遗传
遺傳
유전
HEREDITAS(BEIJING)
2014年
6期
559-566
,共8页
郝佳洁%王春丽%顾文跃%程潇钰%张钰%徐昕%蔡岩%王明荣
郝佳潔%王春麗%顧文躍%程瀟鈺%張鈺%徐昕%蔡巖%王明榮
학가길%왕춘려%고문약%정소옥%장옥%서흔%채암%왕명영
染色体畸变%BAC DNA%染色体臂和区段%荧光原位杂交%食管癌细胞
染色體畸變%BAC DNA%染色體臂和區段%熒光原位雜交%食管癌細胞
염색체기변%BAC DNA%염색체비화구단%형광원위잡교%식관암세포
chromosomal aberration%BAC DNA%chromosome arms and regions%fluorescence in situ hybridization%esophageal cancer cells
染色体畸变是恶性肿瘤细胞的重要遗传学特征,文章旨在应用BAC DNA克隆鉴定食管癌细胞中的染色体臂和染色体区段的畸变。针对染色体各区段选取5~10个1 Mb BAC DNA,分别混合制备成特定染色体区段的BAC DNA混合克隆,然后将染色体臂上覆盖所有区段的上述混合克隆进一步混合制备成特定染色体臂BAC DNA混合克隆。利用简并寡核苷酸引物聚合酶链反应(Degenerate oligonucleotide primed PCR, DOP-PCR)标记染色体臂探针,利用切口平移法(Nick translation)标记染色体区段探针,并对食管癌细胞中期染色体进行荧光原位杂交(Fluorescence in situ hybridization, FISH)分析。正常人外周血淋巴细胞中期染色体FISH结果显示,上述方法标记的探针具有较高的特异性。进一步利用染色体臂混合探针,确定了多个食管癌细胞中的染色体重排所涉及的特定染色体臂;利用染色体区段混合探针,鉴定出KYSE140的t(1q;7q)衍生染色体中1q上的断点范围位于1q32-q41。文章成功建立了1 Mb BAC DNA混合克隆探针标记技术,并鉴定出多个食管癌细胞中的染色体臂和染色体区段畸变,不仅为利用 M-FISH 技术鉴定肿瘤细胞中的染色体畸变提供了更为准确的方法,而且还可能进一步将该法推广应用于恶性血液病的核型分析以及产前诊断。
染色體畸變是噁性腫瘤細胞的重要遺傳學特徵,文章旨在應用BAC DNA剋隆鑒定食管癌細胞中的染色體臂和染色體區段的畸變。針對染色體各區段選取5~10箇1 Mb BAC DNA,分彆混閤製備成特定染色體區段的BAC DNA混閤剋隆,然後將染色體臂上覆蓋所有區段的上述混閤剋隆進一步混閤製備成特定染色體臂BAC DNA混閤剋隆。利用簡併寡覈苷痠引物聚閤酶鏈反應(Degenerate oligonucleotide primed PCR, DOP-PCR)標記染色體臂探針,利用切口平移法(Nick translation)標記染色體區段探針,併對食管癌細胞中期染色體進行熒光原位雜交(Fluorescence in situ hybridization, FISH)分析。正常人外週血淋巴細胞中期染色體FISH結果顯示,上述方法標記的探針具有較高的特異性。進一步利用染色體臂混閤探針,確定瞭多箇食管癌細胞中的染色體重排所涉及的特定染色體臂;利用染色體區段混閤探針,鑒定齣KYSE140的t(1q;7q)衍生染色體中1q上的斷點範圍位于1q32-q41。文章成功建立瞭1 Mb BAC DNA混閤剋隆探針標記技術,併鑒定齣多箇食管癌細胞中的染色體臂和染色體區段畸變,不僅為利用 M-FISH 技術鑒定腫瘤細胞中的染色體畸變提供瞭更為準確的方法,而且還可能進一步將該法推廣應用于噁性血液病的覈型分析以及產前診斷。
염색체기변시악성종류세포적중요유전학특정,문장지재응용BAC DNA극륭감정식관암세포중적염색체비화염색체구단적기변。침대염색체각구단선취5~10개1 Mb BAC DNA,분별혼합제비성특정염색체구단적BAC DNA혼합극륭,연후장염색체비상복개소유구단적상술혼합극륭진일보혼합제비성특정염색체비BAC DNA혼합극륭。이용간병과핵감산인물취합매련반응(Degenerate oligonucleotide primed PCR, DOP-PCR)표기염색체비탐침,이용절구평이법(Nick translation)표기염색체구단탐침,병대식관암세포중기염색체진행형광원위잡교(Fluorescence in situ hybridization, FISH)분석。정상인외주혈림파세포중기염색체FISH결과현시,상술방법표기적탐침구유교고적특이성。진일보이용염색체비혼합탐침,학정료다개식관암세포중적염색체중배소섭급적특정염색체비;이용염색체구단혼합탐침,감정출KYSE140적t(1q;7q)연생염색체중1q상적단점범위위우1q32-q41。문장성공건립료1 Mb BAC DNA혼합극륭탐침표기기술,병감정출다개식관암세포중적염색체비화염색체구단기변,불부위이용 M-FISH 기술감정종류세포중적염색체기변제공료경위준학적방법,이차환가능진일보장해법추엄응용우악성혈액병적핵형분석이급산전진단。
Chromosomal aberration is an important genetic feature of malignant tumor cells. This study aimed to clarify whether BAC DNA could be used to identify chromosome region and arm alterations. For each chromosome region, five to ten 1 Mb BAC DNA clones were selected to construct mixed BAC DNA clones for the particular region. All of the mixed clones from regions which could cover the whole chromosome arm were then mixed to construct mixed BAC DNA clones for the arms. Mixed BAC DNA probes of arms and regions were labeled by degenerate oligonucleotide primed PCR (DOP-PCR) and Nick translation techniques, respectively. The specificities of these probes were validated by fluorescence in situ hybridization (FISH) on the metaphase chromosomes of normal human peripheral blood lymphocytes. FISH with arm-specific mixed BAC DNA probes showed that chromosomal rearrangements and involved chromosome arms were confirmed in several esophageal cancer cells. By using region-specific mixed probes, the breakpoint on 1q from the deriva-tive chromosome t(1q;7q) was identified in 1q32-q41 in esophageal KYSE140 cells. In conclusion, we established an effec-tive labeling method for 1 Mb BAC DNA mixed clone probes, and chromosome arm and region rearrangements could be identified in several esophageal cancer cells by using these probes. Our study provides a more precise method for identifica-tion of chromosomal aberration by M-FISH, and the established method may also be applied to the karyotype analysis of hematological malignancies and prenatal diagnosis.