人宫颈黏液HMGN2鉴定及在宫颈组织的表达  

Characterization of HMGN2 in Human Cervical Mucus and Studying Its Expression in Cervical Tissue

作　　者：王莉莉[1] 何跃东[2] 黄宁[1] 李明[1] 熊文碧[1] 冯云[1] 吴琦[1] 王伯瑶[1] 潘小玲[2] 

机构地区：[1]四川大学华西医学中心基础医学与法医学院感染免疫研究室,成都610041 [2]四川大学华西第二医院,成都610041

出　　处：《生物化学与生物物理进展》2008年第5期577-583,共7页Progress In Biochemistry and Biophysics

基　　金：国家自然科学基金资助项目(39800146,30470763,30671963);纽约中华医学会基金(98-681)~~

摘　　要：为探讨人子宫颈黏液的抗菌机制,采用酸性尿素聚丙烯酰胺凝胶电泳和反相高压液相色谱技术,从人子宫颈黏液酸溶性提取物鉴定出对大肠杆菌氨苄青霉素耐药株ML-35p有较强的抗菌活性的多肽HCP-21和HCP-26.蛋白质N端氨基酸序列测定和精确质谱分子质量测定证明,HCP-21为HMGN2,HCP-26为SLPI片段.提取原代培养宫颈上皮细胞的总RNA,采用RT-PCR技术扩增出一条与HMGN2 cDNA大小相同(270bp)目的基因片段,表明在生理状况下,宫颈上皮细胞即可表达其mRNA.制备HMGN2多克隆抗体,对生理状态下宫颈组织切片和宫颈黏液涂片进行HMGN2分子免疫组化分析表明,该分子主要分布于子宫颈黏膜层,并存在于子宫颈黏液.HMGN2分子在宫颈黏膜上皮组织和黏液中固有表达,可能在子宫颈天然免疫防御中起重要作用.To explore the antimicrobial mechanism of human cervical mucus, HCP-21 and HCP-26 were isolated and purified from acid-soluble extracts of human cervical mucus by acid-urea polyacrylamide gel electrophoresis and reversed-phase high-performance liquid chromatography （RP-HPLC）. Both molecules could effectively kill E. coil ML-35p determined by agarose radial diffusion assay. The N-terminal amino acid sequence of HCP-21 was PKRKAEGDAK and its molecular mass was 9 263.62 identified by amino acid sequencing and mass spectrometry analysis. HCP-21 showed 100% identity to HMGN2 （high mobility group protein N2, HMGN2） fragment 2- 11 by GenBank BLAST searching, and had the same molecular mass as HMGN2. So it was certain that HCP-21 was HMGN2. HCP-26 was SLPI （secretory leukocyte protease inhibitor, SLPI） fragment by N-terminal amino acid sequencing which was SGKSFKAGVC. It was the same with SLPI fragment 26-35 by GenBank BLAST searching. Total RNA was extracted from primary culture cervical epithelial cells, and the specific primer was designed based on HMGN2 eDNA sequence. RT-PCR showed that a fragment about 270 bp was amplified, which was same to HMGN2 eDNA. It was suggested that cervical epithelial cells could express HMGN2 mRNA in physiological condition. The fusion protein GST-HMGN2 was purified by low-pressure chromatography. The antiserum of HMGN2 was prepared from the immune rabbit with the fusion protein. Cervical tissue paraffin section and cervical mucus smear were detected by immunhistochemistry. The staining results showed that HMGN2 mainly distributed in mucosa surface of cervix and existed in cervical mucus. HMGN2 constitutively expressed in cervical mucosa and mucus, so it might play an important role in innate defensive of cervical.

关 键 词：女性生殖道 子宫颈 抗菌肽 HMGN2 

分 类 号：Q786[生物学—分子生物学]