携带miR29c重组腺病毒的制备及其促骨髓瘤细胞凋亡作用

doi:10.3969/cmba.j.issn.1673-713X.2012.01.006

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摘要目的制备携带 miR29c 的重组腺病毒 Ad5F11p-miR29c，评价 miR29c 对骨髓瘤细胞凋亡的影响。方法巢式 PCR 扩增得到 hsa-miR29c，将 hsa-miR29c 基因克隆到腺病毒穿梭质粒 pShuttle-CMV，得到 pShuttle- CMV-miR29c。鉴定正确后，PmeI 线性化并转化含 Ad5F11p 骨架质粒的 BJ5183 感受态细胞，重组获得腺病毒载体。PacI 线性化的重组腺病毒质粒转染 HEK293 细胞，制备重组腺病毒 Ad5F11p-miR29c。以对照病毒 Ad5F11p-EGFP 感染人骨髓瘤细胞系 SKO-007、U266 和 XG7，通过流式细胞术确定最佳感染复数。Ad5F11p-miR29c 以最佳感染复数感染骨髓瘤细胞，流式细胞术检测 miR29c 对细胞凋亡的影响。结果 PCR 扩增获得 miR29c 基因，成功将其连接到腺病毒穿梭质粒 pShuttle-CMV，经测序鉴定正确。采用 Ad-easy 系统，成功构建并制备了 CMV 启动的重组腺病毒 Ad5F11p-miR29c。流式细胞术结果显示，Ad5F11p-EGFP 对 SKO-007 和 U266 细胞的最佳感染复数为 150 MOI；而 XG7 为 100 MOI。Ad5F11p-miR29c 以最佳感染复数感染细胞 48 h 后，细胞凋亡率升高。SKO-007 细胞的凋亡率达到 26.5%，XG7 细胞的凋亡率达到 46.9%。结论成功制备重组腺病毒 Ad5F11p-miR29c，并证实 miR29c 能够诱导骨髓瘤细胞凋亡。

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	张怡堃
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	陈协群
	王立生

关键词 ：微RNAs； ')" href="#">微RNAs； , 细胞凋亡； ')" href="#">细胞凋亡； , 基因表达； ')" href="#">基因表达； , 质粒； ')" href="#">质粒； , 腺病毒科； ')" href="#">腺病毒科； , 骨髓瘤')" href="#">骨髓瘤

Abstract：Objective The aim of this study is to construct a recombinant adenovirus expression vector of Ad5F11p-miR29c and to evaluate its pro-apoptotic effects on myeloma cells. Methods Nest-PCR was applied to get the gene of hsa-miR29c. Then the hsa-miR29c gene was inserted into the pShuttle vector. The resulted pshuttle vector pShuttle-CMV-miR29c was linearied by PmeI, followed by electric transformation into BJ5183 cells containing Ad5F11p, resulting in a recombined adenovirus vector by. The recombined adenovirus vector was linearied by PacI, and then was transfected into HEK293 cells to get Ad5F11p-miR29c. The adenovirus was packaged and produced 8 ~ 12 days after transfection and confirmed by PCR. The optimal MOI was detected by flow cytometry using Ad5F11p-EGFP in multiple myeloma cell lines (SKO-007, U266, and XG7). Flow cytometry was also applied to test the Adenovirus-mediated proapoptotic effects in human multiple myeloma cell lines. Results miR29c gene was obtained by Nest-PCR and ligated into pShuttle-CMV. Recombined adenovirus Ad5F11p-miR29 was successfully constructed using Ad5F11p system, in which the miR29c gene expression was regulated by CMV. Flow cytometry results showed that the optimal MOI in SKO-007 and U266 were both 150, while in XG7 was 100. After 48 h infection of Ad5F11p-miR29c with the optimized MOI, the percentage of apoptotic cells was increased in SKO-007 and XG7 cells. Conclusions We successfully constructed the recombinant adenovirus expression vector Ad5F11p-miR29c and demonstrated that miR29c can induce apoptosis in human myeloma cells.

Key words： MicroRNAs')" href="#">MicroRNAs Apoptosis')" href="#">Apoptosis Gene expression')" href="#">Gene expression Plasmids')" href="#">Plasmids Adenoviridae ')" href="#"> Multiple myeloma')" href="#">Multiple myeloma

收稿日期: 2011-10-24

基金资助:

“重大新药创制”国家科技重大专项（2009zx09503-019）

通讯作者: 王立生 E-mail: wangls@bmi.ac.cn

引用本文:

张怡堃, 王华, 杨萍, 李泽良, 杨月峰, 陈协群, 王立生. 携带miR29c重组腺病毒的制备及其促骨髓瘤细胞凋亡作用[J]. 《中国医药生物技术》杂志, 2012, 7(1): 20-25.
ZHANG Yi-Kun, WANG Hua, YANG Ping, LI Ze-Liang, YANG Yue-Feng, CHEN Xie-Qun, WANG Li-Sheng. Construction of recombinant adenovirus expression vector of Ad5F11p-miR29c and evaluation of its pro-apoptotic effects on myeloma cells. , 2012, 7(1): 20-25.

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