摘要
目的：探讨结肠癌细胞株HT-29中Tn（＋）细胞表达Tn抗原的机制。方法免疫磁珠分选结肠癌细胞株HT-29中HT-29-Tn（＋）细胞与HT-29-Tn（－）细胞；RT-PCR、PCR 扩增Cosmc基因，测序比对是否存在基因突变。野生型Cosmc转染正常及肿瘤细胞；Western blot检测转染后Cosmc蛋白表达状况；荧光分析法检测转染前后T-synthase活性。结果分选后的HT-29-Tn（＋）细胞缺乏T-synthase活性，Cosmc基因编码区缺失，转染WtCosmc可恢复Tn抗原阳性细胞的T-synthase活性，抑制Tn抗原表达。结论 Cosmc基因编码区缺失引起T-synthase活性丧失导致人结肠癌HT-29-Tn（＋）细胞表达Tn抗原。
Objective To explore the mechanism of Tn antigen expression in colon cancer cells HT-29.Methods Tn（＋） and Tn（-） cells were separated from human colon cancer cell line HT-29 by im-mune magnetic beads.Total RNA, genomic DNA （ gDNA） and cytoplasmic proteins in these cells were ex-tracted by using Trizol, DNA preparation kit and nuclear and cytoplasmic extraction reagents respectively.T-synthase activity was measured by a fluorescent assay.Cosmc and T-synthase transcriptional levels were ana-lyzed by RT-PCR using mRNA as the templates.The coding sequence （ CDS） and CpG islands of Cosmc were amplified by PCR using gDNA as the templates.Amplified products were analyzed on 1%agarose gel. The expected bands were purified, and then sequenced to examine Cosmc mutation.Wild type Cosmc （ Wt-Cosmc） were transfected into tumor cell lines and normal cells to define the function of Cosmc.The expres-sions of Cosmc protein in these cells were then examined by Western blot.Results Although no mutation appeared in HT-29-Tn（-） cells, the deletion of CDS and inactivity of T-synthase were observed in HT-29-Tn（＋） cells.Additionally, transfected WtCosmc restored T-synthase activity and then decreased Tn antigen expression in Tn antigen positive cells.Conclusion The absence of CDS in Cosmc gene resulted in the loss of T-synthase activity and consequent Tn antigen expression in HT-29-Tn（＋） cells.
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
2015年第3期219-224,共6页
Chinese Journal of Microbiology and Immunology
基金
国家自然科学基金（30972778）
山东省自然科学基金（Y2007C143,ZR2014HM020）
