Preliminary Observation Of Homozygous Deletions Of RB1CC1 Gene In Disseminated Cells Of Bone Marrow Of Mouse Model Of Oral Carcinoma

Objective To explore the detection method of single disseminated cell genome in bone marrow of mouse model of oral carcinoma and observe the homozygous deletions of RB1CC1 gene of disseminated cells in bone marrow of mouse model of oral carcinoma in the severe abnormal hyperplasia stage.Methods The mouse model of oral carcinoma were induced with 200 μg/ml 4-nitroquinoline-l-oxide (4NQO) in drinking water. The mice were sacrificed regularly and bilateral femur were collected, Single nuclear cells were extracted by Ficoll density gradient centrifugation, and disseminated cells were detected by Immunocytochemistry with epithelial-specific cytokeratin antibodies. The cytokeratin positive(CK+) cells from 35 single nuclear cells smear of bone marrow of mouse oral carcinoma model were isolated by Laser Capture Microdissection(LCM), The DNA of a single cell was amplified by technique of single-cell Whole Genome Amplification(WGA), the second exon, the sixth exon and the seventh exon of RB1CC1 gene were selected for observing, the homozygous deletions of the exons of the CK+ cells were detected by Polymerase Chain Reaction(PCR). Four nomal,4 severe abnormal hyperplasia and 4 squamous cell carcinoma tongue were as control.Results Fourty six CK+cells were isolated by LCM, and 35 CK+ cells from the 35 smear respectively were successfully isolated, the success rate was 76%. Genomic DNA were successfully amplified by single-cell WGA in 3 out of 35, the success rate was 8.6%. Homozygous deletion of RB1CC1 gene were found in the second exon. Two cells of the 3 CK+ cells were found with homozygous deletion in the second exon, and two cases of the four squamous cell carcinoma cases were found with homozygous deletion in the second exon. Four nomal and 4 severe abnormal hyperplasia tongue were not found with homozygous deletion in the second exon. However, the deletions were not found in the rest two exons.Conclusion LCM technology combines single cell WGA technology and PCR technique can be applied to the analysis of gene mutation of disseminated cells in bone marrow of mouse model of oral carcinoma. Homozygous deletion of the second exon of RB1CC1 gene could be found in CK+ cells of bone marrow of mouse oral carcinoma model in the severe abnormal hyperplasia stage. The CK+ cells in the severe abnormal hyperplasia stage may be the disseminated tumor cells.