人白细胞介素10 cDNA重组质粒在细胞和大鼠体内的转染和表达

Transfection and expression of the recombinant plasmid of human IL-10cDNA in cells and rats

目的研究人白细胞介素10(hIL-10)cDNA重组质粒的转染和表达,为以后的基因治疗研究奠定基础。方法构建hIL-10 cDNA真核表达重组质粒pcDNA3-hIL-10后进行酶切和测序鉴定。以SA脂质体介导对照质粒pcDNA3及重组质粒pcDNA3-hIL-10转染COS7细胞和SD大鼠,用ELISA方法检测COS7细胞培养液及大鼠胰腺、肺和肝脏组织中hIL-10的含量。结果用pcDNA3-hIL-10质粒转染COS7细胞48 h后,在培养液中检测到hIL-10蛋白浓度为38 000 pg／mL,而对照组仅为55 pg／mL。hIL-10基因转染24 h后大鼠胰腺、肺和肝脏组织hIL-10的产生量达到峰值,以后逐渐下降,至96 h仍显著高于对照组(P<0．05),1周后降至正常水平。结论本研究构建的pcDNA3-hIL-10重组质粒可以在真核细胞及大鼠体内高效表达。

Purpose To construct the recombinant plasmid of human IL-10cDNA and to observe its transfection and expression in COS7 cells and in the tissues of pancreas, liver and lungs of Sprague Dawley（SD） rats. Methods pcDNA3-hIL-10 plasmid was constructed and confirmed by restriction enzyme mapping and DNA sequencing. COS7 cells and SD rats were transfected by SA liposomes-mediated pcDNA3-hIL-10 or pcDNA3. Levels of hIL-10 in the culture medium of COS7 cells and the tissues of pancreas, liver and lungs were determined by using ELISA kits. Results Forty-eight hours after the transfection of recombinant plasmid pcDNA3-hIL-10 into the COS7 cells, the concentration of hIL-10 in the culture medium of the transfected group was 38 000 pg/mL, while it was 55 pg/mL in the control group. The level of hIL-10 expression in the pcDNA3-hIL-10 group reached maximum at 24 hours post-transfection in pancreas, liver and lungs. Then the hIL-10 level decreased gradually and was still higher than that of the control （P〈0.05） 96 hours after transfection. There was no significant difference between hIL-10 transfected and control groups at 168 hours post-transfection. Conclusions The plasmid pcDNA3-hIL-10 expresses effectively in the transfected mammalian cells and tis- sues.