人降钙素基因在大鼠成肌细胞中的表达与鉴定

Human Calcitonin Gene Expression and Detection in Rat Myoblasts

目的建立稳定表达人降钙素(hCT)基因的细胞株(L6),观察人降钙素基因在大鼠成肌细胞中的表达和分泌情况。方法采用自行构建的5'端融合小鼠抗体轻链基因Igκ的信号肽序列的人降钙素基因分泌性真核表达载体pcDNA3.0-Igκ-hCT,采用脂质体介导方法将人降钙素基因导入大鼠成肌细胞;对照组用pcD-NA3.0空质粒转染。经G418筛选后,采用RT-PCR法和免疫细胞化学法检测人降钙素基因在大鼠成肌细胞中的表达,并用放免法检测细胞培养上清液降钙素的分泌。结果RT-PCR法和免疫细胞化学法均能检测到人降钙素基因在大鼠成肌细胞中的表达;并在连续传代的细胞培养上清液中检测到每106个细胞24h培养液中降钙素分泌量的平均值42.22ng。结论成功转染人降钙素基因真核表达载体pcDNA3.0-Igκ-hCT于大鼠成肌细胞中;该载体可在细胞中持续稳定表达,建立了稳定的人降钙素基因表达系统。

Objective To establish a rat myoblast cell line （L6） with stable expression of human calcitonin （hCT） gene and observe hCT expression and secretion in L6 cells transfected by hCT gene. Methods We constructed a recombinant eukaryotic expression plasmid, pcDNA3.0 -Igκ -hCT, which contains human calcitonin gene and Murine Igκ -chain leader sequence. The recombinant hCT pcDNA plasmid was transfected into L6 cells by cation liposomes and selected by G418. The control group transfected with plasmid pcDNA3.0. The mRNA and protein expression of hCT were detected by reverse transcription -PCR（ RT -PCR） and immunocytochemistry staining respectively, and the hCT secretion in the culture supernatant of 1/5 cells were analyzed by RIA. Results The expressions of hCT mRNA and protein could be detected by RT -PCR and immunocytochemistry staining in the recombinant hCT gene transfected 1/5 cells, but not in pcDNA3.0 transfected cells. During serial passages, we had detected average concentration of hCT, which was 42. 22ng/（ 10^6 - 24h） in the culture supernatant. Conclusion The pcDNA3.0 -Igκ -hCT was successfully transfected into 1/5 myoblast; The vector could express hCT sustainedly. The stable system of hCT gene expression was constructed.