稳定表达人降钙素基因大鼠成肌细胞株的建立

Establishment of rat myoblast line with stable expression of human calcitonin gene

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:实验于2004-06/2005-11在河南省肿瘤病理重点实验室完成。用已构建的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pCDNA3.0空质粒转染。经G418筛选后,用反转录-聚合酶链反应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:①反转录-聚合酶链反应法检测人降钙素基因的表达:反转录-聚合酶链反应扩增后,重组质粒载体转染组成肌细胞总RNA中检测到人降钙素基因的表达,而空载体转染组和未转染成肌细胞均未检测出目的基因(210bp);β-肌动蛋白为内对照约385bp。②免疫细胞化学法测定降钙素的表达:pCDNA3.0-Igκ-hCT转染组成肌细胞浆被黄染,提示有人降钙素表达;而空载体组胞浆染色阴性,无人降钙素表达。③细胞培养上清液中降钙素的含量:pCDNA3.0-Igκ-hCT重组载体转染组(hCT)1～10代细胞培养上清液人降钙素浓度差异无显著性(P>0.05),明显高于同代空载体转染组(pCDNA3.0)(P<0.001)。结论:稳定表达人降钙素基因的大鼠成肌细胞株成功建立,为进一步体内移植治疗骨质疏松症打下基础。

AIM： To establish a rat myoblast line （L6） with stable expression of human calcitonin （hCT） gene and observe the hCT expression and secretion in L6 cells. METHODS： The experiment was conducted in Henan Key Laboratory for Tumor Pathology between June 2004 and November 2005. A recombinant eukaryotic expression plasmid pCDNA3.0-lg l-hCT was constructed, which contained human calcitonin gene and murine IgK-chain leader sequence. The recombinant hCT cDNA plasmid was transferred into L6 cells by cation liposomes and selected by G418. The control group was transfected with plasmid pCDNA3.0. The mRNA and protein expression of hCT were detected by reverse transcription-PCR （RT-PCR） and immunocytochemical staining respectively, and the hCT secretion in the culture supernatant of L6 cells were analyzed by radiommunoassay. RESULTS： （1） Expressions of hCT detected by RT-PCR： hCT expression was detected in the total RNA of the recombinant hCT gene transfected L6 cells by amplified RT-PCR; but no target gene （210 bp） was detected in the pCDNA3.0 transfected cells and untransfected cells; 13-actin served as internal control （385 bp）. （2）Expressions of hCT detected by immunocytochemical staining： Myoblast plasm in the pCDNA3.0-1gK-hCT transfected group was stained yellow, which indicated that there were hCT expressions; but no expression was found in the pCDNA3.0 transfected cells. （3）Content of hCT in culture supernatant of L6 cells： No significant difference was found in the concentration of hCT in the culture supematant during serial passages （1-10 passages, P 〉 0,05）, but it was higher than the secretion of pCDNA3.0 transfected cells （P 〈 0.01 ）. CONCLUSION： The rat myoblast cell line with stable expression of human calcitonin gene is successfully established, which lay a foundation for transplantation therapy of osteoporosis in vivo.