摘要
目的构建小鼠resistin基因表达载体,并将其导入小鼠巨噬细胞系RAW264.7细胞中,从而建立能够高表达resistin的单核巨噬细胞系。方法从小鼠脂肪组织提取mRNA,PCR后连接到真核表达载体进行测序验证,同时构建慢病毒载体,感染至RAW264.7细胞中,通过荧光显微镜观察是否转染成功,ELISA方法检测蛋白分泌水平。结果双酶切结果显示构建的质粒中有大小为345bp的resistin插入条带;经过测序及BLAST比对,插入序列与Genebank中resistin的小鼠CDS序列相同,没有移码突变;荧光显微镜观察病毒感染后的RAW264.7细胞,红色荧光比率达到70%;ELISA检测培养的感染细胞上清中resistin分泌显著增加。结论成功获得了能够分泌resistin蛋白的单核/巨噬细胞。
Objective To construct and transfer mice resistin gene expression vector into RAW264.7 cells to set up mice mononuclear/macrophage cells which could highly express resistin.Methods mRNA was extracted from mouse adipose tissue,and was connected to eukaryotic expression vector after PCR.DNA sequencing and BLAST analysis were performed to verify the sequence of the cloned fragment.Lentivirus vector was constructed and infected into RAW264.7 cells.Fluorescence microscopy imaging was performed to observe the infection efficiency and the secretion of resistin was detected by ELISA.Results The results of double digestion showed that a 345 bp fragment was inserted into the blank vector.The sequence of the fragment was the same as the resistin CDS of mice in Gene bank without frame shift.The fluorescence microscopy imaging showed that the rate of the red fluorescence reached 70%in resistin-expressing-lentivirus infected RAW264.7 cells.Finaly,it was detected that the secretion of resistin increased significantly in the supernatant of infected cells using ELISA assay.Conclusion mononuclear macrophages which could secrete resistin were acquired successfully.
作者
赵鑫月
陈添娇
于皓
李孝琢
尚德淑
ZHAO Xin-yue;CHEN Tian-jiao;YU Hao;LI Xiao-zhuo;SHANG De-shu(Class 81,Phase 102,Shengjing Hospital,Shenyang 110004;Department of Developmental Cell Biology,Key Laboratory of Medical Cell Biolongy,Ministry of Eduration,Shenyang 110122;Class 48,Phase 103,College of Life Sciences,China Medical University,Shenyang 110122,China)
出处
《解剖科学进展》
2020年第5期500-503,共4页
Progress of Anatomical Sciences
基金
辽宁省教育厅重点实验室项目(LZ2015074)
辽宁省教育厅科学研究一般项目(LZ2019029)
“2018年辽宁省大学生创新创业训练计划”资助项目(201810159077)。
