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目的建立乙酰肝素酶可变剪接体Splice 5稳定转染中国仓鼠卵巢(CHO)细胞株,并进行亚细胞定位分析。方法将表达乙酰肝素酶全长的质粒和乙酰肝素酶可变剪接体Splice 5质粒转染入CHO细胞,经G418进行筛选后,挑取单克隆进行培养,RT-PCR检测筛选克隆。用激光共聚焦显微镜测定其亚细胞定位。结果成功获得乙酰肝素酶可变剪接体Splice 5稳定转染株,并研究了其亚细胞定位情况。野生型乙酰肝素酶以颗粒状分布于细胞质中,Splice 5以弥散状分布于细胞质中,且在内质网、高尔基体、溶酶体、细胞膜均有分布。结论 Splice 5和野生型乙酰肝素酶在CHO细胞中分布形式和定位的不同,提示Splice 5可能具有其他的生物学功能。
OBJECTIVE: To establish a stable transfected Chinese hamster ovary (CHO) cell line by using heparanase alternative splicing Splice 5 and perform subcellular localization analysis. Methods The full-length heparanase plasmid and the heparanase splice variant Splice 5 plasmid were transfected into CHO cells. After screening by G418, the clones were selected and cultured. The clones were screened by RT-PCR. Subcellular localization was determined by laser scanning confocal microscopy. Results The hemagglutinin Splice 5 stable transfectant was successfully obtained and its subcellular localization was studied. Wild-type heparanase is distributed in the cytoplasm in the form of granules. Splice 5 is distributed in the cytoplasm in a diffuse manner, and is distributed in the endoplasmic reticulum, the Golgi apparatus, lysosomes and cell membranes. Conclusion The distribution and localization of Splice 5 and wild-type heparanase in CHO cells suggest that Splice 5 may have other biological functions.