JNK3重组腺病毒促进表柔比星诱导的人成神经细胞瘤SH-SY5Y细胞凋亡  

JNK3 recombinant adenovirus promotes apoptosis of human neuroblastoma SH-SY5Y cells induced by epirubicin

作　　者：韩卿[1] 宋方洲[1] 易发平[1] 卜友泉[1] 王治东[2] 李长燕[2] 杨晓明[2] 

机构地区：[1]重庆医科大学生物化学与分子生物学教研室,临床检验诊断学省部共建教育部重点实验室,重庆400016 [2]军事医学科学院放射医学研究所,北京100850

出　　处：《第二军医大学学报》2008年第12期1460-1465,共6页Academic Journal of Second Military Medical University

基　　金：国家自然科学基金(30671008);重庆市自然科学基金重点项目(CSTC2007BA5012);国家外专局项目(20075000019)~~

摘　　要：目的:构建人JNK3基因重组腺病毒,检测其对人成神经细胞瘤SH-SY5Y细胞增殖的影响;以表柔比星作为凋亡诱导剂,检测其对细胞凋亡的影响。方法:以pDBLeu-JNK3质粒为模板PCR扩增人JNK3基因全长,构建重组穿梭载体pAdTrack-CMV-JNK3,线性化后与骨架载体pAdEasy-1在细菌BJ5183内同源重组,在HEK293细胞中进行病毒包装和扩增,经PCR方法鉴定后,用包装后的病毒上清感染人成神经细胞瘤SH-SY5Y细胞,Western印迹方法检测JNK3蛋白的表达;MTT实验检测其对细胞增殖的影响;流式细胞术和琼脂糖凝胶电泳检测表柔比星诱导的细胞凋亡情况。结果:核酸测序和PCR鉴定表明成功构建Ad-JNK3,终点稀释试验测定扩增的腺病毒滴度为6.5×1010pfu/ml;Ad-JNK3在SH-SY5Y细胞中表达JNK3蛋白;MTT检测结果表明Ad-JNK3可抑制SH-SY5Y细胞生长,抑制率为28.08%;流式细胞术结果表明Ad-JNK3可明显促进表柔比星诱导的细胞凋亡,琼脂糖凝胶电泳可观察到DNA梯形条带。结论:重组腺病毒Ad-JNK3能显著抑制SH-SY5Y细胞增殖,促进表柔比星诱导的SH-SY5Y细胞凋亡,为研究JNK3的作用机制及将其用于人成神经细胞瘤的基因治疗提供了条件。Objective： To construct recombinant human JNK3 adenovirus and study its influence on the proliferation of human neuroblatoma SH-SYSY cells, and to study its influence on epirubicin-induced apoptosis. Methods： The full-length JNK3 cDNA fragment was amplified by PCR from the pDBLeu-JNK3 plasmid to construct the shuttle plasmid AdTrack-CMV-JNK3. Then the linearized shuttle plasmid was co-transformed into BJ5183 bacteria with backbone vector pAdEasy-1 to obtain the recombinant adenoviral plasmid Ad JNK3 by homologous recombination. The linearized recombined adenovirus Ad-JNK3 was then transfected into HEK293 cells for packing and amplification. Viral titers were measured by endpoint dilution assay. Ad- JNK3 was identified by PCR. The expression of Ad-JNK3 in SH-SY5Y cells was detected by Western blotting assay. The influence of Ad-JNK3 on the proliferation of SH-SYSY cells was assayed by MTT after cells were infected by 100 pfu/ml adenovirus. The cell apoptosis induced by 0. 5 μg/ml epirubicin was detected by flow cytometry method and agarose gel electrophoresis. Results： The recombinant adenoviral shutter vector AdTrack-CMV-JNK3 and recombinant adenoviral vector Ad-JNK3 were successfully constructed as identified by sequence analysis. PCR assay showed that adenovirus Ad-JNK3 contained JNK3 gene. After amplification in packing cell HEK293, 6. 5× 10^10 pfu/ml titer of Ad-JNK3 was obtained. Western blotting assay showed that JNK3 protein was expressed in SH-SY5Y cells. After infected by 100 pfu/ml adenovirus, the proliferation of SH-SY5Y cells was inhibited by 28.08% with MTT method. Flow cytometry showed that Ad-JNK3 significantly promoted the apoptosis of SH-SY5Y cells induced by epirubicin. The DNA ladder of agarose gel electrophoresis was clearly seen. Conclusion：Ad-JNK3 can obviously inhibit the growth of SH-SY5Y cells and promote apoptosis induced by epirubicin, which provides a solid foundation for further studies on the function of the JNK3 gene and applying it in gene therapy for human neuroblastoma.

关 键 词：JNK丝裂原活化蛋白激酶类 重组腺病毒 表柔比星 人成神经细胞瘤 细胞凋亡 

分 类 号：R739.4[医药卫生—肿瘤] R73-36[医药卫生—临床医学]