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摘要:
目的 构建含CDglyES融合基因的重组腺病毒，体外观察其对人卵巢癌细胞的直接抑制作用及其对血管内皮细胞生长的抑制作用。 方法 用细菌内同源重组方法构建腺病毒穿梭质粒prAdCDglyES。采用脂质体介导转染293包装细胞，扩增获取重组腺病毒rAdCDglyES。采用MTT法观察rAdCDglyES对卵巢癌细胞SKOV-3生长抑制的影响，同时观察其表达产物抑制脐静脉血管内皮细胞ECV-304增殖的作用。 结果 纯化的rAdCDglyES滴度是1×1013.3 TCID50/L，其对SKOV-3细胞生长抑制率是（83.1±6.3）%，与对照组rAd-LacZ（24.1±13.2）%相比有显著性差异（P < 0.01）。浓缩的转染rAdCDglyES的细胞培养上清液抑制ECV-304细胞增殖，抑制率是（78.7±1.6）%，而同样浓缩的转染rAd-CD的细胞培养上清液抑制率是（23.9±9.7）%，二者有显著性差异（P < 0.01）。 结论 研究结果表明重组腺病毒rAdCDglyES具有体外直接和间接抑制人卵巢癌细胞效能。 Abstract:Objective To construct a recombinant adenovirus containing CDglyES fusion gene, which can directly inhibit human ovarian cancer cell and indirectly inhibit vascular endothelial cell growth. Methods We constructed prAdCDglyES using a homologous recombination method in bacteria. The prAdCDglyES was transfected to 293 packaging cells using liposome, in which rAdCDglyES was packaged and amplified. MTT was used to observe the proliferation inhibition effect of rAdCDglyES on human ovarian cancer cells and the growth inhibition effect of expressing products of rAdCDglyES on ECV-304. Results The titer of rAdCDglyES was 1 × 1013.3 TCID50/L, whereas the inhibition rate on human ovarian cancer cell SKOV-3 was (83.1±6.3)%. This result is significantly different from the control rAd-LacZ, which had an inhibition rate of (24.1±13.2)% (P < 0.01). The concentrated culture supernatant from cells transfected with rAdCDglyES can inhibit ECV-304 cell proliferation at a rate of (78.7±1.6)%. This rate is significantly different compared with that of the control with the same concentration of culture supernatant from cells transfected with rAd-CD, with an effect on ECV-304 cell shown by an inhibition rate of (23.9±9.7)% (P < 0.01). Conclusion The results showed that the recombinant adenovirus rAdCDglyES could inhibit human ovarian cancer cells directly and indirectly. -
Key words:
- human ovarian cancer /
- cytidine deaminase /
- endostadine /
- adenovirus /
- gene therapy
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图 1 pAdTrackCMV-CD酶切电泳图
Figure 1. pAdTrackCMV-CD restriction enzyme cutting electrophoresis
1. Marker; 2. Double enzyme cutting pAdTrackCMV-CD; 3、4. pAdTrackCMV; 5. purified CD
图 2 pAdTrackCMV-CDglyES双酶切电泳图
Figure 2. pAdTrackCMV-CDglyES restriction enzyme cutting electrophoresis
1. Marker; 2. Double enzyme cutting pAdTrackCMV-CD; 3. pAdTrackCMV
图 3 PCR鉴定重组腺病毒prAdCDglyES
Figure 3. Recombinant adenovirus prAdCDglyES identified via PCR
1. DL2000 Marker; 2. CD upstream primer and ES downstream primer PCR amplification result; 3. CD upstream and downstream primer PCR amplification result; 4. ES upstream and downstream primer PCR amplification result
图 4 rAdCDglyES/5-FC系统处理的SKOV-3细胞（H & E×400）
Figure 4. rAdCDglyES/5–FC processing SKOV-3 cell (H & E×400)
图 5 SKOV-3细胞中重组腺病毒报告基因GFP的表达（H & E×400）
Figure 5. Expression of the recombinant adenovirus report gene GFP in SKOV-3 cell (H & E×400)
表 1 rAd-LacZ、rAd-CD、及rAdCDglyES对SKOV-3细胞生长的抑制作用 （
Table 1. Inhibition of rAd-LacZ, rAd-CD, and rAdCDglyES on proliferation of SKOV- 3 (
表 2 rAd-CD和rADCDglyES细胞培养上清对ECV-304细胞生长的抑制作用 （
Table 2. Inhibition of culture supernatant from cells transfected with rAd-CD and rAdCDg lyES on proliferation of ECV-304 (
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