Lenti-TK介导间充质干细胞对鼻咽癌CD133^+干细胞的靶向迁移及杀伤作用

Targeted migration and killing effect of mesenchymal stem cells infected with Lenti-TK on CD133^+ cancer stem cells in nasopharyngeal carcinoma

目的:观察慢病毒-胸苷激酶(lentivirus-thymidine kinase,Lenti-TK)/间充质干细胞(mesenchymal stem cell,MSC)对鼻咽癌CD133+干细胞的靶向迁移及杀伤作用。方法:构建包含TK基因的重组慢病毒表达载体Lenti-TK,感染MSC后得到Lenti-TK-MSC,RT-PCR及Western blotting检测Lenti-TK-MSC中HA-TK的表达。免疫磁珠法从鼻咽癌5-8F细胞中分选CD133+细胞;Transwell小室迁移实验检测Lenti-TK-MSC对CD133+5-8F细胞的趋向性;Lenti-TK-MSC联合更昔洛韦(ganciclovir,Lenti-TK-MSC/GCV)与CD133+5-8F细胞共培养,CCK-8试剂盒检测其对细胞的杀伤作用和旁观者效应。结果:成功构建重组慢病毒载体Lenti-TK,其滴度为1×108UT/ml,Lenti-TK(MOI=50)感染MSC 72 h时,感染效率达(95.1±0.1)%。Lenti-TK-MSC迁移至CD133+5-8F细胞组的细胞数明显多于CD133-5-8F细胞组、未分选5-8F细胞组[(83.0±8.7)vs(29.6±5.3)、(38.3±5.2),P=0.000]。Lenti-TK-MSC/GCV处理组与单独GCV处理组、Lenti-TK-MSC/GCV条件培养液(即Lenti-TK-MSC加入1 mg/L GCV培养48 h的培养上清)处理组相比,CD133+5-8F细胞的存活率明显降低[(37.2±2.3)%vs(98.5±3.1)%、(83.8±3.4)%,P=0.000]。Lenti-TK-MSC数量达到混合细胞总数(Lenti-TK-MSC和CD133+5-8F细胞)的20%时,CD133+5-8F细胞存活率为(68.2±2.3)%,表现出明显的旁观者杀伤效应。结论:Lenti-TK感染后MSC对鼻咽癌CD133+5-8F细胞具有靶向迁移及杀伤作用。

Objective： To investigate the targeted migration and killing effect of mesenchymal stem cells （MSCs） infected with Lenti-TK （thymidine kinase） vector on CD133 ＋ cancer stem cells in nasopharyngeal carcinoma. Methods： The recombinant lentiviral expression vector containing TK gene （Lenti-TK） was constructed and transduced into MSC （Lenti-TK-MSC）. Fusion tag-TK （HA-TK） expression was verified by RT-PCR and Western blotting. CD133 ＋cells were sorted from nasopharyngeal carcinoma 5-8F cells by immunomagnetic beads. The chemotactic ability of Lenti-TK-MSC to CD133 ＋5-8F cells was analyzed by Transwell assay. The CD133 ＋ 5-8F cells were co-cultured with Lenti-TK-MSC and GCV to detect its killing effect on cells by CCK-8 Kit. Results： The recombinant lentivirus vector Lenti-TK was success- fully constructed with titer being 1 ×l08 UT/ml. The transduction efficiency of Lenti-TK to MSC was （ 95.1 ± 0.1 ） %, 72 h after transduction at an MOI of 50. The migration number of Lenti-TK-MSC to CD133 ＋5-8F cells was more than that to CD133-5-8F cells and 5-8F cells （ [83.0 ±8.7] vs [29.6±5.3] vs [38.3 ±5.2] ;P =0.000）. The Lenti-TK-MSC/ GCV treatment significantly inhibited the growth of the CD133 ＋ 5-8F cells compared with the GCV group and the Lenti- TK-MSC/GCV condition medium group （the culture supernatant of Lenti-TK-MSC treated with 1 mg/L GCV for 48 h） （[37.2±2.31% vs [98.5±3.11% vs [83.8±3.41%, P=0.000）, with the cell survival being （68.2±2.3）%when the proportion of Lenti-TK-MSC was 20%, which showed a significant bystander killing effect. Conclusion： Lenti- TK infected MSC exert targeted migration and killing effects on CD133 ＋5-8F cells from nasopharyngeal carcinoma.