小鼠口腔癌模型骨髓播散细胞RB1CC1纯合性缺失的初步研究

Preliminary observation of homozygous deletions of RB1CC1 gene in disseminated cells of bone marrow of mouse oral carcinoma model

目的初步探讨小鼠口腔癌模型重度异常增生时期骨髓播散细胞RB1CC1基因纯合性缺失及其意义。方法应用激光捕获显微切割(LCM)技术捕获35张重度异常增生时期小鼠骨髓单个核细胞涂片的单个细胞角蛋白阳性(CK+)细胞,单细胞全基因组扩增技术(WGA)扩增其DNA,PCR检测其视网膜母细胞瘤诱导卷曲蛋白(RB1CC1)的纯合性缺失情况,并与舌部正常、重度异常增生以及鳞癌组织各4例对照。结果 LCM成功捕获CK+细胞35个,WGA成功扩增3个,RB1CC1纯合性缺失情况分别为单个CK+细胞(2/3),舌癌变组织(2/4),舌部正常组织(0/4),重度异常增生组织(0/4)。结论小鼠口腔癌模型重度异常增生时期骨髓CK+细胞有RB1CC1基因纯合性缺失,该时期骨髓CK+细胞可能是播散肿瘤细胞。

Objective To observe the homozygous deletions of RB1CC1 gene of disseminated cells in bone marrow of mouse oral carcinoma model in the severe abnormal hyperplasia stage.Methods The cytokeratin positive （ CK＋） cells from 35 single nuclear cells smear of bone marrow of mouse oral carcinoma model in the severe abnor-mal hyperplasia stage were isolated by Laser Capture Microdissection （ LCM） .The DNA of a single cell was ampli-fied by the technique of single-cell Whole Genome Amplification （WGA） and the homozygous deletions of RB1CC1 gene of the DTC were detected by Polymerase Chain Reaction （ PCR） .Four normal, 4 severe abnormal hyperplasia and 4 squamous cell carcinoma tongue were taken as control.Results Thirty five CK＋cells from the 35 smear re-spectively were successfully isolated by LCM.Genomic DNA were successfully amplified by single-cell WGA in 3 out of 35 cells.Homozygous deletions of RB1CC1 gene were found in 2 of the 3 CK＋cells and 2 in 4 cases of squamous cell carcinoma tongue mucosa.However, the deletion was not found in the rest tissues.Conclusion Homozygous deletion of RB1CC1 gene of the CK＋positive cells in the severe abnormal hyperplasia stage may be the disseminated tumor cells.