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| 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 |
| 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* |
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| (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) |
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| 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6.5×1010 pfu/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。 |
| 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 |
| DOI：10.3724/SP.J.1008.2008.01460 |
| 投稿时间：2008-05-22修订日期：2008-07-21 |
| 基金项目: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). |
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| JNK3 recombinant adenovirus promotes apoptosis of human neuroblastoma SH-SY5Y cells induced by epirubicin |
| HAN Qing1,SONG Fang-zhou1*,YI Fa-ping1,BU You-quan1,WANG Zhi-dong2,LI Chang-yan2,YANG Xiao-ming2* |
| (1.Department of Biochemistry and Molecular Biology,Key Laboratory of Clinical Laboratory Diagnostics of Ministry of Education,Chongqing Medical University,Chongqing 400016,China*2.Institute of Radiation Medicine,Academy of Military Medical Sciences,Beijing 100850) |
| Abstract: |
| Objective:To construct recombinant human JNK3 adenovirus and study its influence on the proliferation of human neuroblatoma SH-SY5Y 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-SY5Y 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 HEK293,6.5×1010 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. |
| Key words: JNK mitogen-activated protein kinases recombinant adenovirus epirubicin human neuroblastoma apoptosis |
