人颗粒溶素在小鼠巨噬细胞中的表达及其致细胞损伤与诱导凋亡的作用

目的构建编码活化的人细胞毒性T淋巴细胞(CTL)表达的天然颗粒溶素(granulysin,GLS)基因的真核表达载体pBudCE4.1/GLS,并转染不同种系来源的小鼠腹腔巨噬细胞。观察GLS在其中的表达及其可能的致细胞损伤与诱导凋亡的作用。方法将人GLS的编码序列从pEGFP-C1/GLS质粒亚克隆入pBudCE4.1真核表达载体中,鉴定正确后转染不同种系来源的小鼠腹腔巨噬细胞。采用RT-PCR及免疫细胞化学染色法检测GLS的表达。通过测定转染细胞培养上清中的乳酸脱氢酶反映细胞的损伤;通过胞核DNA荧光染色法检测细胞的凋亡。结果成功地构建了读码框正确的pBudCE4.1/GLS重组子,并在靶细胞中得到高效表达。表达后48h,观察表达产物对宿主细胞未见有明显的致细胞损伤和诱导凋亡的作用。结论人GLS基因能够在小鼠巨噬细胞中有效表达。该表达产物对小鼠巨噬细胞无明显的致细胞损伤与诱导凋亡的作用,为进一步建立研究GLS免疫学活性的动物模型奠定了基础。

Expression of human granulysin in murine macrophages and its effects on cell damage and apoptosis

AIM: To construct an eukaryotic expression vector pBudCE4.1/GLS encoding human granulysin(GLS) derived from activated CTLs and observe its effects on cell damage and apoptosis when expressed in macrophages of various murine germ lines. METHODS: The gene sequence coding GLS carried on pEGFP-C1/GLS was subcloned into pBudCE4.1 plasmid and then transfected into different murine macrophages. The expression of GLS was detected by RT-PCR and immunocytochemical staining. The impairment and apoptosis of the host cells was assesed by measuring LDH in culture media and fluorescent staining of nuclear DNA, respectively. RESULTS: The pBudCE4.1/GLS recombinant containing accurate ORF of GLS was obtained and successfully expressed in the target cells at 48 h after transfection. No obvious effects on cell damage and apoptosis was detected during this course. CONCLUSION: The gene coding human GLS can be expressed in murine macrophages. The expressed product has no obvious effects on cell damage and apoptosis.[