红、白系细胞具有不同形式的KEL基因mRNA转录本  

Different KEL gene mRNA transcripts in reticulocyte and non-reticulocyte ceils

作　　者：王玲玲[1] 杨颖[1] 王晨[1] 张嘉敏[1] 郭忠慧[1] 李勤[1] 陈和平[1] 朱自严[1] 

机构地区：[1]上海市血液中心,200051

出　　处：《中华医学遗传学杂志》2008年第5期520-526,共7页Chinese Journal of Medical Genetics

基　　金：上海市卫生局科研课题基金（054028）

摘　　要：目的了解不同细胞KEL基因转录本的差异。方法分别抽取K阴性、K阳性、K0的DNA、红系细胞RNA和总RNA，应用逆转录PCR、巢式PCR分别扩增、测序及克隆测序分析KEL基因第1-19外显子以及第2～8外显子。同时，4种单抗标记后流式细胞术检测红、白细胞上的Kell蛋白表达情况。结果红系RNA均为忠实于基因组序列的正常KEL转录本，而K产生4种不同的转录本。但总RNA作模板时，可以发现不同个体产生不同的转录本，以正常、缺失第3外显子、第7外显子之前插入16bp内含子的转录本为最常见，这些异常拼接也见于K0的红系RNA；总RNA第1～19外显子扩增片段虽然电泳条带只有一条，但测序显示为多种序列的混合。流式细胞技术检测证实K0红细胞上无Kell抗原的表达，其余个体的红细胞均有Kell抗原的强表达，但其白细胞上有不同程度Kell抗原的弱表达或无表达。结论KEL基因在不同细胞中存在不同的转录本，可能导致Kell抗原在红、白细胞上有表达或无或弱表达。证实了KEL基因的表达转录研究中红系mRNA比总RNA更可靠。Objective To investigate the difference of the transcripts between reticnlocyte and non-reticulocyte cells in human blood. Methods Genomic DNA, reticulocyte RNA and total RNA of K^- , K^＋ and Kell-null（K0） were extracted, then PCR, reverse transcription-PCR（RT-PCR） and nested PCR followed by sequencing or cloning-sequencing were used to analyze the KEL gene mRNA exons 1-19 and exons 2-8. Four kinds of monoclonal antibodies were labeled to detect the expression of Kell glycoprotein on red cells or leukocytes with flow cytometry. Results In reticulocyte, only one normal KEL transcript faithful to the genomie structure was found in all tested samples except K0 which had 4 different transcripts. Sequence analysis of exons 2-8 of total RNA confirmed the alternative KEL transcripts existed in different samples, mostly caused by abnormal splicing, among them, skipping of exon 3 and a 16 bp insertion of intron 6 at the beginning of exon 7 were the most frequent. Although only one band was observed after amplifying the exons 1-19 from total RNA, the sequencing result showed it was a mixture of different sequences. There was strong expression of Kell glycoprotein on red cells except K0, but no or low expression on leucocytes by flow cytometry. Conclusion Alternative transcripts of KEL gene exist in different cells, which would be responsible for different Kell glycoprotein expression patterns on different cells. This study suggested that retieulocyte RNA was more suitable than total RNA for molecular study of KEL gene transcription.

关 键 词：KEL mRNA转录本 网织红细胞 巢式PCR Kell-null 流式细胞技术 

分 类 号：R686[医药卫生—骨科学]