松果菊苷对人肝癌细胞HepG2生长和缝隙连接细胞通讯的影响

Effects of echinacoside on growth and gap junctional intercellular communication in HepG2 of human hepatoma carcinoma cell

目的观察松果菊苷(ECH)对人肝癌细胞Hep G2生长和缝隙连接细胞通讯(GJIC)的影响。方法 Alamar Blue法绘制Hep G2细胞生长曲线,并检测ECH的细胞毒作用;划痕标记染料示踪技术(SL/DT)法观察空白对照组、API阳性对照组、AGA阴性对照组和ECH组对GJIC的影响;异硫氰酸荧光素(FITC)间接免疫荧光法观察ECH对缝隙连接蛋白Cx43表达的影响。结果 Hep G2细胞在20 000个/m L的细胞密度Alamar Blue还原率与时间有线性关系。ECH(5、10、20、40和80μM)处理Hep G2细胞24 h,细胞存活率分别为(92.69±3.35)%、(88.33±2.12)%、(80.97±2.50)%、(76.95±1.40)%和(69.09±1.11)%(P<0.01)。抑制率最高(80μM浓度下)为30.91%。IC50为467.8μM(368.3μg/m L)。SL/DT显示阳性对照芹黄素和ECH处理24 h后,荧光染料在划痕外4列细胞出现了明显荧光(++++),说明细胞出现染料传输的现象。间接免疫荧光法对Cx43表达进行计分,空白对照组细胞得分为(1.40±0.55)分,ECH组得分为(1.60±0.55)分。结果表明ECH组的Cx43表达增强不明显。结论 ECH对Hep G2细胞有细胞毒作用,同时可增强GJIC功能,但Cx43表达不明显,为ECH抗肿瘤机制提供依据。

Objective To observe the effects of echinacoside （ ECH） on growth and gap junctional intercel-lular communication （ GJIC） in HepG2 of human hepatoma carcinoma cell. Methods The cytotoxicity of ECH was detected through draw the growth curve of HepG2 cells by Alamar Blue method. The effects of blank group, API posi-tive control group, AGA negative control group and ECH group on GJIC were observed by scrape - loading dye trans-fer assay（ SL/DT）. The effect of EHC on expression of Cx43 protein was observed by flourescein isothiocyanate （FITC） indirect immunofluorescence assay. Results There was a liner correlation in alamar blue reduction rate and time of HepG2 cells under 20 000/mL cell density. The HepG2 cells were conducted by EHC （5,10,20,40 and 80 jjlM） for 24 h,and the survival rates of cells were （92. 69 ± 3. 35）% ,（88. 33 ± 2. 1 2 ）% ,（80. 97 ±2. 50） % , （76. 95 ± 1.40） % and （69. 09 ±1. 11）% （ P 〈0 . 0 1 ） . The highest inhibition rate （ under 80 jjlM con-centration） was 30. 91 % . IC50 was 467. 8 jjlM （3 6 8 .3 jjig/mL）. The apigenin and ECH were conducted after treat-ment 24 h, the SL/DT showed positive control, and fluorescent dyes showed obvious fluorescence （ ＋ ＋ ＋ ＋） in the scratches outside four cells, which indicated the cells appear the phenomenon of dye transfer. The expression of Cx43 was scored by indirect immunofluorescence assay. The score of blank control group was （1.40 ± 0. 55 ） and the score of ECH group was （ 1.60 ± 0. 55） . Conclusion ECH had cytotoxic effect on HepG2 cells, and can enhance GJIC function, but the expression of Cx43 was not obvious, which provide the basis for the anti - tumor mechanism of ECH.