融合自杀基因对骨肉瘤细胞作用的实验研究

山东大学学报(医学版) ›› 2009, Vol. 47 ›› Issue (10): 54-59.

融合自杀基因对骨肉瘤细胞作用的实验研究

李伟¹，焦宗乾²，孙成良¹，刘立成¹

1. 山东大学附属省立医院关节外科，济南 250021； 2. 山东省平度市中医院骨外科，山东平度 266700

收稿日期:2008-11-25 出版日期:2009-10-16 发布日期:2009-10-16
作者简介:李伟(1969- ), 男，医学博士、博士后，副主任医师、副教授，硕士生导师，主要从事关节外科基因治疗研究。 Email:greatli2000@yahoocom.cn
基金资助:
国家自然科学基金项目（30672115）；山东省优秀中青年

科学家科研基金（02BS082）

Effect of CD, TK fusion suicide gene on MG63 cells

LI Wei¹, JIAO Zongqian², SUN Chengliang¹, LIU Licheng^1

1. Department of Joint Surgery, Provincial Hospital affilated to Shandong University, Jinan 250021, China；2. Department of Orthopaedics, Pingdu Traditional Chinese Medical Hospital, Pingdu 266700, Shandong, China

Received:2008-11-25 Online:2009-10-16 Published:2009-10-16

摘要/Abstract

摘要：

目的观察单纯疱疹病毒-胸苷激酶/胞嘧啶脱氨酶融合自杀基因（HSV TK/CD）对骨肉瘤细胞增殖的影响。方法将含TK/CD的重组腺病毒载体感染人骨肉瘤细胞系MG63细胞，测定感染效率后，分别给予不同浓度的前体药物和不同感染复数的自杀基因重组腺病毒，计算瘤细胞增殖抑制率，MTT法测定细胞存活率，并用流式细胞术和Hochest 33342 染色法分析杀伤机制。结果含自杀基因的重组腺病毒成功感染MG63细胞；不同感染复数和不同前体药物浓度下，融合自杀基因系统对瘤细胞的杀伤作用明显强于单基因和双基因相加的作用 (P＜0.05)；其杀伤机制为致细胞坏死和细胞凋亡。结论融合自杀基因HSVTK/CD对骨肉瘤细胞系MG63细胞的生长有明显的抑制作用。

关键词: 骨肉瘤, 胸苷激酶基因, 胞嘧啶脱氨酶基因, 融合自杀基因

Abstract:

To observe the effect of herpes simplex virusthymidine kinase/cytosine deaminase fusion suicide gene on MG63 cells. MethodsThe target cells were infected by recombinant adenovirus expressed TK and/or CD and the efficiency of infection was measured with green fluorescent protein. MG63 cells were treated with different prodrug concentrations and multiple of infection(m.o.i). Cell survival rate and proliferation inhibiting rate were measured. The killing mechanism was also investigated with flow cytometry (FCM) and Hochest 33342 fluorescent staining. Results100% MG63 cells could be infected with recombinant adenovirus vector either single or double genes as determined by GFP detection. Survival of MG63 cells in AdCDTK infected group was 31% when GCV was used and 44% to 5Fc. Combination with two prodrugs, cell survival was reduced to 90%, lower than the AdTK(79%), AdCD(63%) or AdTK+AdCD(66%) groups with the same prodrug treatmemt (P＜0.05). Concentration of prodrugs or m.o.i of adenovirus in the fusion gene group was lower than that of the single gene groups with respect to the same inhibition effect on MG63 cells. The infected MG63 cells with the double gene group had greater bystander effect than both AdCD and AdTK groups. The mechanism of killing MG63 cells demonstrated by FCM and fluorescent staining was cytotoxic action and apoptosis. ConclusionThese results prove that the double gene expressed recombinant adenovirus could be used successfully to inhibit the proliferation of MG63 cells and has more powerful killing effect than the single gene group.

Key words: Osteosarcoma; Thymidine kinase gene; Cytosine deaminase gene; Fusion suicide gene

中图分类号:

R684.3

. 融合自杀基因对骨肉瘤细胞作用的实验研究[J]. 山东大学学报(医学版), 2009, 47(10): 54-59.

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