.695和3.406,P均<0.05);以上各细胞蛋白的免疫细胞和免疫印迹信号之间呈正相关(rs分别为0.952、0.915、0.927、0.842、0.879和0.855,P均<0.05)。结论:nLQ可抑制Eca-9706细胞生长及增殖,逆转Eca-9706细胞PTEN的低表达和c-Met及VEGF的高表达,并通过抑制HDAC1和NF-κB及激活PTEN表达的HDAC抑制信号途径而诱导细胞凋亡。

Aim：To explore the mechanism for reverse transformation and apoptosis of Eca-9706 cells induced by nanoliposomal quercetin（nLQ）.Methods：A kind of liposomal quercetin, with chloroform＋DMSO being used as solvents,was prepared through rotary evaporation, and the nLQ was obtained after ultrasonic disruption and filtration.The cultured Eca-9706 cells were treated with nLQ at different concentrations,and cell growth inhibition rate for different exposure time （24,48,and 72 h） was detected by MTT.Eca-9706 cells were allocated into nLQ group （given 40 μmol/L nLQ） and control group.Apoptotic rate in the 2 groups was detected by TUNEL.After 48-hour-exposure,the expression of Cyclin D1,PTEN,c-Met,VEGF,HDAC1 and NF-κB in the 2 groups were detected by immunohistochemistry and immunoblotting.Results：Compared with the control group, the growth-inhibition rate （F24 h=128.041,F48 h=142.683 and F72 h=231.054,P0.001）and apoptotic rate（F=94.770,P0.001） of the groups of Eca-9706 cells had difference. The immunohistochemistry and immunoblotting showed that the expression of PTEN was up-regulated（t=5.352 and 4.308,P0.05）, while the immunohischemistry results of Cyclin D1, c-Met, VEGF,HDAC1 and NF-κB were down-regulated（t=4.006,9.184,13.853,4.698 and 3.575,P0.05）, and their immunoblotting results were also down-regulated（t=3.827,6.399,7.868,4.695 and 3.406,P0.05）.There was positive correlation in data above between immunocytochemistry and immunoblotting（rs=0.952,0.915,0.927,0.842,0.879,and 0.855,P0.05）.Conclusion：The Eca-9706 cell growth and proliferation could be inhibited by nLQ, and the overexpression of c-Met and VEGF and the low expression of PTEN in Eca-9706 cells could be reversed by nLQ. The Eca-9706 cells apoptosis could be induced through inhibiting HDAC1 and NF-κB and activating PTEN expressions.