Preliminary Observation Of Homozygous Deletions Of TP53 Gene In Disseminated Cells Of Submandibular Lymph Nodes Of Mouse Oral Carcinoma Model

Objective:(1) To preliminarily discuss the method to obtain the DNA of disseminated tumor cells (DTC) in submandibular lymph nodes of mouse oral carcinoma lymphatic metastasis model. (2) To observe and compare the homozygous deletions of TP53 gene in DTC of submandibular lymph nodes and their primary tumor in different stages.Methods:(1) The tongue and submandibular lymph nodes tissue were collected respectively from normal oral mucosa, simple-to-moderate dysplasia, severe dysplasia, oral squamous cell carcinoma(OSCC) without lymph node metastases and OSCC with lymph node metastases in mouse, which were induced with 4-nitroquinoline-l-oxide(4NQO) in drinking water. (2) The DNA of the tongue tissue were extracted directly. The serial freezing microtome sections of submandibular lymph nodes tissue were stained by pan-ck antibody, and then the adjacent sections of CK+ cells(DTC) section were stained by HE. The CK+ cells were captured by laser micro cutting technology(LCM). Single-celled whole genome amplification(WGA) was used to amplify the DNA of DTCs. (3) The homozygous deletions of the fifth, sixth, seventh and eighth exons of TP53 gene were detected by polymerase chain reaction(PCR), compared with the normal submandibular lymph nodes and primary tumor of mouse.Results:(1) Eight specimens of CK+ cells (DTC) were acquire in submandibular lymph nodes of mouse in different stages.24 specimens were collected in total of the three groups of DTC cells on each positive specimens were captured by LCM.15 specimens of DNA of CK+ cells in submandibular lymph nodes were acquired by WGA, the total success rate were 62.5%.(2) The homozygous deletions of TP53 gene were found in CK+ cells of submandibular lymph nodes metastasis in different stages:1(1/2) case of exom 5 and 1(1/2) case of exom 8 in severe dysplasia,1 (1/3) case of exon 5 in OSCC without lymph node metastases,2 (2/8) case of exon 5 and 1 (1/8) case of exon 8 in OSCC with lymph node metastases. The homozygous deletions of TP53 gene were also found in primary tumor:1 (1/4) case of exom 5 in OSCC without lymph node metastases,1 (1/4) case of exon 5、6、8, respectively, in OSCC with lymph node metastases, while no homozygous deletion was found in primary tumor of precancerosis.Conclusion:(1) The combination of serial freezing microtome sections, LCM and WGA may be an ideal method to obtain the DNA of DTC accurately. (2) The CK+ cells, which were detected in submandibular lymph nodes in mouse oral carcinoma model, may be DTC. (3) In the process of oral carcinogenesis in mouse, the mutation of TP53 gene may be involved in DTC migration and play an important role in the metastasis of oral cancer.