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目的观察人CCL20基因短发夹RNA表达载体(pSCS-EGFP)转染人胚胎肾细胞(293FT细胞),以及对其表达CCL20 mRNA和分泌CCL20蛋白的抑制作用。方法用脂质体转染法把构建成功的3个pSCS-EGFP质粒(其中pSCS-EGFP-1为CCL20基因错配型,pSCS-EGFP-2和pSCS-EGFP-3均为CCL20基因特异型)转染到293FT细胞,24h后分别用荧光显微镜照相和流式细胞仪检测293FT细胞的转染率。将未转染及分别转染3种质粒的293FT细胞培养48h,用TNF-α和IL-1β刺激培养24h后,分别用荧光定量RT-PCR和ELISA法检测CCL20基因mRNA和蛋白的表达水平,计算其抑制率。结果3种质粒(pSCS-EG-FP-1～3)转染293FT细胞的效率分别为(73.8±5.3)%、(50.0±4.8)%、(56.8±2.9)%。加细胞因子刺激下,3种载体对293FT细胞表达CCL20 mRNA相应的抑制率分别为(18.53±34.44)%、(90.40±3.94)%和(92.50±6.15)%;上清中CCL20蛋白相应的抑制率分别为(14.88±17.39)%、(71.76±8.27)%和(88.56±1.74)%。未加细胞因子刺激下,CCL20 mRNA的相应抑制率分别为(61.37±11.72)%、(84.33±12.78)%和(80.27±15.84)%;CCL20蛋白的相应抑制率分别为(19.91±48.12)%、(-27.87±50.24)%和(87.21±8.36)%。结论特异性CCL20shRNA表达载体能明显下调细胞因子刺激人胚胎肾细胞表达CCL20 mRNA及蛋白,该载体为研究肾移植排斥反应等CCL20相关性肾脏疾病的治疗提供了一定的实验基础。
Objective To observe the inhibitory effect of human CCL20 gene short hairpin RNA (pSCS-EGFP) on human embryonic kidney cells (293FT cells) and its expression of CCL20 mRNA and secretion of CCL20 protein. METHODS: Three pSCS-EGFP plasmids were successfully constructed by lipofectamine transfection (pSCS-EGFP-1 was CCL20 gene mismatch, pSCS-EGFP-2 and pSCS-EGFP-3 were all CCL20 genotypes) The 293FT cells were transfected into 293FT cells. The transfection efficiency of 293FT cells was detected by fluorescence microscopy and flow cytometry respectively 24h after transfection. 293FT cells transfected with three plasmids were cultured for 48h. After cultured with TNF-α and IL-1β for 24 hours, the expression of CCL20 mRNA and protein were detected by RT-PCR and ELISA respectively. Calculate the inhibition rate. Results The efficiency of transfecting 293FT cells with three plasmids (pSCS-EG-FP-1 ~ 3) were (73.8 ± 5.3)%, (50.0 ± 4.8)% and (56.8 ± 2.9)%, respectively. The cytotoxicity of CCL20 mRNA in 293FT cells was (18.53 ± 34.44)%, (90.40 ± 3.94)% and (92.50 ± 6.15)%, respectively. The corresponding inhibition of CCL20 protein in the supernatant The rates were (14.88 ± 17.39)%, (71.76 ± 8.27)% and (88.56 ± 1.74)%, respectively. The corresponding inhibitory rates of CCL20 mRNA were (61.37 ± 11.72)%, (84.33 ± 12.78)% and (80.27 ± 15.84)%, respectively. The corresponding inhibitory rates of CCL20 protein were (19.91 ± 48.12)% , (- 27.87 ± 50.24)% and (87.21 ± 8.36)%, respectively. Conclusion The specific CCL20shRNA expression vector can significantly down-regulate the expression of CCL20 mRNA and protein in human embryonic kidney cells by cytokines. This vector provides experimental basis for the study of CCL20-related renal diseases such as renal allograft rejection.