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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 observe the targeted migration and cytotoxicity of lentivirus-thymidine kinase (Lenti-TK) / mesenchymal stem cells (MSCs) on CD133 + stem cells of nasopharyngeal carcinoma. Methods: Lenti-TK was constructed and transfected into Lenti-TK-MSCs. The expression of HA-TK in Lenti-TK-MSCs was detected by RT-PCR and Western blotting. CD133 + cells were sorted from nasopharyngeal carcinoma 5-8F cells by immunomagnetic beads method; the trend of Lenti-TK-MSC on CD133 + 5-8F cells was detected by Transwell chamber migration assay; Lenti-TK-MSC combined with ganciclovir CCK-8 kit was used to test its cytotoxicity and bystander effect. The CD133 + 5-8F cells were co-cultured with ganciclovir, Lenti-TK-MSC / GCV. Results: The recombinant lentiviral vector Lenti-TK was successfully constructed and its titer was 1 × 108UT / ml. The infection efficiency of Lenti-TK (MOI = 50) was 95.1 ± 0.1% at 72 h. The number of cells transfected with Lenti-TK-MSC in CD133 + 5-8F cells was significantly higher than that in CD133-5-8F cells (83.0 ± 8.7 vs 29.6 ± 5.3, 38.3 ± 5.2), P = 0.000]. Lenti-TK-MSC / GCV treatment group compared with the GCV treatment group alone, Lenti-TK-MSC / GCV conditioned medium (that is, the culture supernatant of Lenti-TK-MSC added 1 mg / L GCV for 48 h) (37.2 ± 2.3)% vs (98.5 ± 3.1)%, (83.8 ± 3.4)%, P = 0.000] in CD133 + 5-8F cells. When the number of Lenti-TK-MSC reached 20% of the total mixed cells (Lenti-TK-MSCs and CD133 + 5-8F cells), the viability of CD133 + 5-8F cells was (68.2 ± 2.3)%, Killing effect. CONCLUSIONS: MSCs have targeted migration and cytotoxicity towards nasopharyngeal carcinoma CD133 + 5-8F cells after Lenti-TK infection.