融合自杀基因抑骨肉瘤生长的实验研究  

Experimental study of fusion suicide gene inhibiting osteosarcoma growth.

作　　者：李伟[1] 沙丹[2] 马春燕[3] 

机构地区：[1]山东大学附属省立医院关节外科 [2]山东大学附属省立医院化疗科 [3]山东大学附属省立医院中心实验室

出　　处：《华中医学杂志》2009年第1期1-5,23,共6页Central China Medical Journal

基　　金：国家自然科学基金资助项目(No.30672115);山东省优秀中青年科学家科研基金(02BS082)

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和对裸鼠骨肉瘤生长的影响。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作用于瘤细胞,测定细胞A值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Hochest33342染色法分析其杀伤机制;人骨肉瘤细胞系MG-63细胞于裸鼠成瘤后将其分为6组(每组8只):PBS对照组(A组);重组腺病毒(Ad)-Lac-Z/GCV+5-FC组(B组);Ad-TK/GCV组(C组);Ad-CD/5-FC组(D组);Ad-TK/GCV+Ad-CD/5-FC组(E组);Ad-TK-CD/GCV+5-FC组(F组)。各组经治疗后观察肿瘤的生长状况及组织病理学改变。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作用和双基因相加的作用,且有显著性差异(P<0.05);杀伤机制为致细胞坏死和细胞凋亡。裸鼠实验中,B组与A组比较,肿瘤体积与肿瘤坏死程度无明显差异(P>0.05);与A、B组相比较,C～F组肿瘤的生长均受到明显抑制(P<0.05),且E、F组与C、D比较有显著性差异(P<0.05)。结论融合自杀基因HSV-TK/CD对骨肉瘤细胞系MG-63细胞和裸鼠骨肉瘤的生长均有明显的抑制作用。Objective To observe the effect of herpes simplex virus-thymidine kinase/cytosine deaminase fusion suicide gene on MG-63 cells and the osteosarcoma growth of nude mouse. Methods The target cells were infected by recombinant adenovirus expressed TK and/or CD and efficiency of infection was measured with green fluorescent protein. MG-63 cells were treated with different prodrug concentration 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. Osteosarcoma model of nude mouse was made with MG-63 cells injected into axillary fossa. Forty eight nude mouse were divided randomly into six groups： A： PBS control group; B： Ad-Lac-Z/GCV ＋ 5-FC group; C： Ad-TK/GCV group; D： Ad-CD/ 5-FC group; E： Ad-TK/GCV ＋ Ad-CD/5-FC group; F： A＆TK-CD/GCV ＋ 5-FC group. Tumor volume, rate of tumor growth inhibiting and histopathology were analysed after each group was treated with gene and prodrug. Results 100% 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 C, CV was used and 44% to 5-FC. Combining 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 prodrug treatmemt（P〈0. 05）. Concentration of prodrugs or m. o. i of adenovirus in 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 double gene group has greater by-stander effect than both Ad-CD and Ad-TK groups. The mechanism of killing cells demonstrated by FCM and fluorescent staining is cytotoxic action and apoptosis. In nude mouse experiment, there was no significant difference in tumor volume and rate of tumor cell death between second group and first group.

关 键 词：骨肉瘤 单纯疱疹病毒-胸苷激酶基因 胞嘧啶脱氨酶基因 MG-63细胞 裸鼠 

分 类 号：R738.1[医药卫生—肿瘤]