中华微生物学和免疫学杂志
中華微生物學和免疫學雜誌
중화미생물학화면역학잡지
CHINESE JOURNAL OF MICROBIOLOGY AND IMMUNOLOGY
2015年
3期
219-224
,共6页
于晓锋%杜镇镇%孙旭红%石传芹%张怀祥%胡涛
于曉鋒%杜鎮鎮%孫旭紅%石傳芹%張懷祥%鬍濤
우효봉%두진진%손욱홍%석전근%장부상%호도
Tn抗原%HT-29细胞%Cosmc%基因突变
Tn抗原%HT-29細胞%Cosmc%基因突變
Tn항원%HT-29세포%Cosmc%기인돌변
Tn antigen%HT-29 cells%Cosmc%Mutation
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。
目的:探討結腸癌細胞株HT-29中Tn(+)細胞錶達Tn抗原的機製。方法免疫磁珠分選結腸癌細胞株HT-29中HT-29-Tn(+)細胞與HT-29-Tn(-)細胞;RT-PCR、PCR 擴增Cosmc基因,測序比對是否存在基因突變。野生型Cosmc轉染正常及腫瘤細胞;Western blot檢測轉染後Cosmc蛋白錶達狀況;熒光分析法檢測轉染前後T-synthase活性。結果分選後的HT-29-Tn(+)細胞缺乏T-synthase活性,Cosmc基因編碼區缺失,轉染WtCosmc可恢複Tn抗原暘性細胞的T-synthase活性,抑製Tn抗原錶達。結論 Cosmc基因編碼區缺失引起T-synthase活性喪失導緻人結腸癌HT-29-Tn(+)細胞錶達Tn抗原。
목적:탐토결장암세포주HT-29중Tn(+)세포표체Tn항원적궤제。방법면역자주분선결장암세포주HT-29중HT-29-Tn(+)세포여HT-29-Tn(-)세포;RT-PCR、PCR 확증Cosmc기인,측서비대시부존재기인돌변。야생형Cosmc전염정상급종류세포;Western blot검측전염후Cosmc단백표체상황;형광분석법검측전염전후T-synthase활성。결과분선후적HT-29-Tn(+)세포결핍T-synthase활성,Cosmc기인편마구결실,전염WtCosmc가회복Tn항원양성세포적T-synthase활성,억제Tn항원표체。결론 Cosmc기인편마구결실인기T-synthase활성상실도치인결장암HT-29-Tn(+)세포표체Tn항원。
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.