融合自杀基因对骨肉瘤细胞作用的实验研究

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。

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

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.