泡球蚴囊液对大鼠肝星状细胞MAPK信号通路5个相关基因的影响

Effect of Echinococcus multilocularis Cyst Fluid on the Expression of Five MAPK-pathway Genes of Rat Hepatic Stellate Cells

目的探讨泡球蚴囊液对大鼠肝星状细胞丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)信号通路5个相关基因的影响。方法采用大鼠肝星状细胞株HSC-T6与不同蛋白浓度的泡球蚴囊液(0.01、0.025、0.05、0.1、0.2、0.4、0.9、1.7、3.4、6.8和13.5 mg/ml)共培养24 h后收集细胞,同时收集对照组(未加泡球蚴囊液干预)细胞,显微镜下观察HSC-T6细胞的形态变化。利用荧光实时定量PCR检测大鼠肝星状细胞的细胞外调节蛋白激酶(ERK)、c-Jun氨基末端激酶(JNK)和丝裂原活化蛋白激酶(p38)的表达情况。结果蛋白浓度为13.5 mg/ml泡球蚴囊液与HSC-T6细胞共培养24 h后,大部分细胞发生固缩,呈脱落前兆。6.8 mg/ml囊液组部分细胞固缩,呈脱落前兆;而部分HSC-T6细胞收缩,呈长梭形,细胞生长出许多细长的伪足,是正常状态的2倍以上。3.4 mg/ml囊液组部分细胞呈收缩的长梭形,生长出许多细长的伪足,但大部分细胞仍呈现为贴壁的胞体较大的不规则多边形,且伸出较短的伪足与其他细胞连接成片状。<1.7 mg/ml囊液组与对照组细胞形态上无差异。实时荧光定量PCR检测显示,泡球蚴囊液浓度>0.4 mg/ml时,ERK1/2、JNK1/2和p38 m RNA水平显著增加;囊液浓度为6.8 mg/ml时,ERK1/2、JNK1/2和p38 m RNA较对照组高表达(P<0.05)。结论浓度为6.8 mg/ml的泡球蚴囊液可显著影响大鼠肝星状细胞ERK1/2、JNK1/2和p38 m RNA水平。

Objective To investigate the effect of Echinococcus multilocularis cyst fluid on five MAPK （mitogen-activated protein kinase）-pathway genes of rat hepatic stellate cell. Methods Rat hepatic stellate cell line， HSC-T6 cells were co-cultured with different protein concentrations of E. multilocularis cyst fluid（0.01， 0.025， 0.05， 0.1， 0.2， 0.4， 0.9， 1.7， 3.4， 6.8， and 13.5 mg/ml） for 24 h. HSC-T6 cells cultured with complete medium served as control group. The morphological change of cells was observed under the microscope. The expression of extracellular signal-regulated kinase （ERK）， c-Jun N-terminal kinase （JNK） and mitogen-activated protein kinase（p38） in HSC-T6 cells was detected by real time fluorescent quantitative PCR. Results After co-cultured for 24 h， most HSC-T6 cells in 13.5 mg/ml group shrank as a precursor to slough off； In 6.8 mg/ml group， some HSC？鄄T6 cells shrank and changed to long fusiform shape with many slender pseudopodia； In 3.4 mg/ml group， most HSC-T6 cells showed as adherent cells with an irregular polygon shape， formed a sheet with short pseudopodia. There was no difference in cell morphology between 〈1.7 mg/ml groups and control group. When the protein concentration was above 1.7 mg/ml， the mRNA level of ERK1/2， JNK1/2， and P38 increased significantly increased. In 6.8 mg/ml cyst fluid group， the mRNA level of ERK1/2， JNK1/2， and P38 was higher than that of the control （P〈0.05）. Conclusion 6.8 mg/ml Echinococcus multilocularis cyst fluid can have a significant impact on mRNA levels of ERK1/2， JNK1/2 and p38 in rat hepatic stellate cells.